Nghiên cứu phản ứng thủy phân các glycozit tự nhiên bằng enzym b glucosidase và đánh giá hoạt tính sinh học của các sản phẩm nhận được tóm tắt LA tiếng anh

phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statisticconsensus is applied to the constructed tree so as to read ma

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GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-

LE THI TU ANH

STUDY OF HYDROLYSIS OF NATURAL GLYCOSIDES

BY β-GLUCOSIDASE ENZYME AND BIOACTIVITIES OF

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The thesis was completed in Graduate University Science and Technology, Vietnam Academy of Science and Technology

Supervisor 1: Assoc.Prof Dr Le Truong Giang

Institute of Chemistry, Vietnam Academy of Science and Technology Supervisor 2: Dr Doan Duy Tien

Institute of Chemistry, Vietnam Academy of Science and Technology

1st Reviewer:

2nd Reviewer:

3rd Reviewer:

The thesis will be defended at Graduate University of Science and

Technology - Vietnam Academy of Science and Technology,

at date month 2018

Thesis can be found in

- The library of the Graduate University of Science and Technology,

Vietnam Academy of Science and Technology

- The National Library of Vietnam

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1 Lê Thị Tú Anh, Đoàn Duy Tiên, Bá Thị Châm, Nguyễn Văn Tuyến, Nghiên

cứu phân lập chủng vi sinh vật thủy phân glycosit thành aglycon có hoạt tính sinh học cao Tạp chí Hóa học, 2016 , 54 (6e2): 84-89

2 Lê Thị Tú Anh, Bá Thị Châm, Nguyễn Thu Hà, Nguyễn Thanh Trà, Nguyên

Văn Tuyến, Nghiên cứu thủy phân astilbin trong rễ Thổ phục linh (Similax glabra) bằng vi sinh vật, Tạp chí Hóa học, 2016, 54 (6e2): 223-227

3 Nguyễn Thị Thu Hà, Phạm Thị Thu Hằng, Nguyễn Thanh Trà, Bá Thị Châm,

Lê Thị Tú Anh, Đặng Thị Tuyết Anh, Nguyễn Hà Thanh, Thành phần hóa

học và hoạt tính ức chế enzym khử HMG-Coenzym A của vỏ đậu xanh (Vigna radiata), Tạp chí hóa học 2017, 55 (4e23), 21-26

4 Nguyễn Thị Thu Hà, Nguyễn Thanh Trà, Bá Thị Châm, Lê Thị Tú Anh,

Đặng Thị Tuyết Anh, Nguyễn Hà Thanh, Thành phần hóa học và hoạt tính

ức chế enzym khử HMG-Coenzym A của lá Sen hồng (Nelumbo nucifera),

Tạp chí hóa học 2017, 55 (4e23), 261-266

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INTRODUCTION

1 The urgency of the thesis

Nowadays, environmental protection has become a necessity in every aspect of life In the field of chemistry, looking for catalytic enzymes, supporting the conversion process, organic synthesis is considered to be environmentally friendly green development Thanks to its superior advantages over other catalysts: they produce very little byproduct, operate at amazing speeds, are usually harmless and do not require expensive and rare elements to produce them… enzyme catalysis not only improves reaction efficiency but also contributes to reducing environmental pollution

β-glucosidases (BGL) are member of cellulase enzyme complex, they catalyze the hydrolysis of the β-glycosidic linkages in carbohydrate

structures Hydrolysis of glycoconjugates such as aminoglycosides, alkyl glucosides, and fragments of phytoalexin-elicitor oligosaccharides is an important application of β-glucosidases

Flavonoids, a group of natural substances with variable phenolic structures, are considered as an indispensable component in a variety of nutraceutical, pharmaceutical, medicinal and cosmetic applications The natural flavonoids almost all exist as their O-glycoside or C-glycoside forms in plants However, their aglycone usually has more activity in comparison with their glycoside forms Therefore, the development of bio-catalyzed hydrolysis of flavonoids glycoside and the study of the activity of these substances are very important to predict potential applications and manufacturing by industry

In the proceeding of research and development of enzyme, the amount

of microorganism must to be cultured Negative effects of these microorganisms on the environment are the reason of the necessary of a disinfection process before disposal

so to ensure an environmentally friendly process,

For research purposes: looking for potential biologically active glycosides, aglycones from plants and developing new research methods – bio-catalysis applied, we select thesis topic: "Study on hydrolysis of natural glycosides by β-Glucoside enzyme and bioactivities of their products" In

this study, P.citrinum were isolated from Clerodendron cyrtophyllum Turcz roots, identified and biosynthesized as β-glucosidase The extracted glycosides from Vietnamese plants are hydrolyzed by this β-glucosidase

