A

MAJOR PROJECT REPORT ON,

JOINT PROJECT WITH AKOLA CHEMICALS LIMITED,
AKOLA
“SYNTHESIS OF PROTEIN FORM HUMAN HAIRS USING
ACID HYDROLYSIS”
SUBMITTED BY
MISS. DIVYA G. DAF

MISS. MANSI A. PONKSHE

MR. SUJAY A. CHOUDHARI

MR. TANMAY M. DALVI

(B .Tech. Chemical Final year)

DR. S.S. TAYDE DP.V.THORAT

(Co- Guide) (Guide & H.O.D)

SHRI SHIVAJI EDUCATION SOCIETY’S, AMRAVATI’S

COLLEGE OF ENGINEERING & TECHNOLOGY, BABHULGAON (JH), AKOLA (M.S) 444104

DEPARTMENT OF CHEMICAL ENGINEERINGSESSION

2018 – 2019
 A
MAJOR PROJECT REPORT ON,
JOINT PROJECT WITH AKOLA CHEMICALS LIMITED,
AKOLA
“SYNTHESIS OF PROTEIN FORM HUMAN HAIRS USING
ACID HYDROLYSIS”
SUBMITTED BY
MISS. DIVYA G. DAF

MISS. MANSI A. PONKSHE

MR. SUJAY A. CHOUDHARI

MR. TANMAY M. DALVI

(B .Tech. Chemical Final Year)

DR. S.S. TAYDE DR. P.V. THORAT

(Co- Guide) (Guide & H.O.D)

SHRI SHIVAJI EDUCATION SOCIETY’S, AMRAVATI’S

COLLEGE OF ENGINEERING & TECHNOLOGY, BABHULGAON (JH), AKOLA (M.S) 44410

DEPARTMENT OF CHEMICAL ENGINEERINGSESSION: 2018 – 2019

 CERTIFICATE
This is to certify that,

MISS. DIVYA G. DAF

MISS. MANSI A. PONKSHE

MR. SUJAY A. CHOUDHARI

MR. TANMAY M. DALVI

(B .Tech. Chemical Final Year)

Have submitted their thesis report on

“SYNTHESIS OF PROTEIN USING HUMAN HAIRS BY ACID

HYDROLYSIS”
During the academic session 2018-2019 in a satisfactory manner for the partial fulfillment
of the requirement for the Degree of Bachelor of Technology (Chemical Engineering) under
affiliation of Sant Gadge Baba Amravati University, Amravati.

DR. S S. TAYDE DR. P V.THORAT

(Co- Guide) (Guide & H.O.D)

SESSION: 2018 - 2019

 ACKNOWLEDEGMENT

We would like to place on record our deep sense of gratitude to Dr. P V. Thorat and Dr. S S.
Tayde, Department of Chemical Engineering, C.O.E.T, Akola, for their stimulating guidance,
continuous encouragement and supervision throughout the course of present work. We express my
sincere gratitude to Mr. Mudaabbir Ul Haque, Akola Chemicals Limited, Akola, for his generous
guidance, help and useful suggestions.

Our sincere thanks to the staff of chemical engineering department both teaching and non-teaching,
without their help it would not have been possible for us to complete this project. Lastly, we would like
to acknowledge all those who directly or indirectly helped us for the successful completion of this
project.

MISS. DIVYA G. DAF

MISS. MANSI A. PONKSHE

MR. SUJAY A. CHOUDHARI

MR. TANMAY M. DALVI

(B .Tech. Chemical Final Year)

 INDEX

CONTENTS PAGE NO.

ABSTRACT IV
Chapter 1 Introduction 01
1.1 General Introduction 01
1.2 Background & History
1.3 Aims & Objectives
1.4 Scope & Limitations
Chapter 2 Literature Review
2.1 Human Hair
2.1.1 Introduction
2.1.2 Composition of Human Hair

2.2 Protein
2.2.1 Introduction
2.2.2 Classification & structures
2.2.3 Protein from Natural Resources
2.2.4 Market & Statistical Data on Protein
2.2.5 Reactions in Protein Recovery
2.3 Amino Acids
2.3.1 Introduction
2.3.2 Amino Acids Composition
2.3.3 Amino Acids derived from Protein
2.4 Hydrolysis
2.4.1 Introduction
Types of Hydrolysis
2.4.2 Alkaline Hydrolysis
2.4.3 Enzymatic Hydrolysis
2.4.4 Acid Hydrolysis
Chapter 3 Experimental Details
3.1 Raw Materials
3.2 Research Methodology
3.3 Experimental Set Up
3.4 Process Flow Diagram
Chapter 4 Testing & Characterization
 4.1 Biuret Test
4.2 Xanthoproteic Test
4.3 Millions Test
4.4 Ninhydrin Test
4.5 Sulfur Test
4.6 Kjeldahl’s Test
4.7 UV Spectrophotometer Test
Chapter 5 Result & Discussion
5.1 Effect of Temperature on Product
5.2 Effect of Concentration
5.3 Effect of Time
5.4 Cost Estimation
Chapter 6 Conclusion
Chapter 7 References
Appendix
 ABSTRACT

Significant amount of hair is being generated in tanning industry. This project mainly focuses on the
production of valuable amino acids from these hair wastes, which are generated from the beam house
process. During liming process the hair removed from the skins(generally from the paste liming/hair saving
method) doesn’t have great end use and hence by hydrolysing the hair and isolating the amino acids
separately they can be used for high end purposes. These hairs comprise predominantly keratin protein,
which have polypeptide chains containing different types of amino acids. Amino acids have high value
applications especially for neutraceutical and pharmaceutical applications. The work plan in meeting the
main objective of this project includes four major steps viz., i) “Pre-treatment of the hair waste” to clean
the hair for the removal of lime, salt etc.ii)The cleaned hair waste need to be further subjected to
“Hydrolysis for the extraction of amino acids” (chemical and or enzymatic method), characterization of the
degree of the hydrolysis and optimising the conditions for the same. iii) “Characterization ofthe
hydrolysate” by using techniques likeHPLC. iv)Development of simpler methods for the isolation of amino
acids.

Keywords: Amino Acids, Hydrolysis, Protein, Keratin.

 CHAPTER 1: INTRRODUCTION

1.1GENERAL INTRODUCTION

Proteins are biological molecules which are polymers of amino-acids. Keratin is a form of
protein which are mainly found in the human hair. Keratin fills up around 70-85% of human hair and one
molecule of keratin measures 10-15 nm. The primary component of keratin is sulfur because of the
presence of the amino acid cysteine. It accounts for 24% of total amino acids present in the human hairs.
An enormous quantity of keratin is available In the form of hair, horns, feathers etc. As keratin is non
biodegradable, it becomes necessary to utilize this protein in some forms and keratin has wide
applications industrially and domestically. This paper deals with the simplified method of extraction of
keratin, lab tests for confirmation and its applications.
The nutritional value is determined by the content of amino acid in the human hair. These
amino acids are methionine and histidine and their number decrease with the age. The main content of
the human hair is keratine which is biopolymer having three dimensional fibrous structure. It consist
small nano amino acids which are polymerized in the known sequence to the molecular weight of
protein of order of 10-100nm.
The distribution of amino acid in hair can divulge information regarding the health and a
means of detecting the diseases by segmentation of hairs as well as attributes of an individual. Therefore,
an nonenzymatic method of hair digestion and profiling is required. In addition to optimizing and
validating a method for measuring the distribution of amino acids in human hair, a robust and
comprehensive approach to objectively compare the most effective means of extracting and manipulating
chromatographic data to obtain the best limits of detection, linearity, and sensitivity are provided.
In this case the keratin is very insoluble so the best way to hydrolyse is by using a very
strong acid or base where the hairs can be broken down in to very small pieces that’s is breakdown of hair
that leads to the formation of protein.
Several important new and relatively recent contributions to the structure of the cell membrane
complex, the composition of the surface layers of hair, the overall structure of the hair fiber and its
follicle have been added to this Chapter. Recent studies revealed details about endogenous and exogenous
hair lipids and the critical involvement of proteins and free lipids in the surface layers of hair
including lipid contributions to the protective properties of the cuticle and the isoelectric point. Advances
in the classification and characterization of the different proteins and genes involved in keratin and
keratin associated proteins in human hair are summarized in this Chapter and the analysis of protein
fragments from hair Human hair is a complex tissue consisting of several morphological
components (see Chap. 1), and each component consists of several different chemical types [1]. Hair is an
integrated system in terms of its structure and its chemical and physical behavior wherein its components
can act separately or as a unit. For example, the frictional behavior of hair is related primarily to the
cuticle,yet, the cuticle, the cortex and its intercellular components act in concert to
determine the softness of hair. The tensile behavior of human hair is determined largely by the cortex, yet
we have learned that the physical integrity of the fiber to combing and grooming forces is also affected by
the non-keratin components of the cuticle and the cell membrane complex. Nevertheless, for simplicity
and ease of discussion, the different types of chemicals that comprise human hair are
generally described separately in this Chapter.
Depending on its moisture content (up to 32% by weight), human hair, consists of
approximately 65% to 95% proteins. Proteins are condensation polymers of amino acids. The structures
of those amino acids that are found in human hair are depicted in Table 2.1. Because of the large number
of chemical reactions that human hair is subjected to by permanent waves, chemical bleaches, alkaline
straighteners and sunlight exposure, many of the proteins are fragmented and several of these amino acids
are converted to amino acid derivatives depicted in Table 2.2. The remaining constituents are water,
lipids (structural and free), pigment, and trace elements that are generally not free, but combined
chemically with side chains of protein groups or with fatty-acid groups of sorbed or bound
lipid. These different components of hair: proteins, lipids, water and trace elements are described
separately in this Chapter while pigments are described in more detail in Chap. 5.Studies of the
proteinaceous matter of human hair may be classified according to the following types of investigation:
Studies of individual or several amino acids, Analysis of types of amino acids, Fractionation and peptide
analysis, Expression of genes, using in situ hybridization or reverse transcriptase- polymerase chain
reaction (RT-PCR) expression by hair follicles or the use of specific protein antibodies or related
techniques.
Most studies of individual amino acids of keratin fibers involve the amino acids cystine or
tryptophan. Quantitation of cystine can be accomplished by chemical analysis of mercaptan with [2, 3] or
without hydrolysis [4] or spectrophotometrically on intact hair [5, 6]. With increasing sophistication in
instrumental analysis, ESCA, SIMS, and different absorbance, reflectance and fluorescence techniques,
spectrophotometric analysis on intact hair is becoming increasingly important.
 Chemical analyses for tryptophan have been described by Block and Bolling [7]
and are all hydrolytic procedures. McMillen and Jachowicz [8] based on prior work in the wool industry
analyzed tryptophan and its kynurenine reaction products by fluorescence spectroscopy using excitation
wavelengths of 290, 320 and 350 nm which provides emission bands at 345, 420 and 465 nm. The
emission band with a maximum at 345 nm corresponds to Tryptophan with an absorption maximum at
about 360 nm. The emission peak at 465 nm from excitation at 320 and 350 nm matches the emission
band of 1kynurenine which has an absorption maximum at about 360 nm.

