Running head: ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 1

β-Glucuronidase Enzyme Reaction Activity Optimization

for the Hydrolysis of Codeine-6-Glucuronide

Advanced Scientific Research Paper

Submitted to the Center for Advanced Studies, Wheeler High School

by

WEINBERG, DAVID

The Center for Advanced Studies

Wheeler High School
Marietta, GA
November 2018
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 2

Abstract
 ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 3

Table of Contents

Chapter 1: Introduction……………………………………………………………………………..
Statement of the Problem…………………………………………………………………...
Purpose of the Study………………………………………………………………………..
Research Questions…………………………………………………………………………
Hypothesis Statement……………………………………………………………………….
Significance of the Study…………………………………………………………………...
Definition of Key Terms……………………………………………………………………
Summary……………………………………………………………………………………

Chapter 2: Literature Review……………………………………………………………………….

Foundational Subproblem 1………………………………………………………………...
Foundational Subproblem 2………………………………………………………………...
Summary…………………………………………………………………………………....

Chapter 3: Research Method………………………………………………………………………..

Research Methods and Design……………………………………………………………...
Population………………………………………………………………………………......
Sample……………………………………………………………………………………....
Materials/Instruments………………………………………………………………………
Operational Definition of Variables………………………………………………………...
Data Collection, Processing, and Analysis…………………………………………………
Assumptions………………………………………………………………………………...
Limitations………………………………………………………………………………….
Delimitations………………………………………………………………………………..
Ethical Assurances………………………………………………………………………….
Summary……………………………………………………………………………………

Chapter 4: Findings…………………………………………………………………………………
Results………………………………………………………………………………………
Evaluation of Findings……………………………………………………………………...
Summary……………………………………………………………………………………

Chapter 5: Implications, Real World Connections, Recommendations, and Conclusions…………

Implications………………………………………………………………………………....
Recommendations…………………………………………………………………………..
Conclusions………………………………………………………………............................

References…………………………………………………………………………………………..

Appendix A…………………………………………………………………………………………
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 4

LIST OF ABBREVIATIONS

GUS Beta-Glucuronidase

LC-MS/MS Liquid Chromatography-Tandem Mass Spectrometry

 ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 5

LIST OF TABLES

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6
 ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 6

LIST OF FIGURES

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6
 ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 7

Chapter 1: Introduction

The topic of this research paper is enzyme hydrolysis, specifically the reaction conditions

and reagents used to obtain the optimal reaction based on recovery of desired chemical

compounds. This study analyzes the same reaction, hydrolysis of codeine-6-glucuronide by

GUS, under sets of varied conditions. The goal of this experimentation is to obtain the highest

possible yield of desired product, free codeine, from the reaction. An expected value for yield

has been calculated based on chemical equivalencies between codeine-6-glucuronide and free

codeine. The use of hydrolysis reactions is widespread in laboratory professions, including

clinical laboratories, so the optimization of reactions is often paramount for accurate results and

clinical reporting.

Introduce topic in short paragraph here

Statement of the Problem

Between 3 enzymes, 2 E. coli-based and 1 abalone-based beta-glucuronidases, which

combination of enzyme source, activity units, temperature and incubation time in sample

preparation by enzyme hydrolysis of codeine-6-glucuronide yields the most accurate results for

percent recovery of analytes upon analysis?

Purpose of the Study

The purpose of this study is to optimize the sample preparation procedures for preparing urine

samples to be tested for drug metabolites. This optimization would be implemented at Newstar

Medical Laboratories, LLC. and possibly other laboratories.

 ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 8

Research Questions (ASPs)

What source of beta-glucuronidase, time of incubation, temperature, and enzyme activity units in

sample preparation afford the most accurate sample analysis by percent recovery of codeine

analytes?

Hypothesis Statements (Identify variables)

An activity of 4,000 units, a time of 2 hours, a temperature of 55°C, and the E. coli-derived beta-

glucuronidase from manufacturer Kura Biotec will result in the most accurate analysis results.