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The flavonoids and their corresponding metabolites are evaluated for bioavailability The fungus after fermentation was studied sterilization by advanced oxidation process

2 The aim of the thesis

Study on applied of enzyme on hydrolysis of natural glycosides to produce new potential biologically active compound

Develop a new methods supporting the conversion process, organic synthesis is considered to be environmentally friendly green development

3 The main contents of the thesis:

- Identification of microorganisms capable of producing β-glucosidase

- Fermentation, evaluation of kinetic parameters of free and fixed

β-glucosidase from P citrinum

- Research on sterilization after fermentation by advanced oxidation

- Study on the extraction of flavonoids glycoside compounds from Vietnamese plants

- Study the hydrolysis of glycoside compounds from plants with glucosidase enzyme

β biological activity of glycoside and aglycone compounds

1.2 Flavonoid compounds

Presentation of flavonoid-related content: baseline, group classification, biosynthesis, reagent identification and bioactivity of the substance group

1.3 Flavonoid glycosides and their aglycon

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Presentation of the content related to the uptake, metabolism of flavonoid glycose from which the potential of the aglycon compared with their glycoside This is followed by an overview of the globally published flavonoid glycozite metabolites

1.4 Biosafety in research

Strict adherence to biosafety procedures is absolutely essential for researchers working with pathogens because the exact transmission pathways of these pathogens are unclear, and specific preventives and therapeutics are generally unavailable It would only take a single mistake

in handling infectious materials to cause a full-on disaster One painful example of this occurred at Beijing's Institute of Virology where a lab researcher was infected by severe acute respiratory syndrome-coronavirus

in a sample that was improperly handled, resulting in the death of the researcher's mother and the infection of several others.Thus, researchers should be particularly careful in handling laboratory-generated organism

CHAPTER 2: EXPERIMENTAL AND RESULTS

2.1 Materials

Residue seeds of Glycine max from Quang Minh vegetable oil joint

stock company, Kim Dong, Hung Yen

Dry leafs of Nelumbo nucifera and seed coat of Vigna radiate from

Hanoi, Bac Giang

Flower of Styphnolobium japonicum (L.) Schott from Nam Dinh

spectroscopic methods such as electrospray ionization mass spectrometry

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(ESI-MS) and high-resolution ESI-MS (HR-ESI-MS),

one/two-dimension nuclear magnetic resonance (NMR) spectra

2.3.4 Method for hydrolysis of glycosides by β-glucosidase: free

enzyme and immobilized enzyme

2.3.5 Sterilization of microorganisms

2.3.6 Biological assays

- DPPH method of antioxidant assay

- Inhibitor enzyme activity of α-glucosidase

- Inhibitor enzyme activity of Angiotensin I

CHAPTER 3: RESEARCH METHODOLOGY 3.1 Isolation and identification of a fungal β-glucosidase

3.1.1 Isolation of a fungal β-glucosidase

The most active β-glucosidase fungus will be used in the next study

3.1.2 Identification of a fungal β-glucosidase

Phenotypic and rDNA internal transcribed spacer sequence analyses

indicated that the isolate belongs to Penicillium citrinum

3.2 Purification and Characterization of a β-Glucosidase

Fermentation condition (pH,carbon source) was optimized for producing the enzyme in shake flask cultures

Kinetic parameters for hydrolysis β-pNG, ability to catalyzes the transglucosidation reaction, dependence of the enzymatic activity on pH

Study on the immobilized BGL-P, performance of immobilized enzyme

is calculated by equation:

Performance of immobilized enzyme (%) = (Et- Es)/Et x100

Et is the enzymatic activity before the immobilization

Es is the enzymatic activity after the immobilization

3.3 Isolation and purification of glycosides from Vietnamese plants

3.3.1 Isolation and purification of glycosides from residue seeds of

Glycine max

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3.3.2 Isolation and purification of glycosides from leave of Nelumbo

nucifera

3.3.3 Isolation and purification of glycosides from coat of green bean

seeds Vigna radiate

EtOH extract

extracted by acetone 3 times

solvent removal by vacuum evaporation

Acetone extract

- Dissolve by EtOAc Extracted by H 2 O

silica gel: EtOAc: H

2 O (97:3) and EtOAc:H

2 O:EtOH (95:3:2)

Sephadex LH-20, EtOH silica gel: EtOAc: MeOH

(96:4) silica gel: EtOAc: MeOH (95:5)

