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MINISTRY OF EDUCATION AND TRAINING
VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
----------------------------
LE THI TU ANH
STUDY OF HYDROLYSIS OF NATURAL GLYCOSIDES BY β-GLUCOSIDASE ENZYME AND BIOACTIVITIES OF
THEIR PRODUCTS
Major: Organic chemistry Code: 62.44.01.14
SUMMARY OF CHEMISTRY DOCTORAL THESIS
Hanoi – 2018
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.
PUBLICATIONS WITHIN THE SCOPE OF THESIS
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.
1
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.
2 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 β-glucosidase enzyme. - biological activity of glycoside and aglycone compounds.
CHAPTER 1: OVERVIEW
Overview of national and international researches related to my study.
1.1 β-D-glucosidase enzyme Presentation of contents related to β-glucosidase: basic contents
related to the definition, classification, reaction mechanism, purification and evaluation of enzyme activity. Next, the content of diversity and the ability of biosynthesis of β-glucosidase in microorganisms, on the improvement of seed sources for the purpose of increasing BGL production and related to commercial BGL production. Finally, on the multidisciplinary application of β-glucosidase.
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
3
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.
The rhizomes of Rhizoma Polygoni cuspidati from Nghia Trai, Hung Yen.
2.2 Chemical and equipments: 2.3. Methods
2.3.1. Methods for isolation, identification of microorganism 2.3.1.1 Method of isolation 2.3.1.2 Method of identification: phenotypic identification,
genotypic identification. 2.3.2 Enzymatic activities and kinetic properties of β-glucosidase:
p-nitrophenyl-β-glucopyranosid (pNPG) method. 2.3.3 Methods for isolation and structural elucidation glycosides:
Chromatographic methods such as thin layer chromatography (TLC), column chromatography (CC). Physical parameters and modern spectroscopic methods such as electrospray ionization mass spectrometry
4 (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
We isolated fungus from roots of Clerodendron cyrtophyllum Turcz . 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 and temperature were investigated.
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
5
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 H2O
EtOAc extract H2O extract
silica gel: EtOAc: H2O (97:3)
and EtOAc:H2O:EtOH (95:3:2)
F1-F2 F3-F4 F7-F10 F5 F6
Sephadex LH-20, EtOH silica gel: EtOAc: MeOH (96:4)
silica gel: EtOAc: MeOH (95:5)
D5.3 (251.2mg)
D6.4 (198.7mg) F1.
1 F1.2
Crystallized CH2Cl
2
D1.1 (12.8mg)
D1.2 (3.4 mg)
Kết tinh CH2Cl
2
3.3.4 Isolation andStyphnolobium japonicum
Characteristic of the compound1H NMR (500 MHz, DMSO6’’’); 3,09J= 7,0 Hz, HH-8); 6,84 (1H, d, (1H, dd,
13C(C-4); 161,2 (C103,9 (C116,2 (C3’’); 70,(CRha-2’’’); 71,3(C6’’’). 3.3.5 Isolation cuspidati
Isolation andStyphnolobium japonicum
Characteristic of the compoundH NMR (500 MHz, DMSO
6’’’); 3,09- 5,00 (proton= 7,0 Hz, HGlc-
8); 6,84 (1H, d, (1H, dd, J=2,0; 8,0 Hz, H
C-NMR (125 MHz, DMSO4); 161,2 (C
103,9 (C-10); 121,1 (C116,2 (C-5’); 121,5(C3’’); 70,3 (CGlc-
2’’’); 71,3(C
Isolation cuspidati
Isolation and purificationStyphnolobium japonicum
Characteristic of the compoundH NMR (500 MHz, DMSO
5,00 (proton-1’’); 6,19 (1H, d,
8); 6,84 (1H, d, J= 8,0 Hz, H=2,0; 8,0 Hz, HNMR (125 MHz, DMSO
4); 161,2 (C-5); 100,1 (C10); 121,1 (C5’); 121,5(C-6’); 101,2 (C
-4’’); 75,8 (C2’’’); 71,3(CRha-3’’’); 71,8 (C
Isolation and purification
purification Styphnolobium japonicum (L.) Schott
Characteristic of the compoundH NMR (500 MHz, DMSO-d6
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, OHNMR (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
6
of glycosides fromSchott
Characteristic of the compound: melting point6): =0,99 ppm (3H, d,
OH ); 5,2 (1H, brs, H1’’); 6,19 (1H, d, J= 2,0 Hz, H
-5’); 7,52 (1H, 6’); 12,58 (1H, s, OH
NMR (125 MHz, DMSO-d6): 6); 164,1 (C
1’); 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
glycosides from
melting point=0,99 ppm (3H, d,
OH ); 5,2 (1H, brs, H= 2,0 Hz, H-6); 6,38 (1H, d,
5’); 7,52 (1H, d, 6’); 12,58 (1H, s, OH-5).
