CHAPTER I

LITERATURE REVIEW

1.1

L-PHENYLACETYLCARBINOL

Phenylacetylcarbinol (PAC) has two forms of enantiomer; one is the R-configuration and another is the S-configuration. (R)-PAC is known as L-phenylacetylcarbinol (LPAC) in its laevo-rotary chiral form or by the IUPAC designated name of 1-hydroxylphenyl-propan-2-one. It is a neutral organic compound of aromatic category due to the presence of the cyclic delocalization. L-PAC is widely used as an intermediate in the synthesis of L-ephedrine and
D-pseudoephedrine,

two pharmaceuticals with nasal

decongestant properties (Oliver et al. 1997). Table 1.1 below lists some of the physical and chemical properties of L-PAC whereas Figure 1.1 shows the chemical formula structure for L-PAC.

Table 1.1 Physical and chemical properties of L-PAC Properties CAS No. IUPAC Name Appearance Molecular formula Elementary composition Molecular weight Density Melting point Boiling point Flash point Solubility Enthalpy of Vaporization Values or Descriptions 53439-91-1 1--hydroxy-1phenyl-2-propanone Powder C9H10O2 C (71.98%), H (6.71%), O (21.31%) 150.17 g mol-1 1.119 1.126 g cm-3 172 oC or 445 K 253 oC or 526 K 109.019 oC 3.969 x 104 mg/L (at 25 oC) 52.865 kJ mol-1 Continue

Continue Special optical rotation Index of Refraction Half life Storage -375.8o 1.542 240 hours -20oC Freezer, under inert atmosphere

Sources: Hussain 2009; ChemSpider 2012

Figure 1.1 Chemical structure of L-phenylacetylcarbinol Source: Chikusa et al. 2001

L-PAC

is transformed biologically through the pyruvate decarboxylase (PDC)-

mediated condensation of added benzaldehyde with acetaldehyde generated metabolically from feedstock sugars via pyruvate (Oliver et al. 1997). The fermentation to produce L-PAC can be achieved by using various types of bacteria and yeasts. Alternatively, it can be synthesized chemically from cyanohydrins but the biotransformation remains as the preferred route for the industry (Shukla & Kulkarni 2000). The biosynthesis pathway of L-PAC in yeast will be discussed in Section 1.3.

1.2

PRODUCT USAGE OF L-PAC

Generally, most

L-PAC

contributes as an intermediate for the production of L-

ephedrine hydrochloride, a well known bronchodilator (Chandrakant et al. 1997). This pharmaceutical has the same effect as Ma Huang in China, have been used for several thousand years as folk remedies for inducing sweat, soothing breath and easing excretion of urine (Rogers et al 1997). Due to its similarity with epinephrine and cardiovascular effects, some claimed that it has the function in control obesity (Astrup et al. 1992). However, due to the control of L-ephedrine by North America,

illegal drug trafficking syndicate of Brazil and Canada targeted processing of amphetamine (United Nation 2009, Li et al 2009).

L-PAC

as the

1.3

SELECTION OF MICROORGANISM

There are a few microorganisms which has been associated with the production of Lphenylacetylcarbinol (L-PAC). Yeast has been commonly linked with high production of L-PAC. Among the species of yeast which are capable of producing L-PAC are Saccharomyces cerevisiae and Candida pseudointermedi (Kumar et al. 2006). Some bacteria strains like Zymomonas mobilis (Shukla & Kulkarni 2000) are also used in the production. Table 1.2 shows the comparison when these yeast species have been used for L-PAC production using molasses as the raw material. Molasses has been chosen as the raw material for our production considering its high potential yield which will be discussed in Section 1.4.

Table 1.2 Name of organism S. cerevisiae C. pseudointermedi S. cerevisiae GCU36

Comparison of types of yeast in L-PAC production Medium used Molasses Molasses Molasses
L-PAC

concentration (gL-1) 1.58 1.47 2.58

Bioconversion (%) 25.00 23.43 33.47

Source: Kumar et al. 2006 & Hussain 2009

Therefore, S. cerevisiae GCU36 has been selected considering its high concentration of product and bioconversion.

1.3.1

Saccharomyces cerevisiae

Saccharomyces cerevisiae is a type of yeast and commonly used in baking and brewing. It is known as Bakers yeast. Being the most commonly studied, it is often used in common fermentations. It has a cell wall made of chitin, has round globular to ovoid in shape yellow-green in colour and about 5 to 10 micrometer in diameter and

reproduces by budding (Ballesta & Larsen 2010). The cell wall lacks of peptidoglycan while its lipid components are ester linked.

S. cerevisiae is classified as saprotroph facultative anaerobe. It is able to break down the food through aerobic and anaerobic respiration; while also able to survive in an oxygen deficient environment for a period of time (Prescott et al. 2002). Figure 1.2 below shows its morphology while the hierarchy of taxonomy is shown in Table 1.3.