1.2 BACKGROUND AND BREIF HISTORY

The source of raw material which is taken here are the human hairs which are mostly wasted
while an haircut or during the time we are giving it to the almighty as token of love. At this time most of
hairs are driven in to vain but this hair contains proteins even our nails and skin also contain protein. This
protein which is available and which can be extracted cannot be wasted as it is, it is required to be
hydrolysed by using strong acids or based and further can be used during to the need to stop the wastage
of human hair the technique to reduce the wastage of human hair we have come uo with the method to
reduce its effect. Proteins were discovered by Jöns Jakob Berzelius in 1838 and are among the most
actively studied molecules in biochemistry.
The word "Protein" is derived from a Greek word "protas" meaning "of primary importance,"
because of the fundamental role of proteins in sustaining life. Proteins were recognized as a distinct
class of biological molecules in the eighteenth century by Antoine Fourcroy and others, distinguished by
the molecules' ability to coagulate or flocculate under treatments with heat or acid. Noted examples at
the time included albumin from egg whites, blood serum albumin, fibrin, and wheat gluten. Proteins
were first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish
chemist Jöns Jacob Berzelius in 1838. Mulder carried out elemental analysis of common proteins and
found that nearly all proteins had the same empirical formula, C400H620N100O120P1S1. He came to the
erroneous conclusion that they might be composed of a single type of (very large) molecule.
The term "protein" to describe these molecules was proposed by Mulder's associate Berzelius;
protein is derived from the Greek word (proteios), meaning "primary", "in the lead", or "standing in
front", in Mulder went on to identify the products of protein degradation such as the amino acid leucine
 for which he found a (nearly correct) molecular weight of 131 Da. The difficulty in purifying proteins in
large quantities made them very difficult for early protein biochemists to study. Hence, early studies
focused on proteins that could be purified in large quantities, e.g., those of blood, egg white, various
toxins, and digestive/metabolic enzymes obtained from slaughterhouses. In the 1950s, the Armour Hot
Dog Co. purified 1 kg of pure bovine pancreatic and made it freely available to scientists; this gesture
helped ribonuclease A become a major target for biochemical study for the following decades.

1.3 AIM AND OBJECTIVE

Aim :-
To prepare biofertilizer which can enhance the growth of the plant or crops.
This liquid protein is easy to use and since in the liquid form can easily dissolve in to the soil.
Objective:-
 to optimize temperature.
 to optimize concentration.
 to optimize time duration.
Specific Objective:-
 to optimize the temperature for hydrolysis reaction
 to optimize suitable concentration for hydrolysis reaction.
 to optimize suitable time for hydrolysis reaction.
 to determine costing of the product.

1.4 SCOPE AND LIMITATION-

Uses of Human Hair

The unique properties of human hair such as its unique chemical composition, slow degradation rate, high
tensile strength, thermal insulation, elastic recovery, scaly surface, and unique interactions with water and
oils, along with its sociocultural roles, haVe led to many diverse uses
 Fashion, Theatre, and Cosmetics Industry

o Wigs, Hair Extensions, Eyelashes, Moustaches, Beards, and Other Beauty Accessories

o This is one of the most ancient and currently the largest of the human hair based industries, with a
constantly increasing scale due to global expansion of the fashion industry.
o Test Material for Hair Care Products

o Human hair swatches are used as test materials for new formulations of shampoos, oils, conditioners,
dyes.
o For Making Cosmetic Brushes

o Scales on hair can hold cosmetic powder particles and apply it uniformly on skin or a surface.

 Agriculture

o As Fertilizer

o Human hair is one of the highest nitrogen-containing (~16%) organic material in nature because it is
predominantly made up of (nitrogen-containing) proteins.
o Pest Control

o Human hair is also known to address problems arising from many animals as well as insect pests,
although by different mechanisms.

 Composite Materials

o Reinforcement of Construction Materials

o Due to high tensile strength and high friction coefficient, human hair has been used for reinforcing clay-
based constructions.
o Molded Furniture and Objects

o A UK-based entrepreneur, Ronald Thompson [38], has developed a method for making composite
materials which includes first weaving human hair into a web or mat and then adding a structural additive
like resin or flexible polymer (preferably a recyclable or biodegradable material).
o Composites for Superconducting Systems

o Superconducting power equipments often use fiber-glass-based composites for cryogenic insulations.
 3.4. Pollution Control and Remediation

o Oil-Water Separation and Oil Spill Remediation

o Human hair surface has a high affinity for oils—much higher than its affinity for water.
o Removing Phenols, Aldehydes, Dyes, and Heavy Metal Pollutants from Water

o Human hair absorbs several chemicals from aqueous solutions.

 3.5. Pharmaceuticals and Biomedical Applications

o Pharmaceuticals

o Human hair proteins typically contain 20 essential amino acids, which can be extracted by complete
hydrolysis of hair .
o Hydrolyzed Hair Keratin

o A mixture of amino acids and polypeptides obtained by the hydrolysis of keratin protein from human
hair, known as hydrolyzed human hair keratin protein (HHKP), is used in hair care products by many
companies .
o Ethnomedicinal Uses

o Several cultures have been using human hair for preparing traditional medicines.
o Suturing Material in Surgery

o Human hair has sufficiently high strength for use as suture in most surgeries.
o Keratin-Based Engineering Biomaterials

o In 2002, Nakamura et al. developed the Shindai method to extract proteins rapidly and efficiently from
human hair, opening possibilities of reengineering human hair proteins into new materials.
o Human Hair Follicle Cell Cultures and Tissue Regeneration

o Biomedical studies show that certain cells from human hair follicles such as outer root sheath cells also
are useful in wound treatments, autologous grafting of chronic wounds , and treatment of alopecia .
o Flexible Microelectrodes

o Human hair by itself is not a good conductor of electricity, but Xu et al. from China have developed a
human hair microelectrode by coating its surface with an ultrathin layer of gold.

 Food Industry

o Many amino acids obtained from human hair such as L-cysteine are also used in the food industry as
leavening agent for pizza dough and doughnuts, for artificial meat flavor, in nutritional supplements, and
so forth.
 Scientific Instrumentation

o Human hair expands in length on absorbing moisture, and this expansion is reversible.

 Textiles, Fiber Stuffing, and Other Artifacts

o Stuffing Toys, Mattresses, and Other Household Items

o Due to its elastic and cushiony nature and good thermal insulation properties, human hair has been used
to stuff household items such as hair-pin cushions and toys in Hawaii and the USA [76, 77]and toys,
furniture, mattresses, quilts, jackets, and so forth, in India ([6], author’s field discussions).
o Fabrics

o High thermal insulation, elasticity, and good tensile strength also make human hair useful for making
various kinds of fabrics.
o Oil Filters

o Tightly woven human hair cloths were used in the 1920s as filters for heavy oils in refineries and
distilleries because these processes involved high pressure that many natural fibers could not withstand .

 Ropes

o Due to good tensile strength, human hair has been used to make ropes in many cultures, for example, to
lift heavy beams and bells in the construction of Japanese temples [82] and for household purposes by
Native Americans .

 Artwork

o Two art traditions evolved in the world around human hair as the key material.

 Miscellaneous Uses

o Human hair is placed with other fibers as nesting material to increase breeding of birds in places where
bird populations are declining.
 CHAPTER 2: LITERATURE REVIEW
2.1 HUMAN HAIR

2.1.1 INTRODUCTION:-

The hair shaft is essentially composed of keratin. Hair keratin is hard, compact and strong. This fibrous
protein is gradually formed inside cells from germinal layer. These cuticle cells are characterized by the
pressence of amorphous keratin while the cortical cells have structure of filaments surrounded by a
keratinic substance that is richer in sulfur and contain amino acid. The keratin of these filaments forms a
helix, With the distance between the turns of 0.51 nanometer and a structure maintained by hydrogen
bonds.
The keratin of these filaments forms helix, with a distance between the turns of 0.51 nanometer and a
structure maintained by hydrogen bonds. This protein plays a key role in the cohesion and physical
properties of hair.The overall human hair composition is 45% carbon, 28% oxygen, 15% nitrogen, 7%
hydrogen, and 5% sulphur.

2.1.2 COMPOSITION OF HUMAN HAIR:-

Hair is comprised of many contributing factors. Proteins, raw elements, amino acids and bonds work
together in forming hair fiber. The dominant contributor in the composition of hair is protein, accounting
for 91 percent of hair fiber.

Amino acids, the building blocks of protein, are made up of COHNS elements, (Carbon, Oxygen,
Hydrogen, Nitrogen and Sulfur).

The percentage of COHNS elements in hair is as follows:

 PERCENTAGE
ELEMENT IN NORMAL
HAIR

Carbon 51%

Oxygen 21%

Nitrogen 17%

Hydrogen 6%

Sulfur 5%

TABLE 2.1:COMPOSITION OF HAIR

These elements form bonds called side bonds which link together the long chain of amino acids known as
the polypeptide chain. This chain forms a helix by creating spiral movement that intertwines.

Millions of polypeptide chains reside in the cortex layer. Side bonds such as hydrogen bonds, salt
bonds and disulfide bonds link together these polypeptide chains. Hair fibers are held in place by the side
bonds which attribute to the elasticity and strength of hair.

A hydrogen bond can easily be broken by water or heat, and is a physical side bond. Collectively,
hydrogen bonds account for one-third of hair’s strength.

Salt bonds are also physical side bonds. Strong acidic or alkaline solutions break salt bonds because
they are affected by changes in pH. Like hydrogen bonds, salt bonds also account for approximately one-
third of hair’s strength.

