GENE MINING AND

CHARACTERISATIONS OF NOVEL
L–PHEYNYL DEHYDROGENASE

Major: BIO Engineering

Supervisor: Dr.Cheng Xinkuan
Name: Vishani Tharindi Punchihewa
Student ID: 19041425
Date: 18/05/2023
 ABSTRACT

L-phenylalanine dehydrogenase (PheDH) is an important enzyme for the biosynthesis

of L-phenylalanine, a chiral amino acid used as a sweetener and a drug intermediate.
This enzyme class is known scientifically as L-phenylalanine:NAD+ oxidoreductase
(delaminating). L-phenylalanine dehydrogenase and PheDH are two more common
names. This enzyme is involved in the metabolism of phenylalanine as well as the
production of phenylalanine, tyrosine, and tryptophan. PheDH also has applications in
the detection of phenylketonuria, a metabolic disorder. In this study, we mined and
characterized a novel PheDH from Geobacillus kautopillus , a thermophilic bacterium.
The benefits of using thermostable enzymes for biotechnological processes at high
temperatures include a decreased risk of contamination by mesophilic microorganisms,
a decrease in the viscosity of reaction medium, an increase in the bioavailability and
solubility of organic compounds, and an increase in the diffusion coefficients of
substrate and products, which leads to faster reaction rates. The novel PheDH showed
high thermostability and substrate specificity, and also OD600 value which can be taken
as the was obtained as 0.665 .Our results demonstrate the potential of the novel PheDH
for industrial applications in amino acid production and medical diagnosis.

Key Words: L-phenylalanine dehydrogenase, enzyme, gene mining, analyze,

characterization

I
 摘 要

L-苯丙氨酸脱氢酶 (PheHD)是 L-苯丙氨酸生物合成的重要酶，L-苯丙氨酸是一

种用作甜味剂和药物中间体的手性氨基酸。这种酶类在科学上被称为 L-苯丙氨
酸：NAD+氧化还原酶（脱氨）。L-苯丙氨酸脱氢酶和 PheDH 是两个比较常见的
名字。这种酶参与苯丙氨酸的代谢以及苯丙氨酸、酪氨酸和色氨酸的产生。PheDH
还可用于检测苯丙酮尿症（一种代谢紊乱）。在这项研究中，我们从嗜热细菌
Geobacillus kautopillus 中挖掘并表征了一种新型 PheDH。在高温生物技术过程
中使用耐热酶的好处包括降低被嗜温微生物污染的风险、降低反应介质的粘度、
增加有机化合物的生物利用度和溶解度，以及增加底物和产物 '底物'和产物的扩
散系数，从而导致更快的反应速率。新型 PheDH 显示出高热稳定性和底物特异
性，并且 OD600 值可以作为 0.665 获得。我们的结果证明了新型 PheDH 的潜力
用于氨基酸生产和医学诊断的工业应用。

关键词：L-苯丙氨酸脱氢酶，酶， 开采的基因挖矿，嗜热，分析，表征

II
 CONTENTS
Chapter 1 Introduction................................................................................1
1.1 Introduction.............................................................................................1