Significance of the Study (Importance, contribution, need, benefits, negatives if never

conducted)

This study looks at what enzyme sources and sample preparation conditions yield the most

accurate results for analysis of drug metabolites, specifically codeine-6-glucuronide, in urine

samples and attempts to optimize these factors. This study is of immediate relevance and

importance in the clinical laboratory industry. Laboratories must try to cut costs wherever

necessary to provide more cost-effective services while maintaining quality. Boison, Dowling,

Johnson, & Kinar (2016) showed that recovery of drug metabolites from horse tissue samples is

increased by the addition of a GUS hydrolysis step, so it has been shown to increase yield.

However, optimization of that hydrolysis is required, and any amount of optimization of

techniques can give laboratories an advantage. This is very useful in an expensive but essential

business like private healthcare. Without efficient methods, costs would be great and patients

would have to pay that difference. Thus, the importance of this study stems from its potential to

increase accuracy and efficiency of reporting and to drive down care costs. NEEDS EDITING
 ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 9

Definition of Key Terms (Alphabetized, used in paper, cited appropriately)

SAME AS IN PROPOSAL?

 Acid/Base Hydrolysis: Acid hydrolysis uses protons from acids as catalysts in

substitution reactions. Base hydrolysis involves the hydroxyl (OH-) group of the

base acting as the substitution for something else in a reaction (S. Vander Wielen,

personal communication, October 11, 2018).

 Aliquot: A known amount of a substance, generally a liquid, that is usually a

fraction of a whole (McNaught, Wilkinson, & Jenkins, 1997).

 Analyte: A part of a substance that is being analyzed (McNaught, Wilkinson, &

Jenkins, 1997).

 Beta-glucuronidase: Beta-glucuronidase is an enzyme that is used in the

hydrolysis of glucuronides from bodily fluids prior to analysis (“β-Glucuronidase

(beta-Glucuronidase)”, n. d.).

 Enzyme Activity Unit: The amount of an enzyme that catalyzes the conversion of

a specific amount of a substance per minute under optimal conditions for the

enzyme (Nomenclature Committee of the International Union of Biochemistry

(NC-IUB), 1979).

 Enzyme Hydrolysis: A reaction in which an enzyme facilitates the breaking of

bonds in molecules in the presence of water (Yang, Dai, Ding, & Wyman, 2011).

 Hydrolysis: Solvolysis by water, solvolysis being the reaction with a solvent

involving the breaking of bonds in the solute (McNaught, Wilkinson, & Jenkins,

1997).
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 10

 Incubation: The catalyzation of a reaction by controlling environmental

parameters around the reaction (R. Godwin, personal communication, September

14, 2018).

 Internal Standard: “A compound added to a sample in known concentration to

facilitate the qualitative identification and/or quantitative determination of the

sample components,” (McNaught, Wilkinson, & Jenkins, 1997).

 Liquid Chromatography: Liquid chromatography is a method where molecules in

a mixture are applied to a solid, or stationary phase, moved along that solid by a

liquid solution, or mobile phase, and separated from each by various interactions

between the individual molecules and the stationary phase (Coskun, 2016).

 Mass Spectrometry: Mass spectrometry is a method of measuring the

characteristics of molecules by ionizing a sample, separating them by mass and

charge using electromagnetic fields, and detecting them and creating a graph

based on the results (Reusch, 2013). Tandem mass spectrometry uses multiple

mass separators to obtain a higher resolution of ions (“Tandem Mass

Spectrometry (MS/MS)”, 2015).

 Metabolite: “A substance made or used when the body breaks down food, drugs

or chemicals, or its own tissue,” (“Metabolite”, n. d.).

 Quality Control: Quality control is an aspect of laboratory management taken to

assure the validity of results and to improve analysis associated with measurement

in the lab (Howanitz & Howanitz, 1983).