Disulfide bonds differ from hydrogen and salt bonds because they are not physical side bonds.
Disulfide bonds are chemical side bonds. Disulfide bonds link together two sulfur atoms attached to
cysteine amino acids within the polypeptide chains. Chemical hair relaxers and permanent waves
chemically alter the hair’s disulfide bond. Disulfide bonds cannot be broken by water or heat
2.2 PROTIEN

2.2.1 INTRODUCTION

Proteins were discovered by Jöns Jakob Berzelius in 1838 and are among the most actively-studied
molecules in biochemistry. The word "Protein" is derived from a Greek word "protas" meaning "of primary
importance," because of the fundamental role of proteins in sustaining life. Proteins were recognized as a
distinct class of biological molecules in the eighteenth century by Antoine Fourcroy and others,
distinguished by the molecules' ability to coagulate or flocculate under treatments with heat or acid. Noted
examples at the time included albumin from egg whites, blood serum albumin, fibrin, and wheat gluten.
Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the
Swedish chemist Jöns Jacob Berzelius in 1838. Mulder carried out elemental analysis of common proteins
and found that nearly all proteins had the same empirical formula, C400H620N100O120P1S1. He came to
the erroneous conclusion that they might be composed of a single type of (very large) molecule. The term
"protein" to describe these molecules was proposed by Mulder's associate Berzelius; protein is derived
from the Greek word (proteios), meaning "primary", "in the lead", or "standing in front", in Mulder went
on to identify the products of protein degradation such as the amino acid leucine for which he found a
(nearly correct) molecular weight of 131 Da.

The difficulty in purifying proteins in large quantities made them very difficult for early protein
biochemists to study. Hence, early studies focused on proteins that could be purified in large quantities,
e.g., those of blood, egg white, various toxins, and digestive/metabolic enzymes obtained from
slaughterhouses. In the 1950s, the Armour Hot Dog Co. purified 1 kg of pure bovine pancreatic and made
it freely available to scientists; this gesture helped ribonuclease A become a major target for biochemical
study for the following decades.

The first protein structures to be solved were hemoglobin and myoglobin, by Max Perutz and Sir
John Cowdery Kendrew, respectively, in 1958. As of 2017, the Protein Data Bank has over 126,060
atomic-resolution structures of proteins
2.2.2 TYPES & CLASSIFICATION OF PROTIENS

Proteins are formed of a large number of amino acid linked together by peptide bonds (polypeptide
chain).

There are four orders of protein structures

1. Primary structure of Proteins

Referred to the number, type and sequence of amino acids in the polypeptide chain. Any change in one of
amino acids in polypeptide chain produces a physiological defect.

2. Secondary Structure of Proteins

The polypeptide chain will be folded to give a specific conformational form which may be:

 Helix-the α
The α-helix is a common secondary structure encountered in proteins of the globular class. The formation
of the α-helix is spontaneous and is stabilized by H-bonding between amide nitrogen and carbonyl carbons
of peptide bonds spaced four residues apart.
 Pleated Sheets- β
β- Sheets are composed of 2 or more different regions of stretches of at least 5-10 amino acids.
The folding of the polypeptide backbone aside one another to form β-sheets is stabilized by H-
bonding between amide nitrogen and carbonyl carbons. β- Sheets are said to be pleated. This is due to
positioning of the α-carbons of the peptide bond which alternates above and below the plane of the
sheet. Hydrogen bonds or disulphide bonds

 Loop sheets:-

Half of the residues in a typical globular protein are present in α helices or β pleated sheets, the remainder
reside in loop or coil conformation which forms the antigen-binding sites of antibodies. These loops should
not be confused with random coils which are biologically unimportant conformations of denatured
proteins.
3. Super secondary structures (motifs)
α helices or β pleated sheets form recognizable super secondary motifs, such as:
 β–α –β: (two strands of β-sheets connected by α helix

 β –hair pin-: two antiparallel β-sheets connected by short regions of loop. Structural motif helix

(HTH) is a major terrn helix-α. It is composed of two DNA capable of binding amino acids joined by a
short strand of helices.

4. Tertiary Structure of Proteins: -

Tertiary structure refers to the complete three-dimensional structure of the polypeptide units of a given
protein. Secondary structures of proteins are coiled to constitute distinct structure.

5. Quaternary Structure of Proteins: -

Many proteins contain 2 or more different polypeptide chains that are held in association by the same
non-covalent forces that stabilize the tertiary structures of proteins. Oligomeric chains are monomer -
structure formed by monomer interaction in an oligomeric protein is known as quaternary structure.

FIG 2.1 : STRUCTURE OF PROTEIN

 CLASSIFICATION OF PROTEINS: -

Proteins can be classified on the basis of their solubility, shape, biological functions, or chemical
composition.

1- According to their solubility:

 Proteins soluble in H2O,or other biological solvents (Albumin –globulin-Histones –Protamin prolamin –
glutelins)
 Proteins not soluble in most protein solvents [albuminoids] as nail and hair.

2-According to their shape:

 Globular proteins: axial ratio is more than 10, more stable as keratin & myosin in muscles.

 Fibrous proteins: axial ratio is less than 10, less stable as albumin & globulin.

3-According to their biologic functions:

 Enzymes e.g. dehydrogenases, kinases
 Storage proteins: ferritin, myoglobin
 Regulatory proteins: DNA-binding protein, peptide hormones o Structural proteins: collagen o
Protective proteins: clotting factors , immunoglobulin o Transport proteins: haemoglobin , plasma
lipoproteins o Motile proteins: actin, tubulin

4-According to their chemical -4composition on hydrolysis, they produce:

Simple proteins, as albumin, globulins, glutellin only amino acids, prolamines, protamine, histones,
albuminoids (scleroproteins).

 They are fibrous proteins. o Insoluble in H2O, dilute acids and alkali, and all neutral solvents. o Not
digested by proteolytic enzymes.
 Found in animal tissues and having supportive and protective function as keratin, elastin, collagen
&gelatin.
 5-Conjugated proteins (compound protein): -

These are formed of protein part and non-protein part.

According to non-protein part, they are divided into:

Glycoproteins and Mucoproteins:

Protein conjugated with carbohydrate e.g. certain hormones [FSH, LH, TSH] &Immunoglobulin

Immunoglobalins:
 Globulins are mainly formed in reticulo-endothelial system in macrophages and lymphocytes. o
Immune system is divided into :
 B-cells (Bone marrow): concerned with circulating humeral antibodies.

 T-cells (Thymus glands): concerned with cell mediated immune response as graft rejection,
hypersensitivity reactions and defense against malignant cells and viral infection.
Lipoproteins: -

 Protein conjugated with lipids either [phospholipids-triglyceride-cholesterol] e.g. Chylomicrons

Phosphoproteins: -
 Protein conjugated with phosphoric acid thruoghhydroxylic group of serine, threonine
&tyrosine
 Casienogen is an example for phosphoproteins (the main protein of milk).
Metalloproteins: -
Proteins conjugated with metals e.g. o Ceruloplasmin = protein + Cu. o Insulin = protein +
zinc.
Chromoproteins: -
 Hemoglobin containing Fe-porphyrin (red color). o Chlorophyll containing Mg-porphyrin
(green color).
 Flavoproteins: These are enzymes containing FMN, FAD (yellow color).
Nucleoproteins: -
 Proteins conjugated with nucleic acids e.g. Histone associated with DNA inchromosomes.
 Derived proteins: - These are the denaturated or hydrolytic products of either simple or
conjugated proteins.
Primary protein derivatives

These results from alteration of proteins from its native state without hydrolysis:

 Metaproteins: Due to the effect of acid or alkali e.g: Acid or alkali metaprotein. o Gelatin
[denaturated collagen].
 Coagulated proteins: Due to the effect of heat e.g. coagulated albumin and globulin.

Secondary protein derivatives:

These are the hydrolytic products of proteins

 Proteoses: - Result from partial hydrolysis of proteins.
 Peptones: - Result from further hydrolysis of proteases and soluble in H2O.
 Peptides:- Resulting from further hydrolysis of peptones.
 Amino acids

2.2.3 PROTIENS FROM NATURAL RESOURCES

• Animal products: meat, milk, milk products, egg, poultry and fish are rich sources of protein containing a
balanced level of amino acids. Ex-Casein, Egg protein, Meat proteins
• Plant proteins: Vegetables, legumes and fruits are good sources of protein. Ex-Soybean proteins,
Wheat protein ,rice proteins, Corn proteins, Sunflower Proteins
Examples: -
Meat Proteins:
 Although tuna, chicken breasts, turkey, and other white fish are very good, low-fat and highprotein
sources lean red meat will provide you with better gains.

 Poultry Proteins:
Poultry can be an excellent source of protein, and is often a primary staple of those trying to lose weight, as
well as competitive athletes, including bodybuilders.
 Fish Proteins:
Fish is high in protein, but low in saturated fat and cholesterol, making it a good substitute for
poultry and meat. However, when selecting fish especially if you plan to eat it uncooked (e.g.,
sushi/sashimi) it is of paramount importance to select a reliable seafood source.
 Dairy Protein:
Milk is a nutrient rich complete ideal food. Milk contains all the essential nutrients required for
growth. Milk is also rich in many vitamins and minerals such as calcium, phosphorus, sodium,
potassium vitamin A, thiamine, riboflavin and nicotinic acid and vitamin B12 which is absent in
vegetarian food items.
 Legumes:
Legumes are a class of vegetables that includes beans, peas and lentils - are typically low in fat,
contain no cholesterol and are high in protein, iron, folate, potassium, magnesium, and phyto
chemicals.

 Soy: The most common source of plant protein regarding consumption is soy protein. Soy has been
consumed for more than 2,000 years by people throughout East Asia.
 Nuts: cashews, almonds, peanuts, pistachios (note that some nuts such as chestnuts and macadamias
are poor sources of protein and others such as Brazil nuts, walnuts, pine nuts, pecan nuts and hazel
nuts are mediocre sources)
 Seeds: pumpkin, sunflower, sesame o Grains: wheat, oats, buckwheat, millet, quinoa, amaranth, pasta,
bread, seitan (wheat protein) (note that rice is a relatively poor source of protein).

2.2.4 MARKET AND STASTICAL DATA ON PROTEIN

GLOBAL OVERVIEW OF THE MARKET

The global protein ingredients market size was valued at USD 23.98 billion in 2015 and is expected
to witness growth at a CAGR of over 7% from 2016 to 2024. One of the key drivers for the growth of the
market is the strong scientific evidence supporting health benefits. Ongoing research conducted by
scientists has proved that protein ingredients are the best source to keep the body fit. Rising consumer
awareness, especially for dietary supplements and functional foods, has been a critical factor for market
development in recent times
FIG 2.2GLOBAL PLANT PROTEIN INGREDIENTS MARKET VOLUME, BY PRODUCT, 2015

2.2.5 REACTIONS INVOLVED IN PROTEIN RECOVERY

Amino acids are covalently bonded together in chains by peptide bonds. If the chain length
is short (say less than 30 amino acids) it is called a peptide; longer chains are called polypeptides or
proteins. Peptide bonds are formed between the carboxyl group of one amino acid and the
amino group of the next amino acid. Peptide bond formation occurs in a condensation reaction
involving loss of a molecule of water.