Chapter 2 Materials and Methods………………………………...6

2.1material and methods……………………………………………………6

Chapter 3 Results and Analysis…………………………………...13

3.1 Results and analysis…………………………………………………..13

Chapter 4 Discussion and Conclusion……………………………20

4.1 Discussion and conclusion…………………………...........................20

Acknowledgement…………………………………………………22

References………………………………………………………….23

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Chapter 1

1.1 Introduction
A phenylalanine dehydrogenase is an enzyme in enzymology that catalyzes the
chemical reaction L-phenylalanine + H2O + NAD+ phenyl pyruvate + NH3 + NADH +
H+. This enzyme's three substrates are L-phenylalanine, H2O, and NAD+, while its four
products are phenyl pyruvate, NH3, NADH, and H+. This enzyme belongs to the
oxidoreductase family, specifically those that operate on the CH-NH2 donor group with
NAD+ or NADP+ as acceptor. This enzyme class is known scientifically as L-
phenylalanine: NAD+ oxidoreductase (deaminating) [1].L-phenylalanine dehydrogenase
and PHD are two more common names. This enzyme is involved in phenylalanine
metabolism as well as the production of phenylalanine, tyrosine, and tryptophan.
From researches the scientists were found Bacillus badius IAM 11059
phenylalanine dehydrogenase was refined to homogeneity from the crude extract of B.
badius using disc gel electrophoresis. The enzyme has a relative molecular mass, MI,
of 310000-360000 and an isoelectric point of 3.5. The enzyme is made up of identical
subunits with M values ranging from 41000 to 42000. The enzyme's substrate
specificity was high for L-Phenylalanine in the oxidative deamination reaction, but low
in the reductive amination reaction with phenyl pyruvate, p-hydroxyphenylpyruvate,
and 2-oxohexanoate. The enzyme gene was cloned into Escherichia coli using the
vector plasmid pBR322.In E.coli, the enzyme was highly expressed [2]
there are some important facts that indicates from the researches.The reversible, and
pyridine nucleotide-dependent oxidative deamination of L-phenylalanine to generate
phenylpyruvate and ammonia is catalyzed by phenylalanine dehydrogenase.
The researchers determined the X-ray crystal structures of the recombinant
enzyme in the complexes E.NADH.L-phenylalanine and E.NAD+ and studied the
steady-state kinetic behavior of the enzyme from Rhodococcus sp. M4. L-3-
phenyllactate, with resolutions of 1.25 and 1.4 A, respectively. Initial velocity, product
inhibition, and dead-end inhibition investigations show that the kinetic process is
ordered, with NAD+ binding before phenylalanine and products released in the order
ammonia, phenylpyruvate, and NADH. The enzyme has no action with NADPH or
other 2'-phosphorylated pyridine nucleotides, but it has a wide range of activity with
NADH analogues.and A coenzyme recycling system is employed with wild-type
phenylalanine dehydrogenase from Bacillus sphaericus and three mutants N145A,
N145V, and N145L to synthesise l-phenylalanine and three non-natural amino acids (p-
F-phenylalanine, pMeO-phenylalanine, and p-CF3-phenylalanine)[3].A variety of
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reaction circumstances are studied through a research. The substitution of serine for
glycine at positions 123 and 124 of phenylalanine dehydrogenases from Bacillus badius
and Bacillus sphaericus, respectively, significantly reduced enzyme activity toward
aromatic amino acids while increasing relative activity toward aliphatic amino acids.
(TACHIBANA et al. 23 March 2009) the mutant from B. badius dehydrogenated
branched-chain amino acids preferentially, whereas the mutant from B. sphaericus
dehydrogenated straight-chain amino acids.
In this study, the PheDH of B. badius was cloned and subjected to site-directed
mutagenesis at the indicated position, followed by kinetic and structural analyses to
identify more exclusive mutants. The results demonstrated that the V144L mutant
significantly enhances specificity toward phenylalanine and decreases specificity
toward L-tyrosine, whereas the V144N mutant decreases specificity toward
phenylalanine and increases specificity toward tyrosine [5].
Furthermore, the mutated V144D had a significantly lower kcat and a lower km
value for phenylalanine than the wild type. The Phe/Tyr specificity constant in V144L
rose more than fourfold when compared to wild type, making it a promising candidate
for more specific PKU identification. And some researches indicates the enzyme is
made up of identical subunits with M values ranging from 41 000 to 42000. (Paradisi et
al 15 August 2006). The enzyme's substrate specificity was high for L- Phenylalanine
in the oxidative deamination reaction, but low in the reductive amination reaction with
phenylpyruvate, p-hydroxyphenylpyruvate, and 2-oxohexanoate. The enzyme gene was
cloned into Escherichia coli using the vector plasmid pBR322. In E. coli, the enzyme
was highly expressed. Purified to homogeneity, the enzyme generated by E. coli
transformant was found to be identical to that of B. badius IAM 11059 in terms of
specific activity, MI, subunit structure, and amino acid content. ( ASANO et al. March
23IJune 2, 1987 - EJB 87 0344)
L-homophenylalanine (L-HPA) is an important building ingredient in the
manufacture of angiotensin-converting enzyme inhibitors and other chiral
pharmaceuticals. Among the processes developed for L-HPA production, bio catalytic
synthesis using phenylalanine dehydrogenase has proven to be the most promising.
However, as with other dehydrogenase catalyzed reactions, the viability of this process
is significantly hampered by insufficient substrate loading and high cofactor costs. A
highly efficient and cost-effective bio catalytic process for LHPA was developed in this
study by combining genetically engineered phenylalanine dehydrogenase and formate
dehydrogenase. The combination of fed-batch substrate input and continuous product
removal boosted substrate loading and cofactor use significantly. Following systemic