Summary
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 11
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 12

Chapter 2: Literature Review

Contains several subheadings, start with introduction, logical structure with varied in-text

citations, minimize lengthy quotes, no block quotes

In order to understand the enzyme hydrolysis process that is essential for production in

many clinical laboratories, one should be knowledgeable about hydrolysis methods and enzyme

functions. In this case, a basic understanding of human metabolism is helpful too, considering

the research is being conducted to inform testing with human urine samples. This literature

review will discuss, as found in peer-reviewed scientific papers, hydrolysis techniques and some

advantages and disadvantages, as well as the function and sources of GUS in the context of

human metabolism and drug testing.

Acid/Base and Enzyme Hydrolyses

Logical structure, don’t summarize, synthesize, conclusion that answers questions, statement of

basis of research, links to other subproblems

EDITS LATER

Hydrolysis involves the breaking of bonds with water. Two efficient hydrolysis methods

are acid/base hydrolysis and enzymatic or enzyme hydrolysis. These techniques of hydrolysis are

important in today’s world, both in science and in industry. Hydrolysis is used to create fuel

(Greenwood, Farrell, Zhang, & O'Hara, 2015) and other products and separate useful

compounds, including those to help fight disease, from other larger substances (Peciulyte,

Karlstrom, Larsson, & Olsson, 2015; Liu, Wen, Zhang, Wei, Deng, Xiao, & Zhang, 2017).

Generally, hydrolysis techniques are used because they are more efficient and provide greater

products yields than other methods of substance separation (Greenwood et al., 2015; Boison et

al., 2016; Liu et al., 2017). Much current research is being conducted to improve hydrolysis
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 13

methods, both to expand their uses to larger scale operations and to optimize their efficiencies

(Greenwood et al., 2015). This section will cover some of the characteristics of acid/base

hydrolysis and enzyme hydrolysis and discuss their relative advantages and disadvantages.

Acid/base hydrolysis involves using acids or bases to catalyze hydrolysis and break

bonds. Acids and bases are somewhat interchangeable in this manner, as the better option

depends on the substance being hydrolyzed. Often this technique is used in large-scale hydrolysis

of tough biofuels and natural substances, especially plant matter (Greenwood et al., 2015; Liu et

al., 2017; Hilpmann et al., 2016). This type of hydrolysis characteristically requires severe

conditions for reactions to take place efficiently. Due to reaction kinetics, the most influential of

these conditions is temperature. In order for acid/base hydrolysis reactions to occur swiftly, high

temperatures are used, often times near or beyond 100°C (Hilpmann et al., 2016; Greenwood et

al, 2015). Yields often improve at these higher temperatures because of those reaction kinetics

(Greenwood et al., 2015). Another main condition affecting acid/base hydrolysis is, of course,

pH. Naturally, these reactions require extremely high or low pHs to proceed (Hilpmann et al.,

2016).

While these reaction conditions can be acceptable in large scale industrial production,

they can often lead to undesired consequences. Hydrolysis at these high temperatures and pHs

can drain resources and money (Greenwood et al., 2015). At temperatures and pHs that are too

high, degradation can occur, leading to the destruction of resources before they can be converted

to useful products. Alternatively, unwanted by-products can be formed in the same way. This can

cause reactions to occur efficiently in only a relatively small window of temperatures and pHs

(Hilpmann et al., 2016). As stated previously, these conditions are obtained and accepted in some

operations, but the production process is obviously very intense.

ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 14

Enzyme hydrolysis, contrastingly, is a more precise technique. It employs enzymes as

catalysts for bond breaking. Uses of enzyme hydrolysis are more exact, including drug analysis

(Boison et al., 2016), and require less severe conditions in order to maximize the activity of the

enzyme and reduce the possibility of degrading the target compounds and enzymes. Increasing

temperature helps increase the reaction rate, as in most cases, but enzymes usually cannot be

subjected to very high temperatures and remain effective due to degradation of the enzyme (Liu

et al., 2017; Hilpmann et al., 2016), although there are exceptions (Hilpmann et al., 2016).