156,5 (C6); 164,1 (C-7); 93,6 (C
2’); 144,7 (C1’’); 74,1 (C
5’’); 66,9 (CGlc-6’’); 98,7 (C4’’’); 68,2 (C
glycosides from
glycosides from flower of
melting point: 242oC=0,99 ppm (3H, d, J
OH ); 5,2 (1H, brs, HRha-1’’’); 5,34 (1H, d, 6); 6,38 (1H, d, d, J = 2,0 Hz, 5).
156,5 (C-2); 133,3 (C7); 93,6 (C-8); 156,4 (C
2’); 144,7 (C-3’); 148,4 (C1’’); 74,1 (CGlc-2’’); 76,4 (C
6’’); 98,7 (C4’’’); 68,2 (CRha-5’’’); 18,6 (C
glycosides from Rhizoma
flower of
C J= 6,5Hz, H
1’’’); 5,34 (1H, d, 6); 6,38 (1H, d, J= 2,0 Hz,
= 2,0 Hz, H-2’); 7,55
2); 133,3 (C-3); 177,4 8); 156,4 (C
3’); 148,4 (C2’’); 76,4 (C
6’’); 98,7 (CRha-1’’’); 70,5 5’’’); 18,6 (C
Rhizoma P
= 6,5Hz, HRha-1’’’); 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
7
`
C8.4 (20mg) C8.5 (15mg)
CC extract (15 g)
F1 F2 F7 (2,0g) F6 F5 (2,4g) F4 F3
Silicagel 0,063 ÷ 0,2
- CH2Cl2 : CH3OH
Crystallized
C2.1
(290mg)
- Silicagel CH2Cl2
/CH3OH
7-2 7-3 7-4 7-5 7-1
Crystallized
C7.3 (155mg)
- Silicagel CH2Cl2
: CH3OH
5-1 5-2 5-3
C5.2 (97mg)
5-4
F8
Crystallized
3.4. Hydrolysis glycoside compounds:
Percentage of hydrolysis [140]: Percentage of hydrolysis (%) =
12 100
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
8 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 Liyana-Pathirama et al. (2005) and Thirugnanasampandan et al. (2008). Two milliliter of different concentrations (0.5 to 128 µg/ml) of each compound in methanol was added to 0.2 ml of DPPH radical solution 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 (%) = OD blank-OD sampleODblank
x100 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]: Reaction at 37o C, pH 7,0, in 30 min. Absorbance of the wells was
measured with a Bioteck spectrophotometer at 410 nm (A). Percentage inhibitor of ACE was calculated using the following
formula:
Wheredifferent 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 isolatewhen tested with βshowed the presence of βafter six days culture. The fulgal isolate C5 experiment.4.1.2. Identification of
Colonies of C5 are fast growing in shades of greenconsisting 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 identifyflanking ITS1/
% inhibitor of ACE Where, A
different concentrations and the blank sample. Captopril was used as positive
CHAPTERThe 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 beta4.1.1 Isolation of fungal beta
Fig 4.1: Colonies of fungal were isolated from root of
We isolated 5 fungi (C1, C2, C3, C4, C5) from Clerodendron cyrtophyllum
glucosidase enzyme. The fungal isolatewhen tested with βshowed the presence of βafter six days culture. The fulgal isolate C5 experiment.