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optimization, 40 g (0.22 mol) of keto acid substrate was transformed to L-HPA in 24 h

and a total of 0.2 mM NAD+ was effectively reused in eight cycles of fed-batch
operation, yielding an average substrate concentration of 510 mM and a productivity of
84.1 g l1 day1 for L-HPA.The current study presents an effective and viable enzymatic
approach for producing L-HPA as well as a universal solution for increasing substrate
loading [7].
They are chosen reductive amination reaction techniques for the manufacture of
homoglutamate in the current work, which is an attractive pathway for the conversion
of 2-ketoadipic acid catalyzed by NADH-dependent phenylalanine-dehydrogenase. To
the best of our knowledge, we are the first to disclose the engineering of the
phenylalanine dehydrogenase enzyme in E. coli for the generation of homoglutamate,
as indicated in scheme. Previously, it was discovered the affinity of TiPDH
transamination of 2-ketoadipic acid as a substrate was insufficient. As a result of our
computational insights into the structure of TiPDH, we proposed site-specific directed
mutagenesis of two key PDH residues using AutoDock Vina docking simulations. It
then explained PDH's binding mode with -Ketoadipic acid and ligands by altering two
key residues in active site of TiPDH, which resulted in the formation of homoglutamate.
Gly114 has been changed to Arg114, and Ala135 has been changed to Arg135. The
Ala135/Arg135 enzyme performed far better than the wild-type enzyme, with a
threefold increase in affinity for the substrate. In addition, we used formate
dehydrogenase (FDH) as a cofactor regenerating enzyme in our system. The system's
equilibrium with co-factor regeneration abundantly included homoglutamic acid
synthesis and CO2 as a reaction byproduct. The biocatalysts utilized in the reductive
amination process are retained in this case to enhance the reaction system's
sustainability [9].
L-PheDH from Sporosarcina ureae was immobilized on DEAE-cellulose after
being treated with 2-amino-4,6-dichloro-s-triazine, hexamethylenediamine, and
glutaraldehyde. The greatest immobilized PheDH activity was determined to be 95.75
U/g support with 56% maintained activity. The optimal pH for immobilized L-PheDH
was changed from 10.4 to 11.0. The immobilized L-PheDH displayed activity
fluctuations near to the maximum value throughout a wider temperature range of 45-
55 °C, but the natural enzyme showed activity variations at 40 °C. The immobilized L-
PheDH also had a higher pH and heat stability than the natural enzyme. K (m) values
of native and immobilized L-PheDH were determined at pH 10.4 and 25 °C as K (m
Phe) = 0.118, 0.063 mM and K (m NAD)(+) = 0.234, 0.128 mM, respectively. The

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flow-injection analysis system detected generated NADH+at the outlet of the packed
bed reactor column. The reactor's conversion efficiency was determined to be 100% in
the range of 5-600 M Phe at 9 mM NAD+ and a total flow rate of 0.1 mL/min. The
reactor was operated for 3 hours every day to analyze 30 samples. The reactor had a
half-life of 15 days. This is something about Immobilization of phenylalanine
dehydrogenase and its application in flow-injection analysis system for determination
[10]
of plasma phenylalanine (et al.tarhan 2011 Jan; 163(2):258-67. doi:
10.1007/s12010-010-9035-8. Epub 2010 Jul 24.).
Here we should analyze the best growing temperature of the phenyl alanine
dehydrogenase according to the different organism. And we can decide what is the most
suitable organism which can be taken as the most stable one.so by gene mining we can
understand and analyze it. To discover new natural products, researchers have
increasingly relied on genome mining methodologies, which traditionally have referred
to exploiting genomic sequence data to identify and forecast genes that encode the
creation of novel substances. While chemical structures can be incredibly different,
nature frequently converges on a few techniques to manufacture the same chemical
building blocks, allowing researchers to use enzyme genetic markers to find new
biosynthetic pathways.
So that is the importance of doing gene mining and it is highly help us to have
better Idea about the genetic structure and identify the gene. And it is important to
understand the characterization of the oxidoreductase because the assessment of
oxidoreductase activity is useful in understanding the metabolic activities of various
organs.so it’s important to analyze the characterizations. Oxidoreductase is a wide set
of enzymes in biochemistry that are engaged in redox reactions in living organisms and
in the laboratory.
So in this study we can focus on the thermos stable L-phenylalanine
dehydrogenase from to produce thermostable extracellular enzymes with essential
biotechnological uses, thermophilic and thermo tolerant bacteria have significant
economic value. Thermophilic activities are known to be connected with protein
thermostability.
The use of thermostable enzymes for biotechnological processes at elevated
temperatures has the following advantages:

1. it reduces the risk of contamination by mesophilic microorganisms;