The head-to-tail arrangement of amino acids in a protein means that there is a amino group
on one end (called the amino-terminus or N-terminus) and a carboxyl group on the other end
(carboxylterminus or C-terminus). The carboxy-terminal amino acid corresponds to the last one
added to the chain during translation of the messenger RNA.
 There are many other properties in which the twenty amino acids differ from one another: some are
bulky, some small; some are capable of donating electrons, others not; some are chemically
reactive. And so forth. Each amino acid represents a different flavor, and the structure and
properties of a protein are defined by the properties and order of its amino acids: its primary
structure.
 There are only twenty amino acids used to synthesize proteins, which limits what proteins are
possible in nature. How constricting is this limitation? Consider the number of possible dipeptides
(two amino acids joined together by a peptide bond). There are 20 possible amino acids in the first
position and 20 possible amino acids in the second position. That makes 202 =
 400 possible dipeptides. Similarly, there are 203 = 8000 possible tripeptides. Proteins range in size
from a smallish 100 amino acids to a 1000. The number of possible proteins in nature is therefore
staggering!
 The synthesis of proteins is the process of combining alpha-amino acids in a linear chain,
connecting alpha-amino groups to carboxylate groups (Figure 1a and 1b). The backbone of this
chain is identical for all proteins. If the R groups were similarly invariable, then all proteins would
be alike, and protein would be able to do only one thing, a not very interesting thing at that.
 Some R groups of amino acids are acidic carboxylic acids, giving rise to negative charges at
physiological pH. Aspartic acid is an example of an acidic amino acid. Some R-groups are basic,
giving rise to positive charges at physiological pH. .
2.3 AMINO ACIDS

2.3.1 INTRODUCTION

Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional
groups, along with a side chain(R group) specific to each amino acid.[1][2][3] The key elements of an amino
acid are carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), although other elements are found in the
side chains of certain amino acids. About 500 naturally occurring amino acids are known (though only 20
appear in the genetic code) and can be classified in many ways.[4] They can be classified according to the
core structural functional groups' locations as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino
acids; other categories relate to polarity, pH level, and side chain group type (aliphatic, acyclic, aromatic,
containing hydroxyl or sulfur, etc.). In the form of proteins, amino acid residues form the second-largest
component (water is the largest) of human musclesand other tissues.[5] Beyond their role as residues in
proteins, amino acids participate in a number of processes such as neurotransmitter transport
and biosynthesis.

In biochemistry, amino acids having both the amine and the carboxylic acid groups attached to
the first (alpha-) carbon atom have particular importance. They are known as 2-, alpha-, or α-amino
acids (generic formula H2NCHRCOOH in most cases,[6]where R is an organic substituent known as a
"side chain");[7] often the term "amino acid" is used to refer specifically to these. They include the
22 proteinogenic ("protein-building") amino acids,[8][9][10] which combine into peptide chains
("polypeptides") to form the building-blocks of a vast array of proteins.[11] These are all L-
stereoisomers ("left-handed" isomers), although a few D-amino acids ("right-handed") occur in bacterial
envelopes, as a neuromodulator (D-serine), and in some antibiotics.[12]

Twenty of the proteinogenic amino acids are encoded directly by triplet codons in the genetic
code and are known as "standard" amino acids. The other two ("non-standard" or "non-canonical")
are selenocysteine (present in many prokaryotes as well as most eukaryotes, but not coded directly
by DNA), and pyrrolysine (found only in some archea and one bacterium). Pyrrolysine and
selenocysteine are encoded via variant codons; for example, selenocysteine is encoded by stop
codon and SECIS element. N-formylmethionine(which is often the initial amino acid of proteins in
bacteria, mitochondria, and chloroplasts) is generally considered as a form of methionine rather than as a
separate proteinogenic amino acid. Codon–tRNA combinations not found in nature can also be used
to "expand" the genetic code and form novel proteins known as alloproteins incorporating non-
proteinogenic amino acids.

Many important proteinogenic and non-proteinogenic amino acids have biological functions. For
example, in the human brain, glutamate (standard glutamic acid) and gamma-amino-butyric
acid ("GABA", non-standard gamma-amino acid) are, respectively, the main excitatory and inhibitory
neurotransmitters. Hydroxyproline, a major component of the connective tissue collagen, is synthesised
from proline. Glycine is a biosynthetic precursor to porphyrins used in red blood cells. Carnitine is used
in lipid transport.

Nine proteinogenic amino acids are called "essential" for humans because they cannot be produced
from other compounds by the human body and so must be taken in as food. Others may be conditionally
essential for certain ages or medical conditions. Essential amino acids may also differ between species.

Because of their biological significance, amino acids are important in nutrition and are commonly
used in nutritional supplements, fertilizers, feed, and food technology. Industrial uses include the
production of drugs, biodegradable plastics, and chiral catalysts.

2.3.3 AMINO ACIDS DERRIVED FROM PROTIENS

There are some 20 amino acids in the proteins that we consume. These amino acids bond together to
form a larger protein molecule. Amino acid being organic compound molecules can form various different
links with each other due to the versatile nature of carbon. This enables the great diversity of proteins that
can be found in nature. These are an essential nutrient in our diet because of the functions they perform.
Structure of Amino Acids:-

FIG 2.3:- STRUCTURE OF ACID

 There are actually thousands of amino acids occurring in nature. But only about 20 amino acids form a part
of the proteins in the human body. These twenty acids will be our focus here. Although all these have varied
structures, the basic structure of amino acid remains uniform.

 All amino acids contain a carbon atom in the middle of the molecule, the alpha-carbon

 This atom is surrounded by three chemical groups.

 One is an amine group -NH2

 The second one is a carboxyl group -OOOH

 The third group is denoted by R. This is the variable radical group and is different for every amino acid.
This R group makes the amino acid unique.

Classification of Amino Acids

Amino Acid can be classified based on their structure and the structure of their side chains i.e. the R-
chains. Now two basic subcategories are

1] Non-Polar Amino Acids

These are also known as Hydrophobic. The R group can be either of Alkyl groups (with an alkyl chain) or
Aromatic groups. The acids falling in this group are stated below. Numbers one to seven are Alkyl and the
last two are aromatic

i. Glycine (H)

ii. Alanine (CH3)

iii. Valine ( CH (CH3)2 )

iv. Methionine ( CH2CH2SCH3 )

 v. Leucine ( CH2CH(CH3)2 )

vi. Isoleucine ( -CH(CH3)CH2CH3 )

vii. Proline (special structure)

viii. Phenylalanine

ix. Tryptophan

2] Polar Amino Acids

If the side chains of amino acid contain different polar groups like amines, alcohols or acids they are polar in
nature. These are also known as Hydrophilic Acids. These are further divided into three further
categories.

a) Acidic: If the side chain contains an extra element of carboxylic acid component these are acid-polar
amino acids. They tend to donate their hydrogen atom. These are:

i.Aspartic Acid ( CH2COOH)

ii.Glutamic Acid ( CH2CH2COOH )

b) Basic: These have an extra nitrogen group that tend to attract a hydrogen atom. The three basic polar
amino acids are

i. Histidine
ii. Lysine ( CH2(CH2)2NH2 )
iii. Arginine
c) Neutral: These are neither acidic nor basic. They have an equal number of amino and carboxyl groups.
Also, they have at least one hydrogen component connected to electronegative atoms. Some of these neutral
acids are

i. Serine ( CH2OH )

ii. Threonine ( CH(OH)CH3 )

iii. Asparagine ( CH2OHNH2 )

iv. Glutamine ( CH2CH2CONH2 )

v. Cysteine ( CH2SH )

vi. Tyrosine
Amino acid can also be classified on the basis of their need to the human body and their availability in the
human body

1] Essential Amino Acids

These are the acids that cannot be synthesized in our bodies. We must rely on food sources to obtain these
amino acids. They are

 Leucine

 Isoleucine

 Lysine

 Theorine

 Methionine

 Phenylalanine

 Valine

 Tryptophan

 Histidine (conditionally essential)

2] Non-Essential
These acids are synthesized in our bodies itself and we need not rely on outside sources for them. They are
either produced in our bodies or obtained from protein breakdowns.

Fig 2.4structure Of The 20 (Alpha Amino Acids Usedin Synthesizing Protien .Below Each Amino
Acid Are Its 3- And 1-Letter Abbreivation S.(Derrived From Else &Baumgardner,Principle Of
Modern Genetivs(1995).West Pub)
2.4 .HYDROLYSIS

2.4.1 INTRODUCTION:-

A hydrolysis process: Hydrolysis is a process whereby chemical bonds are broken by the
insertion of a water molecule. Hydrolysis can be catalyzed by enzymes, metal salts, acids, or bases.
Alkaline hydrolysis relies upon bases—typically, water solutions of alkaline metal hydroxides such
as sodium hydroxide or potassium hydroxide. Heat significantly accelerates hydrolytic processes; in
this way, alkaline hydrolysis uses elevated temperatures (150°C, or ~300°F) to hasten the conversion
of biological material (protein, nucleic acids, carbohydrates, lipids, etc.) into a sterile aqueous
solution consisting of small peptides, amino acids, sugars, and soaps. The only solid by-products of
alkaline hydrolysis are the mineral constituents of the bones and teeth of vertebrates (WR2, 2003).

2.4.2ACID HYDROLYSIS:

Acid hydrolysis is a process in which a protein acid is used to catalyze the cleavage of a chemical
bond via a nucleophilic substitution reaction, with the addition of the elements of water (H2O).When this
process is carried out in the presence a little amount of a non-oxidizing acid like HCl or dilute H2SO4, it is
called acid hydrolysis. Here the acid acts as a catalyst by providing H+ ions to facilitate the intake of H2O
molecules. Thus, an ester can be hydrolyzed to the respective acid and alcohol by boiling with water
containing a little HCl or sulphuric acid.

CH3COOC2H5 + H2O (H+) --------->CH3COOH + C2H5OH

(ethyl acetate) (acetic acid) (ethanol)

Kinetically, acid hydrolysis is a first-order reaction.