Identification ofColonies of C5 are fast growing in shades of green
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. Captopril was used as positive
CHAPTERThe aim of the research is to study the hydrolysis of glycoside
compounds from plants. Therefore, we firstly isolated the fungal
Isolation and properties of fungal betaIsolation of fungal beta
Colonies of fungal were isolated from root of
We isolated 5 fungi (C1, C2, C3, C4, C5) from Clerodendron cyrtophyllum
glucosidase enzyme. The fungal isolatewhen tested with β-pNG method. Analysis of the culture filtrate of C5 showed the presence of βafter six days culture. The fulgal isolate C5
Identification of fungal betaColonies of C5 are fast growing in shades of green
consisting of a dense felt of conidiophores. Microscopically, phialides 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/ITS4 re
% inhibitor of ACE and A blank
different concentrations and the blank sample. Captopril was used as positive
CHAPTER 4. RESULTS AND DISCUSSIONThe aim of the research is to study the hydrolysis of glycoside
compounds from plants. Therefore, we firstly isolated the fungal
Isolation and properties of fungal betaIsolation of fungal beta
Colonies of fungal were isolated from root of cyrtophyllum
We isolated 5 fungi (C1, C2, C3, C4, C5) from Clerodendron cyrtophyllum Turcz
glucosidase enzyme. The fungal isolatepNG method. Analysis of the culture filtrate of C5
showed the presence of β-glucosidaseafter six days culture. The fulgal isolate C5
fungal betaColonies of C5 are fast growing in shades of green
consisting of a dense felt of conidiophores. Microscopically, phialides 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
4 regions for fungal identification
9
= (Acontrol blank were the optical density of the extract at
different concentrations and the blank sample. Captopril was used as positive control
RESULTS AND DISCUSSIONThe aim of the research is to study the hydrolysis of glycoside
compounds from plants. Therefore, we firstly isolated the fungal
Isolation and properties of fungal betaIsolation of fungal beta-glucosidases:
Colonies of fungal were isolated from root of cyrtophyllum
We isolated 5 fungi (C1, C2, C3, C4, C5) from Turcz and screened them for prodution
glucosidase enzyme. The fungal isolatepNG method. Analysis of the culture filtrate of C5
glucosidase with theafter six days culture. The fulgal isolate C5
fungal beta-glucosidases:Colonies of C5 are fast growing in shades of green
consisting of a dense felt of conidiophores. Microscopically, phialides 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 the fungal.
gions for fungal identification
control – Asamplewere the optical density of the extract at
different concentrations and the blank sample. control
RESULTS AND DISCUSSIONThe aim of the research is to study the hydrolysis of glycoside
compounds from plants. Therefore, we firstly isolated the fungal
Isolation and properties of fungal beta-glucosidasesglucosidases:
Colonies of fungal were isolated from root of cyrtophyllum Turcz
We isolated 5 fungi (C1, C2, C3, C4, C5) from 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:Colonies of C5 are fast growing in shades of green
consisting of a dense felt of conidiophores. Microscopically, phialides like appearance (a penicillus).
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
sample)/(Acontrolwere the optical density of the extract at
RESULTS AND DISCUSSIONThe aim of the research is to study the hydrolysis of glycoside
compounds from plants. Therefore, we firstly isolated the fungal
glucosidasesglucosidases:
Colonies of fungal were isolated from root of Clerodendron
We isolated 5 fungi (C1, C2, C3, C4, C5) from 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: Colonies of C5 are fast growing in shades of green
consisting of a dense felt of conidiophores. Microscopically, phialides
DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have
We used 5.8S gene and gions for fungal identification
control– Ablankwere the optical density of the extract at
RESULTS AND DISCUSSION The aim of the research is to study the hydrolysis of glycoside
compounds from plants. Therefore, we firstly isolated the fungal
glucosidases
Clerodendron
We isolated 5 fungi (C1, C2, C3, C4, C5) from roots of and screened them for prodution
C5 gave maximum enzyme pNG method. Analysis of the culture filtrate of C5
activity was 33,628U/ml was identified
Colonies of C5 are fast growing in shades of green consisting of a dense felt of conidiophores. Microscopically, phialides
DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have
We used 5.8S gene and gions for fungal identification. Constructing
blank) were the optical density of the extract at
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
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
DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have
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
DNA sequence analysis methods are objective, reproducible and rapid means of identification, and thus gaining importance and have
We used 5.8S gene and . Constructing
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 maximum sequence replicationsa clear picture for identifying fungal isolate 100 BLAST hits belonrecommending our isolate as a member of this group.