2. it decreases the viscosity of the reaction medium;
3. it increases the bioavailability and solubility of organic compounds
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4. it increases the diffusion coefficient of substrates and products, resulting in

faster reaction rates.
Enzymes are organic molecules present in biological systems that can
catalyze processes. Enzymes as biocatalysts have reduced process costs and
provided as alternatives to synthetic chemicals that may be poisonous and
harmful to the environment [11].Biocatalysts typically operate in a temperature
range, with the ideal temperature being the temperature at which the host
system has the greatest viability. Enzymes perform best in temperatures
ranging from 20 to 40 °C in most biological systems. Some species, however,
can survive in extreme environments such as high temperatures, pH, or salt
concentrations. These organisms are known to produce heat-stable or chemo-
stable enzymes that function in less-than-ideal circumstances.so finding the
thermostable microorganism is the mining part of this paper and through the
experiment ,many of data were obtained.

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Chapter 2

2.1 Materials and Methods

1. With LB medium

liquid:
NaCl 1 g/100 mL
Tryptone 1 g/100 mL
Yeast extract 0.5 g/100 mL
Distilled water 100 mL
solid:
Add agar 2 g/100 mL
2. Extract the genome
Inoculate Bacillus subtilis into LB liquid medium and culture overnight
1. Take 1ml of bacterial culture solution, centrifuge at 12000 rpm for 2 min ,
and absorb the supernatant as much as possible
2. Add 500 μL of Buffer Bs to re suspend the cells, add 50 μL of Lysozyme
( 20 mg/mL ) to mix by pipetting, and place in a 37°C water bath for 60min
3. 12000 rpm , 5 min , centrifuge, discard the supernatant
4. Add 180 μL of Buffer GL, 20 μL of Proteinase K (20 mg/mL) and 10 μL
of RNase (10 mg/mL) to mix well by pipetting, and put it in a water bath at 56°C
for 10 min. At this time, the solution should be clear and transparent. Add 200 μL
Buffer GB and 200 μL absolute ethanol
Treated cells are processed as follows
1. Put the Spin column on the Collection Tube, transfer the solution to the Spin
column, centrifuge at 12000 rpm for 2 min, and discard the filtrate
2. Add 500 μL of Buffer WA to the Spin column, 12000 rpm, 1 min, discard the
filtrate
3. Add 700 μL of Buffer WB to the Spin column at 12000 rpm, centrifuge for 1min,
discard the filtrate
4. Repeat 3
5. Place the Spin column on the collection Tube and let it run at 12 000rpm for
2min.
6. Put the spin column in a new 1.5mL centrifuge tube, add 50~200 μl of
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sterilized water to the center of the spin column membrane, let it stand at room
temperature for 5 minutes , and heat the sterilized water to 65 ℃ in advance
7. Centrifuge at 12000rpm for 2min to elute DNA

3. PCR
PCR ratio (25 μL system)
Taq enzyme 8.5 microliters (which contains four deoxyribonucleotides)
Genome 2 µL
Upstream primer 2 µL (BaPDHF)
Downstream primer 2 µL(BaPDHR)
Sterile water 10.5μL
PCR temperature ( 94°C, 55°C, 72°C)
4. Extract PET28a plasmid
Take 1 mL PET28a glycerol bacterium, add to 100 mL liquid medium, cultivate
overnight

1. Take 1.5-3 mL of overnight cultured bacteria solution, centrifuge at 12000rpm

for 3min, discard the supernatant, and collect the bacteria. Afterwards, the cells were
centrifuged for 1 min , and the culture medium was aspirated
2. Resuspend the bacteria with 250 uL BufferP1
3. Add 250uL BufferP2, gently flip up and down 6-10 times to fully lyse the
bacteria until the solution becomes clear, and let stand at room temperature for 3min
4. Add 350ul BufferP3, immediately flip up and down gently 6-10 times, and place
at room temperature for 8 minutes. Centrifuge at 12000 rpm for 10min at 4°C.
5. Transfer the supernatant to a blue adsorption column and let it stand on ice for
10 minutes. 4℃ 12000 rpm, centrifuge for 1min, pour off the liquid in the collection
tube
6. Add 500 uL deproteinized solution (Buffer DW1), let stand for 5 minutes,
centrifuge at 12000 rpm for 1 minute, and discard the filtrate.
7. Add 500 uL Wash Solution, let stand for 5 min, centrifuge at 12000 rpm for
1min, and discard the filtrate.
8. Repeat the above steps once
9. Centrifuge the empty adsorption column for 1min at 12000 rpm
10. Put the adsorption column into a new EP tube, let it stand for 10 minutes , wait

