The Secondary Structure Analysis and Protein Identification of

Esterase Were Performed by Circular Dichroism

Xinzhi Cao

, Liming Zhong

, Shuyuan Li

, Anqi Zhou

, Kaizheng Zhang

and Zeli Song

Department of Bioengineering, Sichuan University of Science and Engineering, Zigong, Sichuan 643000, China

Wuliangye Group Co. Ltd., Yibin 644007, China

Keywords:

Esterifying Enzyme, Protein Structure, Round Two Chromatographic.

Abstract:

In order to improve the yield of esterase and achieve the purpose of improving liquor quality rate and

aroma, the structure of esterase was studied by circular dichroism method and the protein was identified by

liquid mass spectrometry (LC-MS/MS). The results showed that the content of esterification enzyme Helix

(Helix) was 17.10%, anti-parallel β -folding structure was 29.70%, parallel β -folding structure was 5.40%,

β -rotation structure was 18.60%, irregular coil structure was 37.80%. This indicates that esterase is a kind

of protein with irregular curl. The β -folding structure of esterase accounted for 35.10%, indicating that the

secondary structure of esterase had a certain rigidity, but the β -rotation and random curl structure accounted

for 56.40%, indicating that the various residues in esterase peptide segment had a large degree of freedom,

good flexibility, but poor stability. Protein identification results showed that RHICH Lipase peptide had the

highest content, 906, coverage rate of 76.35%, total theoretical amino acids of 389 protein. The number of

PSEFL DNA-Binding Response regulator peptide was 14, the coverage rate was 32.93%, and the total

number of theoretical amino acids was 246. Then, the number of BURPL 50S ribosomal protein L5 peptide

was 10, the coverage was 18.99%, and the total number of theoretical protein amino acids was 179; Then

there was 9GAMM 30S ribosomal protein S19 peptide, the number was 7, the peptide coverage was 15.22%

and the total number of theoretical amino acids was 92; Finally, the peptide of 9GAMM Succinate

dehydrogenase iron-sulfur subunit was 7, the coverage rate was 16.03, and the total number of protein

theoretical amino acids was 237.

1 INTRODUCTION

Esterase (esterase.C.3.1.1.1), also known as

carboxyl esterase, is an enzyme that can hydrolyze

carboxyl ester bonds and catalyze the synthesis of

low grade fatty acid esters (Chen, 2017), which has

the ability to catalyze ester synthesis and

decomposition. Therefore, liquor industry is used to

call it esterase or ester decomposition enzyme.

Protein refers to the polymer formed by the

connection of 20 different amino acids (Yao, 2006;

Xu, 2011; S Sirén, 2020) The structure of protein

includes the chemical structure and spatial structure

of protein (Xu, 2020; Gao, 2010). The methods

(Zhou, 2021; Wei, 2021) to study the secondary

structure and advanced structure of protein include

X-ray crystal diffraction technology, nuclear

magnetic resonance technology and circular

dichroism technology (Xiao, 2020). But the first two

methods are limited by many factors, it is difficult to

analyze. The analytical scanning of circular

dichroism spectrometer plays an important role in

studying the secondary structure and advanced

structure of proteins, which is a special absorption

spectrum (Huang, 2019). The circular dichroism

spectrum of biological macromolecules such as

proteins detected by circular dichroism

chromatography is used to obtain the secondary

structure of biological macromolecules (Tong,

2018). Therefore, CIRCULAR dichroism is widely

used in protein folding and conformation research

(Liu, 2012).

This paper uses circular dichroism to analyze the

secondary structure of esterase and identify the

esterase protein to provide a theoretical basis for the

application of esterase in wine industry.

Cao, X., Zhong, L., Li, S., Zhou, A., Zhang, K. and Song, Z.

The Secondary Structure Analysis and Protein Identiﬁcation of Esterase Were Performed by Circular Dichroism.

DOI: 10.5220/0012002200003625

In Proceedings of the 1st International Conference on Food Science and Biotechnology (FSB 2022), pages 76-79

ISBN: 978-989-758-638-5

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

2 MATERIALS AND METHODS

2.1

Strains and Chemical Reagents

Staphylococcus aureus: staphylococcus aureus was

isolated and screened from Wuliangye Daqu, and the

strain producing esterification enzyme was

identified as STaphylococcus aureus according to the

morphological characteristics of the fungus colony

and molecular biology.