In 1811 kirchhoff observe that starch was transformed by aqueous mineral acid into glucose and showed
that no acid was used in the process. Braconnot, in 1819 hydrolyzed linen (cellulose) with strong
sulphuric acid, obtaining a fermentable sugar. This use of acid in hydrolysis was rapidly extended to
other classes of organic materials – the esters ,the sugars ,amides etc .-and it was found that, whatever
brought about hydrolysis , acid accelerated the reaction .In addition ,the latter seemed in many cases to
initiate reaction Where water alone failed.The acid hydrolysis of acetate esters has come to be a sort of
proving ground for theories of catalysis. It is customary to attribute the effect of acid to hydrogen ion
contents, and with dilute acid in many reactions, there is at least a rough proportionality between the
velocity of reaction and hydrogen ion concentration. Like many other generalizations in the chemistry
,this relation is more honoured in the branch of observance , and with concentrated acid ,it does not hold
at all. The undissociated acid and negative radical have both been called upon time to time to account for
certain anamolous results.

nd alcohol and its related products, whilHCL and Sulphuric acid are naturally the most commonly used,
though many other have been explored.the formic and tri chloro acetic appear to be over in activity then
would be expected ,where as a oxalic and benzene sulphonic are more active than sulfuric .concentration
from very high to very low are used in both laboratory and commercial practice.

sulfuric acid is particularly useful because it forms ,with many types of organic substances, intermediate
compound that themselves readily undergo hydrolysis. This is exhibited in the acid process of fat
splitting to make fatty acids ,in making alcohol from ethylene, and probably also in hydration of
acetylene to make aldehyde. In all of these, sulfuric acid exhibits a specific action, distinct from its
hydrogen ion concentration, and cannot be replaced by the acids.

The preparation of individual amino acids by the hydrolysis of protein is generally very difficult because
these natural materials are composed of some 20 amino acids having similar chemical properties
generally no one amino acid constitutes more than 10 to 20% of protein. Because of this inherent
difficulty, the individual production of amino acid from kesin and Soyabean proteins awaits further
progress in current research

One protein that is used in multi million pounds quantities annually is wheat gluten- which is value for
its relatively high (30 to 40%) contents of glutamic acid, HOOC(CH2)2CH(NH2)COOH. Monosodium
glutamate is a condiment that is being used in increasing quantities for imparting a meat like a flavor to
soups and other food stuffs. The usual procedure for making of monosodium glutamate involves
hydrolysis of gluten with a constant boiling 20% hydrochloric acid solution; removal of excess HCL by
distillation; and filtration from humin followed by successive crystallization of glutamic acid
hydrochloride, glutamic acid ,and mono sodium glutamate in the presence of decolourising carbon .
 Although acid and alkali may frequently used interchangeably on the same material to give essentially
the same products, this is not invariablly true. The action of acid and on acid to acetoacetic Ester and its
derivatives lead to acetone,carbonic acid ae alkali on the same ester produces acetic acid and alcohol.

2.4.3ALKALINE HYDROLYSIS:

Alkaline hydrolysis is a process in which enzymes facilitate the cleavage of bonds in molecules with the
addition of the elements of water. It is the natural process by which complex molecules are broken down into
their constituent building blocks by the insertion of ions of water (H2O), H+, and OH- between the atoms of
the bonds that held those building blocks together. The process occurs in nature when animal tissues and
carcasses are buried in soil of neutral or alkaline pH. In this case, alkaline hydrolysis is aided by the digestive
processes of soil organisms. Alkaline hydrolysis also occurs in our small intestines after we eat; the complex
molecules of proteins, fats, and nucleic acids are hydrolyzed with the aid of digestive enzymes that function
most efficiently at a slightly alkaline pH (~pH8.0 to 8.5). Historically, alkaline hydrolysis has been used to
study the chemical structure of biological molecules, to prepare skeletal remains for study, and make soaps
from animal fats by cooking the fat with lye to release the fatty acids, then cooling the mixture to precipitate
the fatty acids as their sodium salts.Alkaline hydrolysis ultimately leads to the degradation of proteins—the
major solid constituent of all animal cells and tissues. Sodium or potassium salts of free amino acids are
generated by the hydrolytic reaction, while oligopeptides (small chains of amino acids) are generated as
reaction intermediates. Some amino acids (e.g., arginine, asparagine, glutamine, and serine) are completely
destroyed while others are racemized (i.e., structurally modified from a left-handed configuration to a
mixture of left-handed and right handed molecules).

We may distinguish three different cases of hydroklysis with alkali:

1).the use of low concentrations of alkali in the hydrolysis of esters and similar materials:Here the
hydroxyl ion is supposed to catalyze the reaction as the hydrogen ion thus in catalysis by dilute acid
.since one of the products of reaction is usually an acid that react immediately with the hydroxyl ion
,this case is of significance only in theoretical studies,where an instantaneous value of reaction
velocity is desired .
2}.the use of sufficient costic under pressure in and high concentration to unite with the void
produced.

C6H6Cl + NaOH -------------------> C6H6OH + NaCl + H2O

Sometimes followed by

C6H6OH + NaOH------------> C6H6ONa +H2O

2.4.3ENZYMATIC HYDROLYSIS:

Enzymatic hydrolysis is a process in which enzymes facilitate the cleavage of bonds in molecules
with the addition of the elements of water. Enzymatic hydrolysis of soy protein can enhance or
reduce its functional properties and improve its nutritious value. Soy protein hydrolysates were
primarily used as functional food ingredients, flavor and nutritious enhancers, protein substitute, and
clinical products.Conditions for hydrolysis were usually mild, whereas recently high pressure
treatment attracted moreinterest. Degree of hydrolysis (DH) was usually between 1% and 39.5%. The
main problem associated with proteolytic hydrolysis of soy protein was production of bitter taste,
hydrolysates coagulation and high cost of enzymes. Bitterness reduction can be achieved by control
of DH, selective separation of bitter peptides from hydrolysates, treatment of hydrolysates with exo-
peptidases, addition of variouscomponents [adenosine monophosphate (AMP), some amino acids,
monosodium glutamate (MSG), etc.]to block or mask the bitter taste, and modification of taste
signaling. Hydrolysates coagulation can beresolved by selecting appropriate enzymes and by
applying immobilization technology the production cost can be reduced. Enzymatic hydrolysis also
enhances bioactivity of soy proteins through conversion of glycosides to a glycones, increasing
antioxidant and immune regulatory properties.

2.4.4 MECHANISM OF HYDROLYSIS

Initial, in commercial processes whether a reaction goes fast or slow is extremely important, and thus
the rate at which a chemical reaction approaches equilibrium is significant if the reaction is
thermodynamically possible but proceeds with a velocity that is not economically practical, some means
must be found by which the rate is increased in addition to the variation of temperature, pressure and
concentration ratio as a catalyst may be employed to bring about the desired results. Although reaction
conditions and catalyst are usually determined experimentally, a discussion of typical hydrolytic reaction
from the theoretical point of view made through some light on the mechanism of these reactions and make
the approach to future problems less arbitrary. The data obtained on reaction rates may be interpreted
through either the collision theory or the theory of absolute reaction rates. The former places emphasizes
on the energy of activation as the rate determining factor. This may be related to the temperature (T) and
the rate constant (K) by modified form of the Arrehenius equation:

k = PZe-E/RT ----------------- (1)

where E is the energy of activation, R is the gas constant, Z is the frequency of collision at unit
concentration of reactants and P is a probability factor. This last term accounts for considerable division in
some cases between the theory and experiment. Z may be calculated from kinetic theory. The newer
theory, also called transition-state theory, places emphasis on the free energy of activation and is concerned
with the thermodynamic probability of attaining an “activated Complex”, or transition state. One form of
the rate constant expression is:

k = (klT/h)e-∆F/RT = (KlT/h)K ------------------------------(2)

k = (klT/h)e-∆H/RTe∆S/R -------------------------------(3)

where ∆ , ∆ ∆S are the standard free energy, heat content, and entropy of activation; K is the
equilibrium constant for the activation; and klT/h is a Universal frequency constant. These quantities may
be calculated by the methods of statistical thermodynamics and replacement by Quantum-statistical
expressions gives the rate constant in terms of the potential energy of activation and the partition functions
of the reaction and activated Complex. A comparison of the two shows that of equation 1 may be related to
∆H of equation 3, and since klT/h is constant and Z is of the same order for a given series, P is related to
the entropy term.

Since the collision theory is the older and simpler, equation 1 has been used in the in analysis of most
of the published results.

The order of a reaction and molecularity must be included in the discussing chemical kinetics. The
order of a Kinetic reaction is determined from the mathematical expression showing the dependence of rate
on the concentration of the reactants, and the molecularity by the number of molecules involved in the
reaction.
 CHAPTER 3: EXPERIMENTAL DETAILS

3.1 RAW MATERIALS

The materials required for performing this extraction is Urea, Thiourea, Tris-HCl and
mercaptoethanol. All the chemicals are purchased from Akola Chemicals, Maharashtra. Hair samples are
collected from one volunteer so that we can minimize the experimental errors which may arise due to
gender, age or other factors. The basic laboratory equipment and air-dryer for heating has been provided by
Coimbatore Institute of Technology, Coimbatore.

3.2 METHODOLOGY

It consists of 3 steps which are described below:

I. Pretreatment Of Humar Hairs
Removal of external lipids is the first step towards the extraction of protein. This can be done by
immersing the hair samples in distilled water for one hour to remove any sort of impurities and then
treating it with the mixture of chloroform and methanol for a prolonged period of 24 hours. During this
period, chloroform/methanol mixture (50:50 W/W) effectively removes the lipids in the form of liquid
which can be seen as the oily layer on the top of the solution .

II. Preparation Of Buffer Solution:

Buffer solution is a mixture of Tris-HCl, Thiourea, urea and mercaptoethanol[4]. Based on
stoichiometry[5], 0.25 M of Tris-HCl is prepared by adding 6.057 grams of Tris-HCl in water and making
it to 200 ml. Similarly, 200ml solution of Thiourea and urea is prepared by adding 39.58 and 60 grams
respectively, thus making it to 2.6 M and 5 M respectively. 5% mercaptoethanol solution is made by
adding 10ml of mercaptoethanol to 190 ml of distilled water. This 800 ml mixture of buffer solution is used
for the extraction of protein.

III. EXTRACTION
Hair samples are now cut into smaller pieces of length 1-2 mm using knife or blades and this purified
samples are now immersed in the buffer solution and kept inside the air-oven at the temperature of 50°C
 for a period of 24 hr. After 24 hours, the sample is taken out and filtered through a gravimetric filter paper
and excluding the filtrate, the residue is the obtained protein which is subjected to confirmatory tests.