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for the ethanol to volatilize, add 50μ L of Elution Buffer to the center of the membrane,
let it stand at room temperature for 5-10 minutes , and centrifuge at 12000 rpm for 3
minutes at room temperature , and save the EP tube Liquid ( -20°C)
5. Enzymatic cutting of plasmid and target gene
Plasmid 50 microliter enzyme digestion system
1 µL BamHΙ - HF enzyme
1 µL Xho I enzyme
5 µL CutSmart buffer (1/10th of the system)
10 µL plasmid
33 µL sterile water
Target gene 100 microliter enzyme digestion system
1 µL BamH I -HF enzyme
1 µL Xho I enzyme
10 μL CutSmart buffer (1/10th of the system)
50 μL target gene
38 µL sterile water
6. Production of Competent Cells
1. Take out the competent original bacteria from the refrigerator at -80℃ , take 4
μL and add it to 400μL of anti-antibiotic-free LB medium , shake and cultivate for
30 minutes
2. Streak inoculation on anti-antibiotic LB medium, culture at 37 ° C for 12 h
3. Inoculate the single colony on the petri dish into 50mL of culture solution, shake
at 37 °C for 12 h
4. Take 1 mL of bacterial seed to 50 mL of culture medium, and culture on a
shaking table for 2 h.
5. Transfer the bacterial solution to a 50 ml centrifuge tube, place on ice for 15min,
then. 4℃, 7500 rpm , centrifuge for 10min.
6. Discard the supernatant, blot dry the culture medium, add 10mL of 0.1
micromole /L calcium chloride to the centrifuge tube, shake and mix well, suspend the
bacteria, and ice-bath for 30min.

7, 4 ° C, 7500 rpm , centrifuge for 10 min , discard the supernatant, blot the
remaining culture medium, add 4 mL of ice-cold calcium chloride solution, and suspend
the bacteria. Add 1L of glycerin, divide it into small EP tubes, and put it in a -80°C
refrigerator for later use.

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7. 7. Recombinant plasmid transformation

1、 Take the prepared competent cells in the -80℃ refrigerator, ice bath for
10min, and thaw the cells.
2、 Add 1μL of recombinant plasmid to 100μL of competent cells, shake
well, and place on ice for 30 minutes.
3、 Heat shock at 42°C for 90 seconds, quickly transfer it to ice, and cool
the cells for 5 minutes.
4、 Add 400 μL of anti-antibody-free LB medium, 37 ℃, culture 45 min.
5、 Spread 150 μ L of bacterial liquid on LB solid medium containing
kanamycin, and culture at 37°C for 12 h.
6、 Identification of successfully transformed E. coli.
3-4 colonies on the plate containing kanamycin, carry out colony PCR, and finally
the colony with the target gene band is the successfully transformed Escherichia coli
7、 Induced gene expression
Pick the colony successfully introduced into the recombinant plasmid, inoculate
it into the liquid culture medium, and cultivate it overnight on a shaking table. Then
take 1ml of culture solution and add it to 50 mL of liquid culture solution, culture for
about 2 hours, measure OD600 between 0.6-0.8, make the cells in logarithmic growth
phase, add inducer IPTG, can induce gene expression protein.
The process can be mentioned as here in brief (figure 2.1)

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• cultivate bacillus subtils

and extract genome

• Cultivate bacterial
• using specific primers producing PET2-8a
PCR,amplifies the target plasmid and extract
gene fragment plasmid

• cut with two

• cut with two
endonuclease BamHI-HF
endonuclease BamHI-
Xhol
HF Xhol

Separately purified and then ligated with T4 DNAse

Using the heat shock method to induce the feeding prepared in advanced E-coli

Induce in to LB medium for cultivation, take small amount of bacteria liquid ,spread
on solid medium containing kanamycin, and cultivate overnight,pich the successfully
grown colonies.(figure2.2)

Pick the colony with the target band after PCR culture it to the logarithmic growth
phase and add the inducer IPTG to induce the expression of target gene. The control
group received no inducer

After culturing, the two bacterial solutions were centrifuged to obtain precipitated and
buffer was added to disrupt cells. And the supernatant was obtained by the
centrifugation. The two were compared by the protein electrophoresis. Finally only
the bacteria added with inducer Bio informatics analysis

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Figure 2.1(The process flow chart)