Another characteristic of enzyme hydrolysis is that enzymes, by design, are very specific to

certain substances. Enzymes must be able to chemically bind to other substances, but the

physical structure of enzymes used for hydrolysis must also closely match the structures of the

substances that are hydrolyzed. Sometimes this principle is extended to structures surrounding

the target substances. Even within targets for hydrolysis, variations in structure and the

attachment site of enzymes can impact how effectively enzyme hydrolysis breaks up substances

(Peciulyte et al., 2017).

One other possible method for hydrolysis is to treat substances with acids or bases prior

to enzyme hydrolysis. This could serve to be more efficient for some substances than either

technique used alone. What can occur in a lot of cases of both hydrolysis techniques being used

is that first treating a substance by acid or base hydrolysis allows enzymes to better access sites

or can prepare certain substances for enzyme hydrolysis that would not have been able to without

pretreatment under the severe conditions of acid/base hydrolysis (Peciulyte et al., 2015). This

method of preparing substances for enzyme hydrolysis by a pretreatment technique extends

beyond acid/base hydrolysis to gelatinization and liquefaction (Liu et al., 2017).

ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 15

As shown, both acid/base and enzymatic techniques of hydrolysis have their respective

benefits and situations where they are used. Acid/base hydrolysis is better for larger scale, more

industrial processes where conditions can be made as severe as possible to speed up reaction

rates. Enzyme hydrolysis is preferable for more precise methods as there is less possibility of

degradation or unwanted by-products, even though conditions must be more stringently

regulated. In the context of the research that will be performed in this study, enzyme hydrolysis

is used because the drug analytes that are studied are not stable under severe reaction conditions

and the instruments of analysis are very finely tuned and thus subject to degradation and damage

under those same conditions. Reaction conditions such as temperature and time of reaction will

be manipulated to determine the optimal hydrolysis activity of an enzyme. The specific enzyme

being used, GUS, will be looked at in the next review.

Drug Metabolism and Beta-Glucuronidase

PROOFREAD AND EDIT

Before talking about the enzyme GUS, one has to discuss the functions and processes that

GUS is related to. In human metabolism, one of the main ways that humans excrete waste is

through urine. In order for waste to be excreted in urine, it has to be soluble in urine. Not all

waste products are soluble in urine, so the body has mechanisms to change the solubility of

certain waste products to make them more easily excretable. One of these is a process called

glucuronidation (Liang et al., 2016). Glucuronidation attaches a glucuronic acid – a type of sugar

– substituent to a waste product to make it soluble in urine. One of the main groups of waste

products that are affected by glucuronidation is drugs. Thus, it is very important to understand

glucuronidation as a part of drug metabolism when developing or prescribing drugs (Liang et al.,

2016; Gagez, Rouguieg-Malki, Sauvage, Marquet, & Picard, 2012).

ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 16

In the same vein, being able to separate drugs and drug metabolites from their glucuronic

acid conjugates helps to facilitate drug analysis. GUS, the enzyme used for this research, has the

specific function of cleaving glucuronic acid groups, and sometimes other substrates, from waste

products, including drugs and drug metabolites, carcinogens, and hormones (Sakurama et al.,

2014). Now, other substances are hydrolyzed by GUS: it is used in the body in regular

metabolism to regulate the accumulation of non-harmful substances in the body (Hassan et al.,

2013) and on other substances not related to human metabolism in order to convert them from a

form containing glucuronic acid to another, more useful one without it (Sakurama et al., 2014).

Not every form of GUS – because GUS is actually a class of enzymes – has the same

capabilities. Sakurama et al. (2014) discovered a GUS that has “strict glycon specificity” (p. 7),

meaning that it selectively hydrolyzes only substances with a glucuronic acid group, as opposed

to others that have the capability of hydrolyzing other substrates. The use of GUS that is

implemented in this research is its ability to isolate drugs and drug metabolites from their

glucuronic acid conjugates.