4.2 Purification and properties ofPartial purification of β
precipitation, followed by sephadexfrom Penicillium citrinumdetermined using 4substrate.4.2.1 Properties of BGL
Optimum pH and temperature for enzyme assayβ-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 60activity. 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
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 maximum sequence replicationsa clear picture for identifying fungal isolate 100 BLAST hits belonrecommending our isolate as a member of this group.
Purification and properties ofPartial purification of β
precipitation, followed by sephadexPenicillium citrinum
determined using 4substrate. BGL-
Properties of BGLOptimum pH and temperature for enzyme assay
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 60activity. 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 Kinetic parameters for
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 maximum sequence replications. Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate 100 BLAST hits belonrecommending our isolate as a member of this group.
Fig.Purification and properties of
Partial purification of βprecipitation, followed by sephadex
Penicillium citrinumdetermined using 4-Nitrophenyl β
-P was used at free enzyme anProperties of BGL
Optimum pH and temperature for enzyme assayglucosidase 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 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
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 Kinetic parameters for
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 maximum
Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate 100 BLAST hits belonged to recommending our isolate as a member of this group.
Fig. 4.4: ColoniesPurification and properties of
Partial purification of β-BGL was carried out by precipitation, followed by sephadex
Penicillium citrinum partially purified enzyme (BGLNitrophenyl β
P was used at free enzyme anProperties of BGL-P:
Optimum pH and temperature for enzyme assayglucosidase activity was observed at 40, 50, 60, 70 and 80°C. The
results showed that the BGL activity increased from 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
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 6.0.Kinetic parameters for BGL
10
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 maximum
Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate
ged to Penicillium citrinumrecommending our isolate as a member of this group.
Colonies, phialidesPurification and properties of β-
BGL was carried out by precipitation, followed by sephadex, lyophilized.
partially purified enzyme (BGLNitrophenyl β-D
P was used at free enzyme an
Optimum pH and temperature for enzyme assayglucosidase activity was observed at 40, 50, 60, 70 and 80°C. The
results showed that the BGL activity increased from 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
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
6.0. BGL-P
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 maximum
Neighbour joining tree with bootstrapping gave us a clear picture for identifying fungal isolate C5.
Penicillium citrinumrecommending our isolate as a member of this group.
phialides-glucosidase
BGL was carried out by , lyophilized.
partially purified enzyme (BGLD-glucopyranoside (5 mM) as
P was used at free enzyme and immobilized enzyme.
Optimum pH and temperature for enzyme assayglucosidase activity was observed at 40, 50, 60, 70 and 80°C. The
results showed that the BGL activity increased from The best temperature for BGL
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
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
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
Neighbour joining tree with bootstrapping gave us It is because more than
Penicillium citrinum, thus strongly recommending our isolate as a member of this group.
phialides of C5 glucosidase from culture
BGL was carried out by ammonium sulphate , lyophilized. Activity of the BGL
partially purified enzyme (BGLglucopyranoside (5 mM) as
d immobilized enzyme.
Optimum pH and temperature for enzyme assay glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The
results showed that the BGL activity increased from 5The best temperature for BGL
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
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
phylogenetic tree is crucial in molecular identification, since BLAST al errors. Bootstrap
consensus is applied to the constructed tree so as to read maximum Neighbour joining tree with bootstrapping gave us
It is because more than , thus strongly
from culture ammonium sulphate 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 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
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
phylogenetic tree is crucial in molecular identification, since BLAST al errors. Bootstrap
consensus is applied to the constructed tree so as to read maximum Neighbour joining tree with bootstrapping gave us
It is because more than , thus strongly
from culture ammonium sulphate Activity of the BGL
-P) was glucopyranoside (5 mM) as
d immobilized enzyme.
glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The 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
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
phylogenetic tree is crucial in molecular identification, since BLAST al errors. Bootstrap
consensus is applied to the constructed tree so as to read maximum Neighbour joining tree with bootstrapping gave us
It is because more than , thus strongly
ammonium sulphate Activity of the BGL
P) was glucopyranoside (5 mM) as
glucosidase activity was observed at 40, 50, 60, 70 and 80°C. The 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
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
11
Different concentrations of pNPG (0-25 mM) were used to estimate the kinetic parameters, Km and Vmax using double reciprocal Lineweaver-Burk plot. The results were Km = 0,01µmol và Vmax = 13,91 µmol/min. 4.2.2 Properties of BGL-P immobilized: Immobilization of BGL-P in calcium alginate:
Sodium alginate of 4% concentration and 4% CaCl2 solution were found to be best with respect to immobilization efficiency and calcium alginate beads so obtained were not much susceptible to breakage. BGL-P entrapped in large calcium alginate beads was used successfully for 7 cycles for the conversion of pNPG into product without much damage to the beads under stirring conditions. Immobilization of BGL-P onto spent coffee grounds:
Spent coffee grounds, discarded as environmental pollutants, were adopted as enzyme immobilisation solid carriers instead of commercialised solid supports to establish an economical catalytic system. β-Glucosidase was covalently immobilised onto spent coffee grounds. Conditions were determined to be 40 °C and pH 6 using 4-nitrophenyl β-D-glucuronide as an indicator. Operational reusability was confirmed for 2 batch reactions.
Table 4.3 Kinetic parameters for free BGL-P and immobilized Forms Temperature
(oC) pH Vmax
(µmol/min) Km
(µmol) R2 *
Free forms 60 6.0 13,91 0,011 0,9994 Immobilized in alginat
50 6.0 13,09 0,034 0,9978
Immobilized onto spent coffee grounds
40 6.0 14,45 0,022 0,9992
* is R2 of Lineaweaver and Burk plot 4.2 Chemical structure of isolated compounds
This section presents the detailed results of spectral analysis and structure determination of 20 isolated compounds from 5 plant species: No Symbol Structure Name of compound 1 D1.1
Genistein
12
2 D1.2
Daidzein
3 D5.3 Genistein 7-O-beta-D-glucoside
4 D6.4
Daidzein7-O-beta-D-glucoside
5 S3.1 MB5
catechin
6 S5.2
quercetin-3-O-β-galactoside
7 S7.3
quercetin
8 S8.4
kaemferol
9 S8.5
isorhamnetin-3-O-β-D-glucuronide
13 10 S8.6
quercetin-3-O-β-D-glucuronide
11 MB3
O
OH
HO
OH O
OHO
OH
HOOH
2
3
45
106
7 89
1'
2'
3'
4'
5'
6'
1''
2''
3''
4''
5''
6''
apigenin-6-C-glucoside (vitexin)
12 MB4
apigenin-6-C-glucoside (isovitexin)
13 MB1
luteolin
14 MB2
taxifolin
15 HH1
quercetin-3-O-rutinoside
(rutin)
16 C2.1
resveratrol
17 C5.2
Resveratrol 3 –beta-mono-D-glucoside
(picied)
14 18 C7.3
emodin
19 C8.4
emodin-8-O-β-D-glucopyranoside
20 C8.5
physcion-8-O-β-D-glucopyranoside
Example: Compound MB1: vitexin (MB1) Compound was obtained as a yellow amorphous powder and its
molecular formula MB1was determined as C21H20O10 on the basic of ESI-MS at m/z 433 [M+H]+
and the melting point at 247-249ºC . The 1H-NMR spectrum of MB1 showed a doublet proton at δ 8.01
corresponding to H-2’ and H-6’ proton. Another doublet proton occurs at δ 6.89 corresponding to H-3’ and H-5’. Two protons appeared at δ 6.75 and δ 6.24 as singlets corresponding to H-3 and H-6 protons respectively, one proton anomeric at 4,71 corresponding to H-1’’, which suggested the structure of flavone with one sugar moiety.
The 13CNMR and DEPT spectrum of the compound showed 21 signals for the vitexin. Carbon bonded to the carbonyl group C-4 appeared at δ 182.1. The carbonyl carbon, C-4 resonates around δ 175-178, when the carbonyl is not hydrogen bonded. But in the presence of H-bonding to 5-hydroxy group, it moves downfield to about δ 182. When 3-hydroxy group is alone it resonates at δ 171- 173. When both 3- hydroxyl and 5-hydroxyl groups are present, it resonates at δ176.Carbon bonded to the hydroxyl group C-5, C-7 and C-4’ appeared at δ 160,4, δ 162,8, δ 161.1 respectively. Signals of C-6 from C-8 and signals of C-5 from C-9 are distinguished with the help of 13C-1H coupling data. The degree of coupling identifies each carbon and demonstrates that C-9 resonates at higher field from C-6 while C-8 resonates at higher field from C-6.The degree of coupling identifies each carbon and demonstrates that C-9 resonates upfield from C-5 while C-8 resonates up field in comparison to C-6.