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Isolation and purification

purification Styphnolobium japonicum (L.) Schott

Characteristic of the compound

5,00 (protons CH-OH ); 5,2 (1H, brs, H1’’); 6,19 (1H, d,

= 8,0 Hz,

H-=2,0; 8,0 Hz, H-6’); 12,58 (1H, s, OH

NMR (125 MHz, DMSO

5); 100,1 (C-6); 164,1 (C10); 121,1 (C-1’); 115,2 (C

6’); 101,2 (C4’’); 75,8 (CGlc-5’’); 66,9 (C

3’’’); 71,8 (C

purification

of glycosides from Schott

Characteristic of the compound: melting point

6): =0,99 ppm (3H, d,

OH ); 5,2 (1H, brs, H

1’’); 6,19 (1H, d, J= 2,0 Hz, H

-5’); 7,52 (1H, 6’); 12,58 (1H, s, OH

6); 164,1 (C1’); 115,2 (C-2’); 144,7 (C6’); 101,2 (CGlc-1’’); 74,1 (C

5’’); 66,9 (C3’’’); 71,8 (CRha-4’’’); 68,2 (C

purification of glycosides from

6’); 12,58 (1H, s, OH-5)

 156,5 (C6); 164,1 (C-7); 93,6 (C

2’); 144,7 (C1’’); 74,1 (C5’’); 66,9 (CGlc-6’’); 98,7 (C

6’’); 98,7 (C4’’’); 68,2 (CRha-5’’’); 18,6 (C

glycosides from Rhizoma

5’’’); 18,6 (C

Rhizoma P

= 6,5Hz, HRha1’’’); 5,34 (1H, d,

-= 2,0 Hz, 2’); 7,55

3); 177,4 8); 156,4 (C-9); 3’); 148,4 (C-4’); 2’’); 76,4 (CGlc-

1’’’); 70,5 5’’’); 18,6 (CRha-

Polygoni

1’’’); 5,34 (1H, d,

= 2,0 Hz, 2’); 7,55

3); 177,4

9); 4’); 1’’’); 70,5

olygoni

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Qc: the amount of hydrolyzed product

Qo: the amount of glycoside initially put into the reaction

M1: molecular weight of glycoside

M2: molecular weight of hydrolysis product

3.5 Disinfection of study microorganisms using Advanced oxidation

processes

3.5.1 Prepaire of Advanced oxidation processes: electro-disinfection 3.5.2 Studies on the Electrochemical Disinfection of B cereus as an indicator

3.5.2.1 Studies on the effect of electric current on the disinfection

3.5.2.2 Studies on the effect of pH of electrolysis water on the disinfection

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3.5.3 Applied the Electrochemical Disinfection on P citrinum

3.6 Bioactivity of glycosides and the products of hydrolysis

3.6.1 Antioxidant activity by DPPH assay [117-119]

Compound was determined by modified methods of Pathirama et al (2005) and Thirugnanasampandan et al (2008) Two milliliter of different concentrations (0.5 to 128 µg/ml) of each compound

Liyana-in methanol was added to 0.2 ml of DPPH radical solution Liyana-in methanol (final concentration of DPPH was 1.0 mM) The mixture was shaken vigorously and allowed standing for 60 min in the dark The absorbance

of the resulting solutions, the blank and the control were measured at 517

nm using Bioteck spectrophotometer Standard antioxidant compound resveratrol was used as positive control DPPH scavenging activity of the compound was calculated using the following formula:

DPPH scavenging activity (%) = ODblankOD-blankOD samplex100

Where OD sample and OD blank were the optical density of the extract

at different concentrations and the blank sample

The effective concentration providing 50% inhibition (EC50) was calculated from the graph of percentage inhibition against each extract concentrations

3.6.2 α-Glucosidase inhibition assay:

The enzyme solution contained 20 μl α-glucosidase (0.5 unit/ml) and 120 μl 0.1 M phosphate buffer (pH 6.9) p-Nitrophenylα-D-glucopyranoside (5 mM) in the same buffer (pH 6.9) was used as a substrate solution