Acetonitrile, formic acid and ammonium

bicarbonate were all ms grade. Dithiothreitol and

iodoacetamide were analytically pure. Trypsin

sequencing grade.

2.2

Circular Dichroism Detection

The sample was dissolved in water at a

concentration of 0.2 ug /uL. The initial wavelength

was set at 180 nm, the end wavelength was set at

260 nm, the step size was 1 nm, the collection time

was 1 s/ point, and the cuvette width was 0.1 cm.

The blank control solution was sampled with 300 uL

and deducted after measurement. Then test the

sample with 300 uL, and save the data after test.

2.3

Protein Identification

2.3.1 LC - MS/MS Detection

Packed with Acclaim PepMap RPLC C18, 5 μm,

100A; 150 μm I.D. × 150 mm, Packed with Acclaim

PepMap RPLC C18, 1.9 μm, 100A; Mobile phase A:

0.1% formic acid; Mobile phase B: 0.1% formic

acid, 80% ACN; Flow rate: 600 nL/min; Analysis

time of each component: 60 min.

2.3.2 Mass Spectrometry Conditions

Primary mass spectrometry parameters: Resolution:

70,000; AGCtarget: 3 e6; MaximumIT: 100 ms;

Scanrange: 300 to 1400 m/z.

Secondary mass spectrometry parameters:

Resolution: 17,500; AGCtarget: 1 e5; MaximumIT:

50 ms; TopN: 20; An NCE/steppedNCE: 28.

2.3.3 Database Search

The mass spectrometry raw file retrieves the target

protein database using Byonic.

3 RESULTS AND DISCUSSION

3.1

CD Spectrum of Esterase in Far

ULTRAVIOLET Region

The UV CD spectrum of esterification enzyme was

shown in Fig.1. According to Fig.1, there was a

positive polarization peak at 195 nm and a small

shoulder at 187 nm, while the control group had a

wide negative shoulder at 210 nm and a wide

negative peak at 220 nm.

The abscissa represents the scanning wavelength and the

ordinate represents the ellipticity

Figure 1: Far-ultraviolet (180-260 nm) scanning of

esterification enzyme.

3.2

Secondary Structure of

Esterification Enzyme

The secondary structure of the sample was fitted

with CDNN software, including Helix, Antiparallel,

Parallel, beta-turn and RNDM.coil.

The analysis results were as follows:

esterification enzyme helix structure 17.10%,

antiparallel structure 29.70%, parallel β -folding

structure 5.40%, β -rotation structure 18.60%,

random coil structure 37.80%. This indicates that

esterase is a kind of protein with irregular curl.

3.3

Esterase Protein Identification

Raw files generated by esterase through LC-MS/MS

data collection were opened with Xcalibur, and total

ion flow chromatography fig. 2 could be seen as

follows:

Table 1: Calculation of secondary structure ratio in esterification enzyme.

The sam

le Helix Anti

arallel Parallel Beta-Turn Rndm. Coil

Esterifying enzyme 17.10% 29.70% 5.40% 18.60% 37.80%

The Secondary Structure Analysis and Protein Identiﬁcation of Esterase Were Performed by Circular Dichroism

Figure 2: Total ion flow chromatogram of esterification enzyme.

The results of protein identification were

retrieved by software Byonic database as shown in

the attached page:

From the attached pages: The results showed that

the top ten proteins with high scores were RHICH

Lipase, PSEFL DNA-Binding Response regulator,

BURPL 50S ribosomal protein L5 and 9GAMM 30S

Ribosomal protein S19, 9GAMM Succinate

dehydrogenase iron-sulfur subunit, 9GAMM

Succinate--CoA ligase [ADP-forming] Subunit beta,

ARHIZD Lipase, BURPL 50S Ribosomal protein

L14, 9GAMM 2-methylisocitrate lyase, 9GAMM

Sulfate Adenylyl transferase subunit 2. The number

of RHICH Lipase peptide was 906, the coverage rate

was 76.35%, and the total number of theoretical

amino acids was 389. The number of peptide in

PSEFL DNA-Binding Response regulator was 14,

the coverage rate was 32.93%, and the total number

of theoretical amino acids was 246. Then the

number of peptides in the BURPL 50S Ribosomal

protein L5 was 10, the coverage was 18.99%, and

the total number of theoretical amino acids in the

protein was 179; Then 9GAMM 30S ribosomal

protein S19 had a peptide number of 7 and a peptide

coverage of 15.22% with a theoretical total of 92

amino acids; Fifth, the number of peptide of

9GAMM Succinate dehydrogenase iron-sulfur

subunit was 7, the coverage rate was 16.03, and the

total number of protein theoretical amino acids was

237; The sixth score was 9GAMM Succinate-- the

maximum number of peptides in CoA ligase

[ADP-forming] subunit beta was 2, the coverage

rate was 7.22%, and the total number of theoretical

amino acids in protein was 388. In the seventh place,

ARHIZD Lipase had a maximum of 16 peptides, a

coverage rate of 9.77%, and a total of 389

theoretical amino acids; For the eighth BURPL 50S

ribosomal protein L14, the maximum number of

peptides was 9, the coverage rate was 22.95%, and

the total number of theoretical amino acids was 122;

The number of 9GAMM 2-methylisocitrate lyase is

3, the coverage rate is 5.19%, and the total number

of protein theoretical amino acids is 289; The

number of peptides in 9GAMM Sulfate adenylyl

transferase subunit 2 is 3 at most, the coverage rate

is 8.50%, and the total number of theoretical amino

acids in protein is 306.

4 CONCLUSIONS

Using circular dichroism to analyze esterase, the β

-fold structure of esterase accounted for 35.10%, β

-rotation and random curl structure accounted for

56.40%, indicating that esterase is a protein mainly

with random curl, and each residue in esterase

peptide segment has a large degree of freedom, good

flexibility, poor stability. Protein identification

results showed that RHICH Lipase had the highest

protein content, with 906 peptides, 76.35% coverage

rate and 389 theoretical amino acids. PSEFL

DNA-Binding Response Regulator had 14 peptides,

the coverage rate was 32.93%, and the total number

of theoretical amino acids was 246. Then, the

number of peptide fragments in the BURPL 50S

RT: 0.04 - 57.36

5 10 15 20 25 30 35 40 45 50 55

Time (min)

100

105

Relative Abundance

16.04

31.66

32.40

30.27

27.50

22.10

35.4327.84

23.70

38.60

39.08

33.18 36.62

21.54

24.98

44.12

16.84

19.29

54.56

43.81

40.46

44.65

48.21

47.56

54.79

48.85

45.43

50.74

56.64

8.06

15.52

12.8210.84

NL:

3.24E10

TIC MS

化󲺠󲺧

FSB 2022 - The International Conference on Food Science and Biotechnology

Ribosomal protein L5 was 10, the coverage was

18.99%, and the total number of theoretical amino

acids in the protein was 179. Then the number of

peptides in 9GAMM 30S ribosomal protein S19 was

7, the coverage rate was 15.22%, and the total

number of theoretical amino acids in protein was 92.

Finally, the peptide number of 9GAMM Succinate

dehydrogenase iron-sulfur subunit was 7, the

coverage rate was 16.03, and the total number of

protein theoretical amino acids was 237. Different

methods and molecular mechanisms are used to

change the properties of esterification enzyme.

Therefore, protein identification combined with

esterification enzyme can provide ideas for the

experimental design of improving the properties of

esterification enzyme, so as to promote the

high-quality development of wine industry.

AUTHOR INFORMATION

Xinzhi Cao (1965-), male, professor, ph. D., mainly

engaged in food biotechnology, E-mail addresses:

caoxinzhi@163.com

ACKNOWLEDGMENTS

Fund Project: Cooperation project of Wuliangye

Yibin Co., Ltd (CXY2019ZR012

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The Secondary Structure Analysis and Protein Identiﬁcation of Esterase Were Performed by Circular Dichroism