Figure 2.2

Here we using bioinformatics analysis and for it as the analyzing platform the NCBI,
and the DNAMAN software are used to analyze. All L-phenylalanine dehydrogenase
from different organizations are searched by using NCBI web site. The growth
temperature of these organizations are googled. The top 8 L-phenylalanine
dehydrogenase with the highest growth temperature of these organizations are selected
from the results of the NCBI. And, their amino acid sequences are downloaded. The
growth temperature of the organizations, the amino acid composition of L-
phenylalanine dehydrogenase also needs to be considered. The composition of the 10
amino acid sequence (derived from high growth temperature) should be analyzed, and
multiple sequence alignment and phylogenetic tree construction should be done with
the DNAMAN software. Previously downloaded 10 amino acid sequences are put into
a txt file. Naming the each amino acid sequence in fasta format is done. And these are
the amino acid sequences that are obtained and numbered them as amino acid sequence
1, amino acid sequence 2, amino acid sequence 3….etc.

 amino acid sequence 1- L-phenylalanine dehydrogenase precursor

[Rhodococcus sp. (in: high G+C Gram-positive bacteria)]
 amino acid sequence 2- L-phenylalanine dehydrogenase [Geobacillus
kaustophilus GBlys] 368 aa protein
 amino acid sequence 3- L-phenylalanine dehydrogenase [Geobacillus
thermoleovorans CCB_US3_UF5] 380 aa protein
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 amino acid sequence 4- L-phenylalanine dehydrogenase [Rhodococcus sp.

AW25M09] 357 aa protein
 amino acid sequence 5- L-phenylalanine dehydrogenase [Bacillus sp.
UTB2301] 369 aa protein
 amino acid sequence 6- L-phenylalanine dehydrogenase [Geobacillus
thermodenitrificans NG802]380 aa protein
 amino acid sequence 7- L-phenylalanine dehydrogenase [Thermoactinomyces
intermedius]366aa protein
 amino acid sequence 8- L-phenylalanine dehydrogenase [Rhodococcus sp.
MTM3W5.2]357 aa protein
the amino acid sequences of above are downloaded and saved in a text file.
Then the DNAMAN software was opened and . Click in turn: File---open---
the above txt file of amino acid sequence and Click in turn: Sequence---
Multiple sequence alignment
Please select protein and click file to open the above txt file again. And then

by clicking “下一步” and “下一步” and “下一步” and “完成 and c Click

the output---Graphic (EMF) File--- Fill in the file name as amino acid
sequence.and the multiple sequence alignments were obtained. Different
colors on multiple sequences indicate different conservations. So then from
the results of the multiple sequence alignments we can obtained the homology
tree by Click the output---tree---homology tree--. The homology tree can be
drawn.
Finally the best selected enzyme’s amino acid sequence was analyzed
using the “expasy”.and the data was analyzed by using it.this will be the
analysis of physicochemical properties of L-pheynylalanine dehydrogenase.

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Chapter 3

3.1 Result and analysis

The concentration of the plasmid also measured as the 104.3 µgmL(figure 3.1)
From the experiment we mainly measure the value of OD600 which determined
an arbitrary value that helps the cell growth stage, when compared to a standard
or control reference. By comparing a measured result to a control, it is possible
to estimate the growth stage and the total concentration of cells. And confirm
the cells in the logarithm phase and that’s the inducer can be added to make the
gene express.by confirming the cells are in logarithmic groth phase ,when the
cell groth and the reproduction rate was very fast and it was the best time to
induce the expression.we got the OD600 value as the 0.665 (figure 3.1)

Figure 3.1 figure 3.2

Differences of amino acid sequence of L-pheynylalanine from different microbial

sources is shown by the results of multiple sequence alignment (figure 3.3). This is used
to analyze protein domains, and also individual amino acid sequence and tertiary and
secondary structures

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Table 1.growth temperatures of the selected microorganisms

L-PHENYL ALANINE FROM Growth
DIFFERENT MICTROBIAL SOURCES temperature(°C)
precursor [Rhodococcus sp. (in: high 10-40
G+C Gram-positive bacteria)]

Geobacillus kaustophilus GBlys]368 aa 60-74

protein

Geobacillus thermoleovorans 35-65

CCB_US3_UF5]380 aa protein
Rhodococcus sp. AW25M09]357 aa 30-50
protein
Bacillus sp. UTB2301]369 aa protein 37-55

Geobacillus thermodenitrificans NG80- 55-65

2]380 aa protein
Thermoactinomyces intermedius]366 aa 40-55
protein
Rhodococcus sp. MTM3W5.2]357 aa 25-37
protein
The results of the growing temperature were found from the google and From this
chart (Table 1). We can obtain the best pheynylalanine dehydrogenase in Geobacillus
thermoleovorans mictrobial source.and this can be regarded as the high thermos stable
microorganism and from this we can obtain the high thermostable l-phenylalane
dehydrogenase.and disulfide bonds are a key factor of thermal stability of enzyme.in
addition cysteine is the key to forming disulfide bonds.so high thermostability means
the highest cysteine content.so the best phenylalanine was selected with it.
Multiple alignments of eight amino acid sequences of the characterized L-
phenylalanine dehydrogenase from different bacteria in this work and in previous
literature. Identical residues are black and conserved residues are other colors.