Figure 1.2

Scanning electron micrograph showing the morphology of a typical S. cerevisiae

Table 1.3

The taxanomy classification for S. cerevisiae Fungi Ascomycota Saccharomycetes Saccharomycetales Saccharomycetaceae Saccharomyces S. cerevisiae

Kingdom Phylum Class Order Family Genus Species

Source: Ballesta & Larsen 2010 It is also important to note that S. cerevisiae is not normally pathogenic to human. It is rarely reported that the colonization of S. cerevisiae in human tissue can cause any diseases (Ballesta & Larsen 2010). S. cerevisiae is considered as safe under the USFDA designation list as GRAS (FDA 2011). It is also safe for use (Agents that

are not associated with disease in healthy human adults or Risk Group 1) under the NIH Guidelines for Research (NIH 2011). The optimum level for S. cerevisiae is at 4.5 while the acceptable pH value for the growth is between 2.4 and 8.6. S. cerevisiae can tolerate up to 40C of temperature (Prescott et al. 2002). The mutation of Saccharomyces cerevisiae can be performed by using ultraviolet radiations and nitrous acid. Saccharomyces cerevisiae GCU36 was chosen considering the high L-PAC which can be economical for our production (Hussain 2009).

1.3.2

Biosynthesis Pathway of L-PAC

The biosynthesis begins with the action of pyruvate decarboxylase (PDC) which catalyzes the conversion of pyruvate to acetaldehyde with the resultant loss of a molecule of CO2. Pyruvate is the end product of glycolysis, from the conversion of sugar and is allowed to accumulate exogenously during the exponential phase of yeast growth. This reaction requires the co-factors thiamine pyrophosphate (TPP) and magnesium ion. PDC then catalyzes the condensation of acetaldehyde and pyruvate to form acetoin, and by analog also causes condensation of added benzaldehyde to produce L-PAC. It is seen that the process is itself divided into two stages first is to let the yeast grow and followed by a bioconversion stage where benzaldehyde is added (Oliver et al. 1997). The biosynthesis is illustrated in Figure below.

Figure 1.3 Biosynthesis of L-phenylacetylcarbinol

1.4

SOURCE OF RAW MATERIALS

Beet molasses is a byproduct of beet sugar refining. It contains about 50% sucrose and categorized as one of the high sugar-content compounds. Other than that, it is a valuable raw material in animal feed industry, yeast, citric acid, alcohol production, and pharmaceutical industry (Asadi 2007). Figure 1.4 below shows the image of beet molasses.

Figure 1.4

Beet Molasses

Source: HariniEthimax 2012

In production of L-phenylacetylcarbide, beet molasses is chosen as a raw material with Saccharomyces Cerevisae GCU36 as the combination of this produce a relatively high yield of L-phenylacetylcarbide. Based on Table 1.2, the yield of Lphenylacetylcarbide is 2.58g/L. Besides producing high yield, beet molasses is an easily obtained and relatively economic raw material in Malaysia. Table 1.4 shows the quality standards for components and properties of molasses. Table 1.4 Quality Standards for Nonfood-Grade Molasses

Quality Standards for Nonfood-Grade Molasses Sucrose 46.0-52.0% Ash 10.0-12.0% Protein 8.0-10.0% Betain 4.0-6.0% Water 18.0-20.0% pH 7.0-7.5% Density (80% DS) 1400kg/m3 Source: Asadi 2007

REFERENCES

Anon. 2009. Precursors and Chemicals Frequently Used in the Illicit Manufacture of Narcotic Drugs and Psychotropic Substances. United Nation: United Nation Publication.

Astrup, A., Buemann, B., Christensen, N. J., Toubro, S., Tboebek, G., Victor, 0. J.,& Quaade, F. 1992. The effect of epehedrine/caffein mixture on energy expenditure and body composition in obese women. Metabolism., 41: 686-688.

Chandrakant M. Tripathi, Suresh C. Agarwal, & Samar K. Basu. 1997. Production of L-Phenylacetylcarbinol by fermentation. Journal of Fermentation and Bioengineering 84: 487-492.

ChemSpider. 2012. Phenylacetylcarbinol. http://www.chemspider.com/ChemicalStructure.9106838.html [5 March 2013].

Li Dan, Lin Jian-qun, Lin Hui-bin, Lin Jian-qiang, & Qu Yin-bo. 2009. Advances in bioconversion production of L-phenylacetylcarbinol. The Chinese Journal of Process Engineering 9.

Chikusa, Y., Hirayama, Y., Ikunaka, M. & Matsumoto, J. 2001. Process for producing l-erythro-(1r,2s)-2-amino-1-phenylpropan-1-ol. patents/EP1142864A1?cl=en [2 March 2013]. http://www.google.com/

Kumar,M.R., Chari,M.A. & Narasu, M.L. 2006. Production of L-phenylacetylcarbinol (L-PAC) by different novel strains of yeasts in molasses and sugar cane juice as production medium. Research Journal of Microbiology 1(5): 433 437.

Oliver, A.L., Roddick, F.A., & Anderson, B.N. 1997. Cleaner production of phenylacetylcarbinol by yeast through productivity improvements and waste minimisation. Pure & Applied Chemistry 69(11): 2371-2385.

P.L. Rogers, H.S. Shin and B. Wang. 1997. Biotransformation for L-ephedrine production. Advance Biochemical Engineering Biotechnology 56:33-59.

Shukla, V.B. & Kulkarni, P.R. 2000. L-phenylacetylcarbinol (L-PAC) biosynthesis and industrial applications. World Journal of Microbiology and Biotechnology 16(7): 499-506.