This is something that GUS excels at. GUS is used often in clinical laboratories in sample

preparation before drug analysis. The isolation of drugs and drug metabolites allows for clearer

recognition by machines of drug presence in human urine samples. Taylor et al. (2017)

mentioned that “an enzymatic hydrolysis step using ß-Glucuronidase may be implemented to

increase the sensitivity of [benzodiazepines]” (p. 2). Taylor et al.’s study was done with clinical

laboratory testing in mind. In fact, they showed an increase in positive screens for

benzodiazepines, a class of depressant drugs, of 42.5% with the addition of GUS in sample

preparation (Taylor et al., 2017). Much current research is occurring in optimization of sample

preparation using GUS in clinical laboratories, including this study. Morris et al. (2014) showed
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 17

that a specific form of GUS can give the same amount of hydrolysis “at [room temperature]

equivalent to measurements at 55°C” and “was considerably faster” compared to another form

(p. 3). They also showed that this form was less damaging to instruments over time as many

samples were run through them. These different forms of GUS are another area of ongoing

research and optimization.

GUS is harvested or manufactured from a variety of sources. Humans are one such

source (Hassan, Waheed, Grubb, Klei, Korolev, & Sly, 2013), although human GUS is not used

in clinical settings. Other sources are more easily harvestable, such as abalone (Morris et al.,

2014). Abalone-based GUS was used often in clinical laboratories, but other sources have

become more prominent recently. Bacterial GUS is even more easily acquired than abalone

GUS, so it has become more pervasive. The main source of GUS from bacteria is Escherichia

coli and other closely related bacteria; it was thought to be essentially the only bacterial source of

GUS (Krahulec, Szemes, & Krahulcová, 2010). However, Krahulec et al. (2010) discovered

another source of GUS in bacteria. Sakurama et al. (2014) continued this, finding other bacterial

sources, as did other groups, and still more sources of GUS are being sought out. In reality,

human GUS is very similar to bacterial GUS (Hassan et al., 2013), but obviously it is easier to

obtain GUS from bacteria than from humans. The desire for better sources of GUS has begun the

development of synthetic analogues to natural GUS. Manufacturers like IMCSzyme and Kura

Biotec have developed recombinant GUS, and research has begun on characterizing these

recombinant enzymes and evaluating their usefulness (Morris et al., 2014).

GUS is obviously an important enzyme because of its role in the laboratory. Since

glucuronidation is such a ubiquitous mechanism in metabolism, it is important to understand

glucuronidation and GUS to be able to monitor metabolism. Since healthcare, including drug
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 18

testing in clinical laboratories, is so imperative and expensive, it is important as well to use the

right materials and to optimize resources and processes to provide efficient results and care at

low cost. This research uses GUS in sample preparation to hydrolyze codeine-6-glucuronide.

GUS source, time of incubation, temperature, and enzyme activity units are manipulated to

determine which combination of these factors leads to the most efficient and most accurate

results for drug concentration in samples.

Summary
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 19

Chapter 3: Research Method

This chapter concerns the research process, including specific details on data collection

and analysis. Also covered will be samples for statistical purposes, materials and instruments, the

scope of the study, and any assumptions made concerning the research. The question the

researcher is trying to answer is: What source of GUS, time of incubation, temperature, and

enzyme activity units in sample preparation afford the most accurate sample analysis by percent

recovery of codeine analytes? The hypothesis formulated for this question is: An activity of

4,000 units, a time of 2 hours, a temperature of 55°C and the E. coli-derived GUS from

manufacturer Kura Biotec will result in the highest percent recovery. The aim of this chapter is

to ensure the replicability of the study for any future researchers that may take interest in this

area of study.

Research Methodology and Design

Accurately describes, substantiate appropriateness of the design, statement of why design was

chosen or was appropriate, explain how you were able to accomplish the study goals

PROOFREAD AND EDIT (CODEINE FROM CERILLIANT)

The researcher conducted the data collection for this research at Newstar Medical

Laboratories, LLC. The results and methods of similar studies were reviewed in the literature

review to inform this study’s methodology. The researcher prepared samples to start with the

IMCSzyme E.coli-based GUS, beginning trials with activity, then temperature and time of

incubation. Then the researcher conducted trials with the Kura Biotec E.coli-based GUS. MORE