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Figure 3.3a figure 3.3b

Multiple alignments of eight amino acid sequences of the characterized L-
phenylalanine dehydrogenase from different bacteria in this work and in previous
literature. Identical residues are black and conserved residues are other colors (figure
3.3a)
Search for bacterial genomic sequences by NCBI may find over 200 sequences of
pheynylalanine dehyrogenase family proteins. Homology tree showing the relationship
between different amino acid sequences. The tree was constructed using
DNAMANsoftware. Values at nodes indicate significance in bootstrap analysis. The
homology tree (figure 3.3b) also showed that the sequence 2 and sequence 3 were
very close , with a high level of Geobacillus kaustophilus GBlys]368 aa protein (90%-
100%) . the close relationship to Geobacillus kaustophilus GBlys]368 aa protein was
obtained from the amino acid sequence 3 which means Geobacillus thermoleovorans
CCB_US3_UF5]380 aa protein and amino acid sequence 6 (Geobacillus
thermonitrificans) and there was a relationship between bacillus sp.and the
Geobacillus sp.that can be obtained. And the amino acid sequence 8 ( Rhodococcus sp.
MTM3W5.2]357 aa protein) was isolated from the miccroorganism of best
phenylalanine dehydrogenase.the closely related group could be idientified using
homology tree. And the best one was obtained from it.there are many simmillarities
between Geobacillus sp.(Geobacillus kaustophilus GBlys]368 aa protein, Geobacillus
thermoleovorans CCB_US3_UF5]380 aa protein, Geobacillus thermodenitrificans
NG80-2]380 aa protein) which belong to same genus.and the thermal stability is also
good and also when comparing the Geobacillus kaustophilus GBlys] and amino acid
sequence 5 which means Bacillus sp. UTB2301]369 aa protein.also (70%-80%) similar
to the best one which was obtained.

According to the data the best selected enzyme was idientified as Geobacillus
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 Undergraduate Thesis of Tianjin University of Science and Technology for Cohort 2019

kaustophilus.Here we mainly analyzed the Protein molecular weight, amino acid

composition (figure 3.4), isoelectric point, half-life, atomic composition, hydrophobic
region, transmembrane region, phosphorylation site, secondary structure. Homology
modeling to obtain the tertiary structure of the protein .The data which were obtained
by 'expasy’ were like follows

Figure 3.4

Number of amino acids: 368

Molecular weight: 40116.82
Theoretical pI: 6.34
Total number of negatively charged residues (Asp + Glu): 49
Total number of positively charged residues (Arg + Lys): 46
Atomic composition:

Carbon C 1780
Hydrogen H 2821
Nitrogen N 495
Oxygen O 532
Sulfur S 14

Formula: C1780H2821N495O532S14
Total number of atoms: 5642
Aliphatic index: 86.44
Grand average of hydropathicity (GRAVY): -0.168

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 Undergraduate Thesis of Tianjin University of Science and Technology for Cohort 2019

Figure 3.6 Compute pI/Mw is a tool which allows the computation of the
theoretical pI (isoelectric point) and Mw (molecular weight) of L-pheynylalanine
dehydrogenase.

Figure3.7 The secondary structure of proteins

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Undergraduate Thesis of Tianjin University of Science and Technology for Cohort 2019

Figure 3.8 secondary structure of proteins

Figure3.9 functional domains of proteins

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 Undergraduate Thesis of Tianjin University of Science and Technology for Cohort 2019

Figure 3.10 tertiary structure of proteins

The Geobacillus kaustophilus PDH projected model has a similar fold to the
Rhodococcus enzyme, consisting of a Rossmann fold domain and a substrate-binding
[18]
domain .The Rossmann fold domain binds the NAD+ cofactor and has a parallel
beta-sheet bordered by alpha-helices. The L-phenylalanine substrate is bound by the
[19]
substrate-binding domain, which has a mixed alpha/beta structure . The active site
lies at the interface of the two domains and consists of two catalytic residues (figure
3.7).The resultant theoretical values verify that the identified and unmodified proteins
are consistently positioned on a 2D PAGE gel1 (figure 3.6).
The enzymes have a Rossmann fold domain and a substrate-binding domain. A parallel
beta-sheet bordered by alpha helices makes up the Rossmann fold domain, which binds
the NAD+ cofactor. The combined alpha/beta structure of the substrate-binding domain
binds the L-phenylalanine substrate. Lys78 and Asp1182 are two of the catalytic
residues that are present at the active site, which is situated at the intersection of the
two domains [21].
The tertiary structure of protein redicted tertiary structure of PDH from Geobacillus
kaustophilus, the SWISS-MODEL output (figure 3.10).