3.3EXPERIMENTAL SETUP

Human hair (of a Indian woman) was washed with ethanol; external lipids were removed using a mixture
of chloroform/methanol (2: 1, v/v) for 24 h. The delipidized hair (20 mg) was mixed with a solution (5 ml)
containing 25mM Tris–HCl, pH 8.5, 2.6 M thiourea, 5 M urea and 5% 2-mercaptoethanol (2-ME) (Shindai
method) or 25mM Tris–HCl, pH 9.5, 8 M urea and 5% 2-ME (conventional method) at 50 °C for 1—3 d.
The mixture was filtered and centrifuged at 150003g for 20 min at room temperature. The obtained
supernatant was used as a hair protein fraction. The pellet was recovered, washed with distilled water and
used as an extracted hair sample.

Determination of Extracted Proteins:

 Dry Weight Method:
A hair protein fraction was dialyzed against 2 l of distilled water with 5—7 changes, lyophilized, and then
dried sufficiently in a silicagel box. Alternatively, an extracted hair sample (residue fraction) was washed
with distilled water and then dried in a silicagel box. The amounts of a hair protein fraction and of an
extracted hair sample (residue fraction) were obtained by weighing the dried samples using an electronic
balance.
 Bradford Method: Protein amounts were determined by the colorimetric method of Bradford15) using the
Bio-Rad protein assay (Bio-Rad).

Gel Electrophoresis/Two Dimensional Electrophoresis (2de-Ep):-

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the
method of Laemmli16) with an 8—18% slab gel. Proteins in the gel were stained with 0.1% Coomassie
brilliant blue RMay 250, 10% acetic acid and 40% ethanol for 1 h and destained in 10% acetic acid and
40% ethanol. 2DE was performed according to the method developed by O’Farrell17) using 2% ampholine
(pI 3.5—10).
Western Blot Analysis
Proteins separated on SDS polyacrylamide gel were transblotted onto a nitrocellulose membrane in a
solution containing 20mM Tris–glycine, pH 8.3 and 10% methanol. The membrane was blocked with 5%
BSA in TBS-T (25mM Tris–HCl, pH 7.2, 50mM NaCl and 0.5% Tween-20) at room temperature and
reacted with 1: 1000 phosphotyrosine (Wako), 1: 1500 phosphoserine (Affiniti), and 1 : 2000
phosphothreonine (Sigma) antibodies for 1 h at room temperature. After extensive washing in TBST, the
membrane was incubated with 1: 30000 peroxidaseconjugated secondary antibodies against rabbit
immunoglobulin in TBS-T for 1 h at room temperature. The blots were visualized with the Super Signal
CL-HRP Substrate System (Pierce).

Light Microscopy
Morphological changes of the hair surface were examined using a light microscope (Nikon, Labophot-2).

3.4 PROCESS FLOW DIAGRAM:-

FIG 3.1:-PROCESS FLOW DIAGRAM

 CHAPTER 4 :- TESTING AND CHARACTERIZATION

4.1. BIURET TEST

 The biuret test, also known as Piotrowski's test, is a chemical test used for detecting the presence
of peptide bonds. In the presence of peptides, a copper(II) ion forms mauve-colored in an alkaline
solution. Several variants on the test have been developed, such as the BCA test and the Modified
Lowry test.
 The biuret reaction can be used to assess the concentration of proteins because peptide bonds occur
with the same frequency per amino acid in the peptide. The intensity of the color, and hence the
absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer–
Lambert law.
 Despite its name, the reagent does not in fact contain biuret ((H 2 N-CO-) 2 NH). The test is named so
because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.

Procedure:-

An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide)
followed by a few drops of aqueous copper(II) sulphate. If the solution turns purple, protein is present. 5–
160 mg/ mL can be determined. Peptides with the correct length of at least 3 amino acids are necessary
for a significant, measurable colour shift with these reagents.

4.2. XANTHROPROTEIC TEST

The xanthoproteic reaction is a method that can be used to determine the amount of protein soluble in
a solution, using concentrated nitric acid. The test gives a positive result in amino acids
carrying aromatic groups, especially in the presence of tyrosine. If the test is positive the proof is
neutralized with an alkali, turning dark yellow. The yellow colour is due to xanthoproteic acid which is
formed due to nitration of certain amino acids, most common examples being tyrosine and tryptophan. This
helps in determining the presence or absence of proteins. Reaction of nitration of tyrosine as an example
xanthoproteic reaction
Procedure:-

Add 1 ml of concentrated HNO 3 to 1 ml of the test sample. Heat the mixture and cool it. Slowly
add sodium hydroxide (40% w/v in water) solution until the mixture becomes alkaline and a colour change
is noted. If the colour changes from yellow to orange, this indicates the presence of an aromatic amino
acid.

4.3. MILLIONS TEST

Millons reagent is an analytical reagent used to detect the presence of soluble proteins. A few drops
of the reagent are added to the test solution, which is then heated gently. A reddish-brown coloration or
precipitate indicates the presence of tyrosine residue which occur in nearly all proteins.

Millons test is not specific for proteins (it detects phenolic compounds), and so must be confirmed by
other tests for proteins such as the biuret test and the ninhydrin reaction. The reagent is made by
dissolving metallic mercury in nitric acid and diluting with water. The test was developed by the French
chemist Auguste Nicolas Eugene Millon (1812–1867).

4.4 NINHYDRIN TEST:-

This test is a general test and thus given by all amino acids. This test is due to a reaction between a
amino group of free amino acid and ninhydrin. Ninhydrin is a powerful oxidizing agent and its presence,
amino acid undergo oxidative deamination liberating ammonia, CO2, a corresponding aldehyde and
reduced form of ninhydrin ( hydrindantin). The NH3 formed from a amino group reacts with another
molecule of ninhydrin and is reduced product ( hydrindatin) to give a blue substance diketohydrin (
Ruhemanns complex). However, in case of imino acid like proline and hydroxyproline, a different product
having a bright yellow color is formed. Asparagine, which has a free amide group, reacts to give a brown
colored product.
4.5 SULFUR TEST:-

This is the confirmation of Keratin. As keratin is mainly composed of cysteine, a sulphurous amino
acid, this test is performed to confirm the presence of sulphur. Few drops of NaOH solution is added to the
test solution, followed by the addition of lead acetate solution. Sulphur present in the keratin forms Na2S,
which inturn reacted with lead acetate to form lead sulphide. This is confirmed by the appearance of black
colour. All the above tests confirmed the presence of Keratin in the extracted product.

4.6. KJELDAHLS TEST:-

The Kjeldahl method or Kjeldahl digestion in analytical chemistry is a method for the quantitative
determination of nitrogen contained in organic substances plus the nitrogen contained in the inorganic
compounds ammonia and ammonium (NH 3 /NH 4 + ). Without modification, other forms of inorganic
nitrogen, for instance nitrate, are not included in this measurement. This method was developed by Johan
Kjeldahl in 1883 . The method consists of heating a sample at 360–410°C with concentrated sulphuric
acid(H 2 SO 4 ), which decomposes ("digests") the organic sample by oxidation to liberate the
reduced nitrogen as ammonium sulphate. Catalysts like selenium, Hg 2 SO 4 or CuSO 4 are often added to
hasten the digestion. Na 2 SO 4 is also added to increase the boiling point of H2SO 4 . Digestion is complete
when the liquor clarifies with the release of fumes.A distillation system depicted below is built.

The end of the condenser is dipped into a known volume of standard acid (i.e. acid of known
concentration). A weak acid like boric acid (H3BO3 ) is often used. HCl , H2SO 4 or some other strong
acid can be used instead, but this is less commonplace. The sample solution is then distilled with a small
amount of sodium hydroxide (NaOH). can also be added with a dropping funnel NaOH reacts
the ammonium (NH4 + ) to ammonia(NH3 ), which boils off the sample solution. Ammonia bubbles
through the standard acid solution and reacts back to ammonium salts with the weak or strong acid.
Ammonium ion concentration in the standard acid solution, and thus the amount of nitrogen in the sample,
is measured via titration. If boric acid (or some other weak acid) was used, direct acid-base titration is done
with a strong acid of known concentration. HCl or H 2SO4 can be used. Indirect back titration is used
instead if strong acids were used to make the standard acid solution: strong base of known concentration
(like NaOH) is first added in excess, and then the excess is titrated with a strong acid of known
concentration. Titration of ammonia absorbed in boric acid solution thus has an advantage that only one
standard solution is needed. One of the suitable indicators for these titration reactions is Tashiro's
indicator .

In practice, this analysis is largely automated; specific catalysts accelerate the decomposition.
Originally, the catalyst of choice was mercuric oxide. However, while it was very effective, health
concerns resulted in it being replaced by cupric sulfate. Cupric sulfate was not as efficient as mercuric
oxide, and yielded lower protein results. It was soon supplemented with titanium dioxide, which is
currently the approved catalyst in all of the methods of analysis for protein in the Official Methods and
Recommended Practices of AOAC International.

4.7 UV SPECTROMETER TEST:-

UV-Vis is a fast, simple and inexpensive method to determine the concentration of an analyte in
solution. It can be used for relatively simple analysis, where the type of compound to be analyzed
(‘analyte’) is known, to do a quantitative analysis to determine the concentration of the analytes. In UV-
Vis, a beam with a wavelength varying between 180 and 1100 nm passes through a solution in a cuvette.
The sample in the cuvette absorbs this UV or visible radiation.

I0 is the radiation coming in,

I the radiation coming out

The amount of light that is absorbed by the solution depends on the concentration, the path length of the
light through the cuvette and how well the analyte the light absorbs at a certain wavelength. The
transmittance I/I0 is an indication of the concentration of the analyte in the sample. I/I0 is defined as the
transmittance (or transmission) T. If there is no absorption of the light passing through the solution, the
transmittance is 100%.
The most-used term in UV-Vis spectrometry to indicate the amount of absorbed light is the absorbance,
defined as:
A = - log10 T = -log10 (I/I 0 ).
1/T = 10(A)
The UV-Vis spectrum shows the absorbance of one or more sample component in the cuvette when we
scan through various wavelengths in the UV/Vis region of the electromagnetic spectrum.
 The x-axis (horizontal) shows the wavelength.
 The y-axis (vertical) shows the dependent variable; the absorbance.
 CHAPTER 5:- RESULTS & DISCUSSION
5.1 EFFECT OF TEMPRATURE ON PRODUCT

Effect of temperature on human hair (when time &concentration parameter are kept constant)

HAIR HCL(2N) TIME EFFECT OF TEMPRATURE ON HAIR

QUANTITY QUANTITY (Hr) 55⁰C 75⁰C 90⁰C 110⁰C
35gm 800ml 12 No Partial Complete Mixture
digestion digestion digestion bubbles
out
TABLE 5.1:-EFFECT OF TEMPRATURE ON PRODUCT

In the above case we have done 4 batches where the volume of hcl , Quantity of hair and time is kept
constant then at different temperatures the decomposition of hairs is observed which occur differently.
Hence the temperature at which the complete digestion occur is at 90⁰C which is the optimum temperature
condition.