10 μl of test samples, dissolved in DMSO at various concentrations, were mixed with enzyme solution in microplate wells and incubated for 5 min at 37°C 10 μl of substrate solution were added and incubated for an additional 30 min The reaction was terminated by adding 100 μl of 0.2

M sodium carbonate solution Absorbance of the wells was measured with a Bioteck spectrophotometer at 405 nm, while the reaction system without compound was used as control The system without α-glucosidase was used as blank, and acarbose was used as positive control

3.6.3 An angiotensin converting enzyme inhibitor [124-126]:

measured with a Bioteck spectrophotometer at 410 nm (A)

Percentage inhibitor of ACE was calculated using the following formula:

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different concentrations and the blank sample

Captopril was used as positive

The aim of the research is to study the hydrolysis of glycoside compounds from plants Therefore, we firstly isolated the fungalglucosidase

4.1 Isolation and properties of fungal beta

4.1.1

Fig 4.1

We isolated 5 fungi (C1, C2, C3, C4, C5) from

Clerodendron cyrtophyllum

beta-glucosidase enzyme The fungal isolate

when tested with β

showed the presence of β

after six days culture The fulgal isolate C5

experiment

4.1.2 Identification of

Colonies of C5 are fast growing in shades of green

consisting of a dense felt of conidiophores Microscopically, phialides like a brush

DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have commonly been used to identify

flanking ITS1/

% inhibitor of ACE Where, A

CHAPTER

The aim of the research is to study the hydrolysis of glycoside compounds from plants Therefore, we firstly isolated the fungalglucosidase

Isolation and properties of fungal beta

4.1.1 Isolation of fungal beta

Fig 4.1: Colonies of fungal were isolated from root of

glucosidase enzyme The fungal isolate

when tested with β

showed the presence of β

experiment

Identification of

consisting of a dense felt of conidiophores Microscopically, phialides like a brush-like appearance (a penicillus)

DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have ommonly been used to identify

flanking ITS1/

% inhibitor of ACE

A sample and A different concentrations and the blank sample

CHAPTER

The aim of the research is to study the hydrolysis of glycoside compounds from plants Therefore, we firstly isolated the fungal

Isolation of fungal beta

Colonies of fungal were isolated from root of

when tested with β-pNG method Analysis of the culture filtrate of C5 showed the presence of β

Identification of fungal beta

consisting of a dense felt of conidiophores Microscopically, phialides

like appearance (a penicillus)

flanking ITS1/ITS4 re

% inhibitor of ACE

and A blank

CHAPTER 4 RESULTS AND DISCUSSION

Isolation of fungal beta

cyrtophyllum

Clerodendron cyrtophyllum Turcz

pNG method Analysis of the culture filtrate of C5 showed the presence of β-glucosidase

fungal beta

4 regions for fungal identification

= (Acontrol

blank were the optical density of the extract at different concentrations and the blank sample

Captopril was used as positive control

RESULTS AND DISCUSSION

Isolation of fungal beta-glucosidases:

cyrtophyllum

Turcz and screened them for prodution glucosidase enzyme The fungal isolate

pNG method Analysis of the culture filtrate of C5

glucosidase with theafter six days culture The fulgal isolate C5

fungal beta-glucosidases:

DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have ommonly been used to identify the fungal

gions for fungal identification

were the optical density of the extract at different concentrations and the blank sample

control

Isolation and properties of fungal beta-glucosidases

glucosidases:

cyrtophyllum Turcz

and screened them for prodution glucosidase enzyme The fungal isolate C5 gave maximum enzyme

pNG method Analysis of the culture filtrate of C5

with the activity was after six days culture The fulgal isolate C5

glucosidases:

DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have

the fungal We used 5.8S gene and gions for fungal identification

were the optical density of the extract at

glucosidases

glucosidases:

Colonies of fungal were isolated from root of Clerodendron

and screened them for prodution

C5 gave maximum enzyme pNG method Analysis of the culture filtrate of C5

activity was after six days culture The fulgal isolate C5 was identified

glucosidases:

We used 5.8S gene and gions for fungal identification

glucosidases

Clerodendron

We isolated 5 fungi (C1, C2, C3, C4, C5) from roots of

and screened them for prodution

activity was 33,628U/ml was identified

We used 5.8S gene and gions for fungal identification Constructing

The aim of the research is to study the hydrolysis of glycoside

compounds from plants Therefore, we firstly isolated the fungal

β-Clerodendron

roots of and screened them for prodution of

33,628U/ml

in next

mostly consisting of a dense felt of conidiophores Microscopically, phialides

We used 5.8S gene and

Constructing

The aim of the research is to study the hydrolysis of glycoside

roots of

of C5 gave maximum enzyme pNG method Analysis of the culture filtrate of C5

33,628U/ml

in next

mostly consisting of a dense felt of conidiophores Microscopically, phialides

We used 5.8S gene and

Constructing

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phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statistic

consensus is applied to the constructed tree so as to read maximum sequence replications

a clear picture for identifying fungal isolate

100 BLAST hits belon

recommending our isolate as a member of this group

4.2 Purification and properties of

Partial purification of β

precipitation, followed by sephadex

from Penicillium citrinum

determined using 4

substrate

4.2.1 Properties of BGL

Optimum pH and temperature for enzyme assay

β-glucosidase activity was observed at 40, 50, 60, 70 and 80°C The results showed that the BGL activity increased from

which decrease in

activity was 60

activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also prevent contamination to allow the reaction to proceed at higher range of temperature

As far as pH is concerned, the plot obtained by the expected bell curve and maximum activity was observed in the pH range of 5.0 to 6.5 and the BGL-P was optimized at pH

Kinetic parameters for

consensus is applied to the constructed tree so as to read maximum sequence replications

Purification and properties of

glucosidase activity was observed at 40, 50, 60, 70 and 80°C The results showed that the BGL activity increased from

which decrease in

activity was 60

activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of temperature

As far as pH is concerned, the plot obtained by the expected bell curve and maximum activity was observed in the pH range of 5.0 to 6.5 and the

P was optimized at pH

consensus is applied to the constructed tree so as to read maximum sequence replications Neighbour joining tree with bootstrapping gave us

determined using 4-Nitrophenyl β

-P was used at free enzyme an

Properties of BGL

which decrease in activity was observed

activity was 60oC Temperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of

P was optimized at pH

consensus is applied to the constructed tree so as to read maximum

Neighbour joining tree with bootstrapping gave us

100 BLAST hits belonged to

Fig 4.4: Colonies

Partial purification of β-BGL was carried out by

precipitation, followed by sephadex

Penicillium citrinum partially purified enzyme (BGL

Nitrophenyl β

P was used at free enzyme an

Properties of BGL-P:

activity was observed

Temperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of

P was optimized at pH 6.0

Kinetic parameters for BGL

ged to Penicillium citrinum

Colonies, phialides

β-BGL was carried out by precipitation, followed by sephadex, lyophilized

partially purified enzyme (BGLNitrophenyl β-D

P was used at free enzyme an

activity was observed

Temperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of

6.0

BGL-P

a clear picture for identifying fungal isolate C5

P was used at free enzyme and immobilized enzyme

The best temperature for BGLTemperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of

phylogenetic tree is crucial in molecular identification, since BLAST search alone cannot overcome possibilities of statistical errors Bootstrap consensus is applied to the constructed tree so as to read maximum

It is because more than

Penicillium citrinum, thus strongly

phialides of C5

glucosidase from culture

BGL was carried out by ammonium sulphate

, lyophilized Activity of the BGL partially purified enzyme (BGL

glucopyranoside (5 mM) as

d immobilized enzyme

glucosidase activity was observed at 40, 50, 60, 70 and 80°C The results showed that the BGL activity increased from 5

The best temperature for BGLTemperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of

phylogenetic tree is crucial in molecular identification, since BLAST

al errors Bootstrap consensus is applied to the constructed tree so as to read maximum

, thus strongly

from culture

ammonium sulphate Activity of the BGL partially purified enzyme (BGL-

glucopyranoside (5 mM) as

glucosidase activity was observed at 40, 50, 60, 70 and 80°C The

50 to 70°C after The best temperature for BGLTemperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of

, thus strongly

from culture

ammonium sulphate Activity of the BGL

-P) was glucopyranoside (5 mM) as

0°C after The best temperature for BGL-P Temperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of

, thus strongly

ammonium sulphate Activity of the BGL

P) was glucopyranoside (5 mM) as

0°C after

P Temperature is an important factor for enzymatic activity Activity of enzyme at higher temperature range is an advantageous factor for the saccharification of biomass and can also event contamination to allow the reaction to proceed at higher range of

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