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 Undergraduate Thesis of Tianjin University of Science and Technology for Cohort 2019

CHAPTER 4
4.1 Discussion and Conclusions
Here the Geobacillus kaustophilus of L-pheynylalanine dehydrogenase was selected as
the best L-phenylalanine dehydrogenase and that can be most thermostable one and the
relationship between the best microorganism and the others was analyzed and from that
we can have the better idea about suitability of the microorganism and also the OD600
value also in logarithmic phase .so that will be helpful for the growth. OD600
measurements are commonly used to determine the growth stage of a bacterial culture;
these measurements help guarantee that cells are harvested at an optimal phase that
corresponds to an acceptable density of living cells.so the value was 0.665 and it can
be considered as good number.so from this study we could analyze the homology tree
and the differences and the relationship between the best phenylalanine
dehydrogenase.and tertiary structure of protein of the Geobacillus kaustophilus similar
to the Roddhococcus sp.according to the previous researches.so from the bioinformatics
analysis the similarities can be compared too. As the conclusion In this study, we
created and tested a new bifunctional PheDH with the microorganism with highest
cysteine content and the highest growing temperature. The bifunctional PheDH-FDH
demonstrated notable stability in mildly acidic and alkaline circumstances (pH 6.5-9.0)
as well as exceptional enantiomer selection by enzymatic conversion with a high
molecular conversion rate (99.87%) in catalyzing phenyl pyruvic acid to L-
phenylalanine [20]. The findings suggested that PheDH with the microorganism which
was obtained might be used in medical diagnosis and pharmaceutical area for phenyl
pyruvic acid analysis and L-phenylalanine biosynthesis. When comparing the growing
temperature of the microorganisms the genus of geobacillus’s temperatures were
highest averagely they have 55-65 Celsius degrees and from that we can get an idea
about the most suitable and the thermostable microorganism of the L-phenylalanine
dehydrogenase. In conclusion, this study demonstrated a novel strategy for gene mining
and characterization of novel L-phenylalanine dehydrogenase by constructing a
bifunctional PheDH-FDH fusion protein for bioconversion of L-phenylalanine with
coenzyme regeneration. This work provides a new insight into the design and
application of fusion enzymes for biocatalysis and biosynthesis.
The methods of manufacture and difficulties encountered in the synthesis of these
enzymes are covered in this to comprehend the nature of enzyme activity, a comparison
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 Undergraduate Thesis of Tianjin University of Science and Technology for Cohort 2019

of temperature. The paper concludes with a brief discussion of thermostable catalysts'

future. These organisms and the enzymes they produce are extremely heat stable, a
feature known as thermostability. The ability of a substance to withstand changes in its
physical/chemical structure at extremely high temperatures is referred to as
thermostability. Processes such as breakdown and polymerization can cause structural
changes, yet thermostable enzymes can withstand these effects. These enzymes have
the benefit of working well at very high temperatures, catalyzing reactions at high
temperatures in the commercial manufacturing of diverse goods. Their thermostability
is due to their structure, which enables exclusive access to active areas, improving the
enzyme's specific activity.

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 Undergraduate Thesis of Tianjin University of Science and Technology for Cohort 2019

ACKNOWLEDGEMENT

I would like to express my deep gratitude to Professor Cheng Xinkuan, my research

supervisors, for his patient guidance, enthusiastic encouragement and useful critiques
of this research work. I would also like to thank Ms. Li Yaru, for her advice and
assistance in keeping my progress on schedule. My grateful thanks are also extended to
my co laboratory partner Tian Yaru for her help in doing the experiments. Thanks all
my teachers who taught us past four years. Without you all I will not be able to complete
this successfully.

At last but not the least, I would like to thank my family: my parents Mrs. Chintha
And Mr.Thamitha, for giving birth to me at the first place and supporting me spiritually
throughout my life.and thank you my beloved Mr.Tharinda for being my side always.
And thanks all my family and friends, colleagues ,loved ones and relatives. I can’t
express my gratitude enough.

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 Undergraduate Thesis of Tianjin University of Science and Technology for Cohort 2019

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