5.2 EFFECT OF CONCENTRATION ON PRODUCT

Effect of concentration of HCL on human hair (when time &temp parameter are kept constant)

HAIR TEMPRATURE TIME(Hr) EFFECT OF CONCENTRATIUON ON HAIR

QUANTITY
0.25 0.35 1 2

35gm 90-95⁰C 12 COMPLETE COMPLETE Mixture Mixture

DIGSTION DIGESTION bubbles out bubbles
out

TABLE 5.2:- EFFECT OF CONCENTRATION ON PRODUCT

In the above condition the quantity of hair, optimum temperature and time is kept constant and at
different concentration the decomposition of hair is observed the range in which the complete digestion
5.3 EFFECT OF TIME DURATION ON PRODUCT
HAIR TEMPRATURE CONCENTRATION EFFECT OF TIME ON HAIR
QUATITY
6Hr 12Hr 18Hr 24Hr
35gm 95-100⁰C 0.35 No effect Complete Complete Hair get
digestion digestion completely
broken

TABLE 5.3 :-EFFECT OF TIME PERIOD ON PRODUCTS

In this condition the quantity of hair, optimum temperature , optimised concentration are maintained . The
only thing which is varied time period. At a temperature range of 12-18 Hr the complete digestion of the
hair occur.

5.4 COST ESTIMATION

S.No. Material Quantity Orignal cost Required cost

1. Hydrochloric Acid 0.8 L ₹460 per lit ₹368

2. Sodium Hydroxide 1.38 L ₹740 per kg ₹59.2

3. Copper Sulfate 0.002L ₹400 per liter ₹0.8

4. Conc. Nitric Acid 0.002 L ₹626 per liter ₹1.252

5. Mercuric Sulfate 0.001 kg ₹3900 per 0.5 ₹7.8

kg
6. Lead Acetate 2 ml ₹430 per liter ₹0.86

TABLE 5.4:-MATERIALS USED &THEIR PRIZES

Electricity required:-

S.No. Equipment Units cost per unit Orignal cost

1. D 0.25 ₹12 ₹3
igestor
TOTAL COST = ₹ 440.912/-

 Overhead cost =10% of total cost

=10*(440.912\100)
= Rs.44.0912/-

 Testing cost =3% of total cost

= 3*440.912\100
= Rs.13.22

 Overall total cost =440.912 + 44.912 +13.22

= Rs.498.2232/-

 Product cost =overall total cost output

=498.2232\0.236

Rs.2,111.11 Rs\kg of liquid protein

 CHAPETR 6:- CONCLUSION

 From various batches and experiments we have tried to get the optimum conditions for temperature,
time and concentration using Acid hydrolysis

1. Optimum temperature range:- 95 to 100 °c

2. Optimum concentration:-0.25 to 0.35 N

3. Optimum time :-8 to 10 hours

HAIR QUANTITY TEMPRATURE CONCENTRATION TIME

35gm 95-100⁰C 0.35N 12-18Hr

TABLE 6.1:-OPTIMISED CONDITIONS

The optimised condition obtained which is obtained from above batches are given in the above table.
At this condition the complete digestion of the hair will occur and we can form the Liquid Protein.