MORE MORE
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 20

The researcher prepared the first set of samples with one source of GUS and manipulated

variables at between five and eight data points, depending on the condition variable. However,

the researcher only manipulated one variable at a time. For example, the researcher began with

the IMCSzyme GUS and manipulated activity units. In this case, activity units were changed as

incubation time and temperature were held constant at 1 hours and 55C. The same procedure was

followed for manipulating other condition variables. The researcher collected results for samples

with a single GUS source, and then work began on samples with a different source. This

separation ensured that inherently different activity levels for enzymes didn’t confound results

for preparation condition manipulations. Incubation of all experimental sample occurred in a

Benchmark Incu-Mixer MP 4-plate. Experimental sample reactions for IMCSzyme occurred in

50 µL of synthetic urine, which simulates the actual chemical environment of human urine, due

to volume constraints. All other experimental reactions, however, occurred in 100 µL. The

researcher prepared/ran all experimental samples in triplicate.

An LC-MS/MS instrument analyzed the experimental samples, after preparation under

the various combinations of factors. This method first separates analytes from each other and

then individually measures their masses so they can be identified. LC-MS/MS has been validated

many times as an analytical method and is taught in chemistry courses. The instruments used at

Newstar Medical Laboratories, a Shimadzu 20-Series LC setup and an AB Sciex API Triple-

Quad 4500 MS, are valid and reliable for clinical use (CITATIONS?). Analysis results obtained

from these instruments are sent to and displayed on a paired computer with software for

quantitative analysis, specifically MultiQuant 3.0.????, also from AB Sciex. Also used as part of

the LC-MS/MS analysis was Analyst software, provided by AB Sciex.

ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 21

In analyzing each batch of samples to obtain quantitative results, the researcher prepared

a set of samples alongside experimental samples to prepare the instruments for analysis. Blanks,

samples containing only mobile phase, went first so that the mobile phase running through the

LC would equilibrate with the stationary phase. Calibrator samples injected into the instrument

next. These set up a calibration curve on the paired software that serves as the reference for

assigning samples concentrations based on LC-MS/MS analysis. Quality controls followed

calibrators and have a known concentration of analyte. They are used in clinical practice to

verify that the calibration curve is accurate before any patient samples are run. After running

quality controls, the LC-MS/MS injected experimental samples for analysis. All experimental

samples contained, because of prior preparation, a concentration of codeine-6-glucuronide

equivalent to a known concentration of codeine. This served as the reference value against which

the researcher compared experimental samples. Each sample created contained internal

standards. These are solutions of analytes of a known concentration that are slightly modified to

distinguish them from target analytes. They serve to reduce the confounding of response results

from LC-MS/MS. By creating a ratio of responses between target analytes and internal

standards, one can protect against data skewing because of common instrument issues. Because

both the targets and internal standards are affected, no change is recorded in the response ratio.

Once the researcher obtained results for the experimental samples, the researcher

compared them with the expected concentrations of codeine in samples to determine the percent

recovery of analytes and thus the effectiveness of hydrolysis under experimental conditions. The

researcher recorded, tabulated, and illustrated these data graphically, when appropriate. To

quantify analysis results, the researcher used descriptive statistics, including mean, standard

deviation, and percent coefficient of variance. The industry standard of an acceptable difference
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 22

in percent recovery from expected is plus or minus 20%. This standard allowed the researcher to

assess the viability of experimental conditions for sample analysis for actual urine samples. The

researcher then analyzed the results to determine which method for sample preparation yields

codeine recovery percentages closest to the expected values.

Population

Description of population as appropriate, estimated size, relevant characteristics, describe why

population is appropriate in relationship to study problem/purpose, distinguish between

population and sample

Sample

Identify sampling method, explain selection of relevant sample, including known population

characteristics, Quantitative study: minimum sample size with sampling error considered,

representativeness, and assumptions of statistical tests, as appropriate describe how existing data

were originally collected and for what purpose, must be sufficient description for replicability

Materials/Instruments

Description of data sources (archived data, published instruments, materials, interview protocol,

apparatus), specific types of validity and reliability, include appendices as needed, materials

developed for the study, apparatus including make, mode, how it is used, outcomes it provides,

validity, reliability

Operational Definition of Variables (Qualitative and Mixed Design?)

ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 23

Data Collection, Processing, and Analysis

Describe in enough detail for replication, describe steps taken to complete the study, provide

specific details related to the execution of the study, describe types of data and statistical

analysis/software, describe analysis strategy used to test hypotheses, must be appropriate for

statistical test

???????? PROOF AND EDIT

The data that the researcher will need to determine the optimal method of sample

preparation are percent recovery of codeine and percent difference from expected concentrations

of codeine. The researcher will obtain these values by entering experimental samples into an LC-

MS/MS for analysis. Software compatible with the LC-MS/MS will calculate and display results

for percent recovery, from which the researcher will calculate coefficient of variation as well.

Data obtained from LC-MS/MS will transfer to software on the laboratory’s computers for

further analysis and calculations, so the researcher will not have to compute percent recovery

manually. However, the researcher will need to compute the coefficient of variation among

triplicated samples. This will show the precision of the preparation method in hydrolyzing

codeine-6-glucuronide. The researcher will create graphical representations of the various

manipulated variables plotted against percent recovery to show the impact of conditions on

enzyme efficiency. The researcher will determine the optimal method of sample preparation by

determining the closest percent recovery of codeine analytes to the expected value. He will also

factor in the coefficients of variation in weighing whether or not a particular average percent

recovery is reliable.
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 24

Assumptions

Assumptions about population and design and rationale, support for assumptions

 The researcher assumes that the instruments used for analysis are tuned and provide

accurate results for percent recovery.

 The researcher assumes that they will have access to enough enzymes/supplies to prepare

enough samples for the study.

 The researcher assumes that the equipment used for sample preparation and analysis are

available for use when the researcher needs them.

 The researcher assumes that the enzymes obtained from manufacturers function as

expected and as advertised by the manufacturers.

 The researcher assumes that the quality control samples from manufacturers provide

accurate calibration for experimental samples.

Limitations

Study limitations, potential weakness to interpretation and validity, context of design, how

threats to validity were addressed as much as possible

 A limitation of the study is in the “cleanliness” of the enzymes used. Different sources of

GUS yield different levels of “cleanliness” in the enzyme. E. coli sources are generally

considered the cleanest and abalone is less so. However, these factors and their impacts

on sample hydrolysis are outside of the researcher’s control.

ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 25

 A limitation of the study is in the time frame the researcher will have to collect data.

Because of time constraints, the researcher will not have time to investigate the effects of

longer incubation times on metabolite recovery results or any long-term cost benefits of

one enzyme over another.

Delimitations

Scope of study, choices made to narrow

 This study does not include any tests on enzymes that are not GUS. Within the confines

of laboratory where the research will be performed, GUS is the only enzyme available for

study.

 This study does not perform any tests on variables that are not time of incubation, activity

units, or temperature. Tests on other variables, such as pH, are possible but not within the

scope of this research.

 This study obtains results for validation of preparation techniques from liquid

chromatography-mass spectrometry. The mass spectrometer is specifically of the triple-

quadrupole variety. Other techniques, such as gas chromatography, could be used to

obtain the same or similar results, but the study will only be performed using instruments

available in the lab where the research will be performed.

Ethical Assurances

Compliance with standards for conducting research as appropriate to design, how assurances for

approval were obtained

???????????
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 26

Summary

Key points, make PAST TENSE after research is complete

ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 27

Chapter 4: Findings

Brief overview of purpose, brief overview of the chapter, organize based on research

question/hypotheses, APA for how to report results

Results

Report results without discussion, in paragraph format, appropriate description, results presented

in logical fashion, answer questions as stated, address violations of assumptions, make decisions

based on results of statistical analysis (significance), APA to present results in text, tables,

figures, provide sufficient information for reader to make own judgement regarding

interpretation, refer to tables and figures in text

Evaluation of Findings

Briefly report what findings mean, interpret results in light of context, expected vs unexpected

and explanations for unexpected/conflicting results, brief interpretation within study context and

profession, originality of contribution, figures that explain should be right by the paragraph, cite

if not own creation, discuss findings in terms of practical utility, how profession/field of study

are impacted by inquiry, avoid drawing conclusions beyond what can be interpreted directly

from study results, data must be explained thoroughly, most important tables/graphs should be

right next to paragraphs explaining results, specific and concise, all other data in appendices

Summary
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 28

Chapter 5: Implications, Real World Connections, Recommendations, and Conclusions

Brief review of problem, purpose, method, limitations, ethics, conclude with brief overview of

the chapter

Implications

Each research question and hypothesis (when appropriate) individually, draw logical

conclusions, discuss impact of limitations on interpretation, put results in context of study,

literature, profession, contribution of practical utility

Real World Connections

All real world connections for practical applications, supported with findings, connections for

future research

???????????? RATIONALE OF STUDY BELOW

The purpose in conducting this study is to optimize the enzyme hydrolysis of codeine-6-

glucuronide by beta-glucuronidase. All analytical laboratories must prepare samples before

analysis, and hydrolysis reactions are commonly used to convert compounds into more easily

analyzed forms. Codeine-6-glucuronide was chosen for this research because it is known for

being difficult to hydrolyze, even over other drugs in other classes (R. Godwin, personal

communication, October 9, 2018). Clinical laboratories that evaluate the presence of drugs and

drug metabolites in patient samples must ensure that sample preparation methods provide

adequate conversion of glucuronide conjugates to parent drugs, like codeine, in order to obtain
ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 29

adequate response from instruments to confirm or deny the presence of drugs. This research

serves to optimize the procedures for such preparation. Enzyme source, incubation time, enzyme

activity units, and temperature will be examined because they are influential and cost-inducing

factors in sample preparation (Morris, Chester, Strickland, & McIntire, 2014, pp. 1-2; Taylor,

Flint, Ma, Hill, Clark, & Strathmann, 2017, p. 2). Using beta-glucuronidase as the enzyme for

analysis is also important because it is designed to hydrolyze glucuronide-containing drug

metabolites, which are pervasive in human metabolism (Hassan, Waheed, Grubb, Klei, Korolev,

& Sly, 2013, p. 1; Liang et al., 2015, pp. 2-3). The researcher undertook this research and the

internship where this research was performed in order to further their knowledge of the

laboratory industry and the field of analytical chemistry, and because it provided the opportunity

to apply such knowledge.

IMPORTANCE OF STUDY BELOW

This study looks at what enzyme sources and sample preparation conditions yield the

most accurate results for analysis of drug metabolites in urine samples and attempts to optimize

these factors. This study is of immediate relevance and importance in the industry of clinical

laboratories. Laboratories must try to cut costs wherever necessary to provide more cost-

effective services while maintaining quality. Boison, Dowling, Johnson, & Kinar (2016) showed

that recovery of drug metabolites from horse tissue samples is increased by the addition of a

GUS hydrolysis step, so it has been shown to increase yield. However, optimization of that

hydrolysis is required, and any amount of optimization of techniques can give laboratories an

advantage. This is very useful in an expensive but lucrative business like private healthcare.

Thus, the importance of this study stems from its potential to increase accuracy and efficiency of

reporting and to drive down care costs.

ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 30

Recommendations

Recommendations for practical applications, supported with findings, recommendations for

future research

Conclusions

Summarize key points of chapter

 ENZYME REACTION OPTIMIZATION FOR HYDROLYSIS 31

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Appendix A: Title

Insert content here

Not needed for all papers, usually added for data/quotations too lengthy for inclusion in text, not

listed as chapters, divided by letters on separate pages, must meet margin guidelines, list each

appendix separately in Table of Contents, tables and figures must be numbered, captioned, and

listed in List of Tables or List of Figures, all material must be distinct, legible