 In this project we have made a biofertilizer from human hairs which is waste
 We have succeeded to make liquid protein at the rate of 2,111Rs\L
 CHAPTER 7:-REFERENCES
1. KIRK AND OTHMER, Encyclopedia of the Chemical Technology, Fidth Edition ,Pg. No. 226-249
2. 8Akira NAKAMURA Makoto ARIMOTO, Keiji TAKEUCHI, and Toshihiro FUJII, Rapid Extraction
Procedure of Human Hair Proteins and Identification of Phosphorylated Species, Biol. Pharm. Bull. 25(5)
569—572 (2002)
3. Hari HarParshad Cohly1, Rahul Thomas2 and Raj Kumar2, | EXTRACTION AND SOLUBILIZATION
OF HUMAN HAIR KERATIN FROM HAIR CUT Waste, Department of Biology, Jackson State
University, Jackson, MS 39217, USA 2Dayalbagh Educational Institute, Dayalbagh, Agra, India Pg. No.
33
4. Paweł STAROŃ, Marcin BANACH1ZygmuntKOWALSKI and Anita STAROŃ, HYDROLYSIS OF
KERATIN MATERIALS DERIVED FROM POULTRY INDUSTRY, 0.2429/proc.2014.8(2)050
2014;8(2)
5. Korniłłowicz-Kowalska T, Bohacz J. Dynamics of growth and succession of bacterial and
fungalcommunities during composting of feather waste. Biores Technol. 2010; 101: 1268-1276.
DOI:10.1016/j.biortech.2009.09.053.
6. Wang YX, Cao XJ. Extracting keratin from chicken feathers by using a hydrophobic ionic liquid.
ProcBiochem. 2012; 47: 896-899. DOI: 10.1016/j.procbio.2012.02.013.
7. Grazziotin A, Pimentel FA, Sangali S, de Jong EV, Brandelli A. Production of feather protein hydrolysate
by keratinolytic bacterium Vibrio sp. kr2. Biores Technol. 2007;98:3172-
3175DOI:10.1016/j.biortech.2006.10.034
8. Grazziotin A, Pimentel FA, de Jong EV, Brandelli A. Nutritional improvement of feather protein by
treatment with microbial keratinase. Animal Feed Sci Technol. 2006; 126: 135-144. DOI:
10.1016/j.anifeedsci.2005.06.002.
9. Cheng S, Lau K, Liu T, Zhao Y, Lam P, Yin Y. Mechanical and thermal properties of chicken feather
fiber/PLA green composites. Composite: Part B. 2009; 40: 650-654.
DOI:10.1016/j.compositesb.2009.04.011.
10. Xia Y, Massé DI, McAllister TA, Kong Y, Seviour R, Beaulieu C. Identity and diversity of archaeal
communities during anaerobic co-digestion of chicken feathers and other animal wastes. BioresTechnol.
2012; 110: 111-119. DOI: 10.1016/j.biortech.2012.01.107101
11. Staroń P, Banach M, Kowalski Z. Keratin - Origins, properties, application. Chemik. 2011; 65: 10: 1019-
1026.
12. Costa JC, Barbosa SG, Sousa DZ. Effects of pre-treatment and bioaugmentation strategies on the
anaerobic digestion of chicken feathers. Biores Technol. 2012; 120: 114-119. DOI:
10.1016/j.biortech.2012.06.047.
13. Fakhfakh N, Ktari N, Haddar A, Mnif IH, Dahmen I, Nasri M. Total solubilisation of the chicken feathers
by fermentation with a keratinolytic bacterium, Bacillus pumilus A1, and the production of protein
hydrolysate with high antioxidative activity. ProcBiochem. 2011; 46: 1731-1737. DOI:
10.1016/j.procbio.2011.05.023.
14. Brebu M, Spiridon I. Thermal degradation of keratin waste. J Anal Appl Pyrolysis. 2011; 91: 288-295.
DOI: 10.1016/j.jaap.2011.03.003.
15. Fujii T, Murai S, Ohkawa K, Hirai T. Effects of human hair and nail proteins and their films on rat mast
cells. J Mater Sci Mat Med. 2008; 19: 2335-2342. DOI: 10.1007/s10856-007-3341-x.
16. Reichl S, Borrelli M, Geerling G. Keratin films for ocular surface reconstruction. Biomaterials.2011; 32:
3375-3386. DOI: 10.1016/j.biomaterials.2011.01.052.
17. Barone JR, Schmidt WF. Effect of formic acid exposure on keratin fiber derived from poultry feather
biomass. Biores Technol. 2006; 97: 233-242. DOI: 10.1016/j.biortech.2005.02.039.
18. Wrześniewska-Tosik K, Marchut-Mikołajczyk O, Mik T, Wieczorek D, Pałczyńska M. Mats for removing
technical oil contamination. Fibr Text East Europe. 2012; 6B: 101-106.
19. Regulation of the European Parliament and of the Council (EC) No 1069/2009 of 21 October 2009
20. U.SakthipriyadharshiniPreparation of Amino Acids From Keratin: An Effective Utilization of Hair Wastes
Generated From Tannery Pg. No. 1
21. C.R. Robbins, Chemical and Physical Behavior of Human Hair, Chemical Composition of Different Hair
Types, DOI 10.1007/978-3-642-25611-0_2, # Springer-Verlag Berlin Heidelberg 2012.
22. Rogers GE (2004) Hair follicle differentiation and regulation. Int J Dev Biol 48:163–170 Zahn H et al
(1963) Anwendungschwefelchemischeranalysen-methoden auf dauergewellteshaar. J SocCosmetChem
14:529–543
23. Stein H, Guarnaccio J (1960) The determination of sulfhydryl groups in reduced hair
keratin.AnalChemActa 23:89
24. Leach SJ (1960) the reaction of thiol and disulfide groups with mercuric chloride and methylmercuric
iodide in fibrous proteins. Austral J Chem 13:547–566
25. Robbins CR (1967) Infrared analysis of oxidized keratins. Text Res J 37:811–813Robbins CR, Bahl M
(1984) Analysis of hair by electron spectroscopy for chemical analysis.J SocCosmetChem 35:379–390
26. Block RJ, Bolling D (1952) the amino acid composition of proteins and foods. Charles C.Thomas,
Springfield, IL
27. McMullen R, Jachowicz J (1998) Thermal degradation of hair. I: effect of curling irons. JCosmetSci
49:223–244
28. Moore H et al (1958) Chromatography of amino acids on sulfonated polystyrene resins:asfbimproved
system. Anal Chem 30:1185–1190
29. Sagal J Jr (1965) Acid and base binding behavior of white and pigmented human hair. TextRes J 35:672–
673
30. Robbins CR, Kelly CH (1969) Amino acid analysis of cosmetically altered hair. J SocCosmetic Chem
20:555–564
31. Corfield MC, Robson A (1955) the amino acid composition of wool. Biochem J 59:62–68
32. Robbins CR, Kelly CH (1970) Amino acid composition of human hair. Text Res J40:891–896.
33. Ward WH, Lundgren HP (1955) the formation composition and properties of the keratin, In: Advances in
protein chemistry, vol 9, and references therein. Academic Press, New
YorkVenkatesh.T1Anbarasu.A2Subramanian.O.S3 Lab-Scale Extraction, Confirmation and Applications
ofProtein from Human Hairs Volume 3[8], pp: 4008-4011, August 2015
34. Advanced organic chemistry, ArunBahl and B.S.Bahl, ISBN 13: 9788121935159.
35. Structure and chemistry of Keratin fibers, Bradbury JH, Adv Protein Chem, 27(1973) 111-211.
36. Industrial applications of keratin – A review, R.Karthikeyan et al., Jr of Scientific and Industrial
Research, Vol 66, September 2007, 710-715.
37. A Rapid Extraction Procedure of Human Hair Proteins and Identification of Phosphorylated Species,
Akira Nakamura et al., Biol. Pharma. Bull. 25(5) 569-572 (2002).
38. Chemical Process Calculations, D.C.Sikdar, ISBN-13:978-8120347823.
39. Biochemical Methods by S.Sadasivam and A.Manickam,SecondEdition,New Age International
Publishers,Page-22.
40. Lab Manual in Biochemistry, Immunology and Biotechnology, Arti Nigam and Archanaayyagari, Tata
McGraw-Hill Education.
41. Chemical Reaction Engineering, Octave Levenspiel, ISSN: 978-0471254249.
42. A biotechnological process for treatment and recycling poultry feathers as a feed ingredient, A.Bertsch et
al., Bioresource Technology, 96 (2005) 1703-1708.
43. Biodegradation of keratin waste: Theory and practical aspects, Kowalska Teresa et al., Waste
Management, 31 (2001) 1689-1701.
44. Brazilian keratin hair treatment: a review, Journal of Cosmetic Dermatology, Vol 12, Issue 2,Pages 144-
148, June 2013.
45. A Review of Keratin-Based Biomaterials for Biomedical Applications, Jillian G. Rouse et al., Materials
2010, 3, 999-1014.
46. ROBERT L. HrLLt AND WILLIAM R. Schmidt, The Complete Enzymtic Hydrolysis of Proteins, Vol.
237, No. 2, February 1962.Vol. 237, No. 2, February 1962
47. OCTAVE LEVIENSPIEL, Chemical Reaction Engineering, Third Edition, Pg. No. 611, Chapter 27.
48. K. A. GAVHANE, Chemical Reaction Engineering-II, NiraliPrakashan, Pg. No. 9.1, Chapter 9.
49. Kuehler, C.A. and Stine, C.M. (1974). Effect of enzymatic hydrolysis on some functional properties of
Whey J. Food Sci. 39, 379-382
50. Protein, from Wikipedia, theencyclopedia
51. Laboratório de Bioquímica e MicrobiologiaAplicada, Departamento de Ciência de Alimentos (ICTA),
Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves 9500, 91501-970 Porto Alegre,
RS, Brazil Production of Proteolytic Enzymes by a Keratin-Degrading AspergillusNiger Volume 2011,
Article ID 487093, 9 pages
52. Hideto TAKAMI, t Satoshi NAKAMURA, Rikizo AONO, and Koki HORIKOSHIDegradation of Human
Hair by a Thermostability Alkaline Protease from Alkaliphilic Bacillus sp. No. AH- 101Biosci. Biotech.
Biochem., 56 (10), 1667-1669, 1992
53. H. Takami, T. Akiba, and K. Horikoshi, Appl. Microbiol. Biotechnol., 28, 120-124 (1989).
54. H. Takami, T. Akiba, and K. Horikoshi, Appl. Microbiol. Biotechnol., 33, 519-523 (1990).
55. H. Takami, T. Akiba, and K. Horikoshi, Biosci. Biotechnol.Biochern., 56, 333-334 (1992).
56. R. J. Yu, S. R. Harmon, and F. Blank, J. Bacteriol., 96, 1435-1436(1968).
57. W. Ebeling, N. Hennrich, M. K1ocknow, H. Meltz, H. D. Orth,and H. Lang, Eur. J. Biochern., 47, 91-97
(1974).
58. T. Nakanishi and T. Yamamoto, Agric. Bioi. Chern., 38, 2391-2397(1974).
59. H. P. Baden, in "Hair Research," ed. by C. E. Orfanos, W.Montagna, and G. Stuttgen, Springer-Verlag,
Berlin, New York,1981, pp. 73-75.
60. J. M. Gillespie and R. C. Marshall, "Hair Research," ed. by C. E.Orfanos, W. Montagna, and G. Stuttgen,
Springer-Verlag, Berlin,
61. Mariana Călina,b Diana Constantinescu-Aruxandei a, Elvira Alexandrescu aIulianaRăut
aMihaelaBadeaDoni aMelania-Liliana Arsene a, Florin Oancea a, LuizaJecu a,, Veronica Lazăr b
Degradation of keratin substrates by keratinolytic fungi M. Călin et al. / Electronic Journal of
Biotechnology 28 (2017) 101–112
62. Kreplak L, Doucet J, Dumas P, Briki F. New aspects of the α-helix to β-sheet transition in stretched hard
α-keratin fibers. Biophys J 2004;87:640–7
63. Bragulla HH, Homberger DG. Structure and functions of keratin proteins in simple, stratified, keratinized
and cornified epithelia. J Anat 2009; 214: 516–59.
64. Lange L, Huang Y, Busk PK. Microbial decomposition of keratin in nature — A new hypothesis of
industrial relevance. ApplMicrobiolBiotechnol 2016; 100: 2083–96.
65. Jin H-S, Park SY, Kim K, Lee Y-J, Nam G-W, Kang NJ, et al. Development of akeratinase activity assay
using recombinant chicken feather keratin substrates.PLoS One 2017;12:e0172712.
http://dx.doi.org/10.1371/journal.pone.0172712.
66. Gupta R, Ramnani P. Microbial keratinases and their prospective applications: An overview.
ApplMicrobiolBiotechnol 2006; 70: 21–33.
67. Jaouadi B, Abdelmalek B, JaouadiZaraî, Bejar NS. The bioengineering and industrialapplications of
bacterial alkaline proteases: The case of SAPB and KERAB in:Carping Angelo, editor. Progress in
molecular and environmental bioengineering — fromanalysis and modeling to technology applications.
InTech; 2011. p. 445–66.
68. Brandelli A. Bacterial keratinases: Useful enzymes for bioprocessingagroindustrial wastes and beyond.
Food Bioproc Tech 2008; 1: 105–16.
69. Jillian G. Rouse and Mark E. Van Dyke, A Review of Keratin-Based Biomaterials for Biomedical
Applications Materials 2010, 3, 999-1014;
70. Langbein, L.; Rogers, M.A.; Winter H.; Praetzel, S.; Schweizer, J. the catalog of human hair keratins. II.
Expression of the six type II members in the hair follicle and the combined catalog of human type I and II
keratins. J. Biol. Chem. 2001, 276, 35123−35132.
71. Langbein, L.; Schweizer, J. Keratins of the human hair follicle. Int. Rev. Cytol. 2005, 243, 1−78.
72. Schweizer, J.; Langbein, L.; Rogers, M.A.: Winter, H. Hair follicle-specific keratins and their diseases.
Exp. Cell Res. 2007, 313, 2010−2020.
73. Zhen, L.S. Ben Cao Gang Mu; the Time Literature & Art Press: Changchun, Jilin, China, 2005.
74. Hofmeier, J. Horn-lime plastic masses from keratin substances. German Pat DE184915, 18 December
1905.
75. Breinl, F.; Baudisch, O. The oxidative breaking up of keratin through treatment with
Hydrogen peroxide,Z Physiol. Chem. 1907, 52, 158−169.
76. Neuberg, C. Process of producing digestable substances from keratin. US Pat. 926,999, 6 July 1909.
77. Lissizin, T. Behavior of keratin sulfur and cystinsulfur in the oxidation of these proteins by potassium
permanganate I. Biochem. Bull. 1915, 4, 18−23.
78. Yutaka Shimomura and Masaaki Ito,Human Hair Keratin-Associated Proteins, J
InvestigDermatolSympProc 10:230 –233, 2005.
79. Powell BC, Rogers GE: Sequence, expression, and evolutionary conservation of a gene encoding a
glycine/tyrosine-rich keratin-associated protein of hair. J BiolChem 268:4511–4518, 1993
80. Dawber RPR, Price VH: Trichothiodystrophy: An ultrastructure study of the hair follicle. Br J Dermatol
113:273–280, 1985
81. Kariya N, Shimamura Y, Itom: Size polymorphisms in the human ultrahigh sulfurhair keratin-associated
protein 4, KAP4, gene family. J Invest Dermatol 124:1111–1118, 2013
82. Advanced organic chemistry, Arun Bahl and B.S.Bahl, ISBN 13: 9788121935159.
83.Structure and chemistry of Keratin fibers, Bradbury JH,
 APPENDIX
Amino-acid 3-letter 1-letter Structure Properties
name code code
Alanine Ala A Non-polar; Hydrophobic

Arginine Arg R Positively charged (basic amino acids; non-acidic

amino acids); Polar;
Hydrophilic; pK=12.5

Asparagine Asn N No charge (non-acidic amino acids); Polar;

Hydrophilic

Aspartate Asp D Negatively charged (acidic amino acids); Polar;

Hydrophilic; pK=3.9

Cysteine Cys C No charge (non-acidic amino acids); Non-polar;

Hydrophilic

Glutamate Glu E Negatively charged (acidic amino acids); Polar;

Hydrophilic; pK=4.2

Glutamine Gln Q No charge (non-acidic amino acids); Polar;

Hydrophilic

Glycine Gly G No charge (non-acidic amino acids); Non-polar;

Hydrophilic

Histidine His H Positively charged (basic amino acids; non-acidic

amino acids); Polar;
Hydrophilic; pK=6.0
 Isoleucine Ile I Non-polar; Hydrophobic

Leucine Leu L Non-polar; Hydrophobic

Lysine Lys K Positively charged (basic amino acids; non-acidic

amino acids); Polar;
Hydrophilic; pK=10.5

Methionine Met M Non-polar; Hydrophobic

Phenylalanine Phe F Non-polar; Hydrophobic

Proline Pro P Non-polar; Hydrophobic

Serine Ser S No charge (non-acidic amino acids); Polar;

Hydrophilic

Threonine Thr T No charge (non-acidic amino acids); Polar;

Hydrophilic

Tryptophan Trp W No charge; Non-polar; Hydrophobic

Tyrosine Tyr Y No charge (non-acidic amino acids); Polar;

Hydrophilic