The HMBC correlations HMBC between
(δC160,4)/C(δC 160,4)/C(δH4,71) and Cposition of glucose at 2’, 6’(δ(δC121,1)/Clink between CHMBC correlation from10 (δC104,7)
Thus, the structure of
4.3 Hydroly4.4.1 Hydrol4.4.1.1
Quercetinenzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected. design was processed and analyzed using
Figure
The HMBC correlations HMBC between 160,4)/C-6 (160,4)/C-7 (4,71) and C-
position of glucose at δH8,01) and C
121,1)/C-4’ link between CHMBC correlation from
104,7) . Thus, the structure of
and the important HMBC correlations of Hydrolyzation of glycosides from Vietnamess plants by BGLHydrolyzation by free BGL
1.1 Hydrolyzation of Quercetin-
enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected.
esign was processed and analyzed using
Figure 4.74: The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.
The HMBC correlations HMBC between 6 (δC98,4)/C7 (δC 162,8
-7 (δC 162,8)/Cposition of glucose at C-
) and C-2 ( (δC161,1)
link between C-1’ and CHMBC correlation from
Thus, the structure of
HO
HO
OH
HO
6
7
2''
3''
Figureand the important HMBC correlations of zation of glycosides from Vietnamess plants by BGLzation by free BGL
Hydrolyzation of -3-O-beta
enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected.
esign was processed and analyzed using
The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.
The HMBC correlations HMBC between 98,4)/C-10 (δC162,8)/C-8 (δ
162,8)/C-8 (-8 of A ring.
2 (δC164,2), 161,1) suggested the
1’ and C-2. The structure of C ring was confirmed by HMBC correlation from H-3 (δH
Thus, the structure of MB1
O
OH O
O
OH
HOOH
2
3
45
10
89
1'
1''
2''
4''
5''
6''
Figure 4.44. and the important HMBC correlations of zation of glycosides from Vietnamess plants by BGLzation by free BGL
Hydrolyzation of quercetinbeta-galactoside was hydrolysed by different BGL
enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected.
esign was processed and analyzed using
The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.
15
The HMBC correlations HMBC between C104,6), between δC 104,7)/C8 (δC 104,7
8 of A ring. The HMBC correlations between 164,2), between
suggested the The structure of C ring was confirmed by
H6,75) to
MB1 was determined and named
OH2'
3'
4'
5'
6'
HO
4.44. Chemical structure and the important HMBC correlations of zation of glycosides from Vietnamess plants by BGLzation by free BGL-P :
quercetin glycosidegalactoside was hydrolysed by different BGL
enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected.
esign was processed and analyzed using
The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.
The HMBC correlations HMBC between OH104,6), between
)/C-10 (δ104,7)/C-9(δThe HMBC correlations between
between H-3’, 5’ (suggested the the structure of B ring and the
The structure of C ring was confirmed by C-2 (δC164,2)/C
was determined and named
OHO
OH O
OHO
OH
HOOH
45
106
7
8
9
1''
2''
3''
4''
5''
6''
Chemical structure and the important HMBC correlations of zation of glycosides from Vietnamess plants by BGL
glycosides: galactoside was hydrolysed by different BGL
enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected.
esign was processed and analyzed using Modde 5.0 software.
The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.
OH-5 (δH104,6), between H-6 (δ
δC 104,7), betweenδC 156,1) comfirmed the
The HMBC correlations between 3’, 5’ (δH
the structure of B ring and the The structure of C ring was confirmed by
164,2)/C
was determined and named
O
O
2
3
1'
2'
3'
4'
5'
6'
Chemical structure and the important HMBC correlations of MB1zation of glycosides from Vietnamess plants by BGL
galactoside was hydrolysed by different BGL
enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected.
Modde 5.0 software.
The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.
H13,15) and CδH6,24) and C), between) comfirmed the
The HMBC correlations between H 6,89) and C
the structure of B ring and the The structure of C ring was confirmed by
164,2)/C-4 (δC182,1)/ C
was determined and named vitexin.
OH4'
MB1 zation of glycosides from Vietnamess plants by BGL
galactoside was hydrolysed by different BGLenzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h, 6h; the actual performance of the reactions were collected. Experimental
Modde 5.0 software.
The regression plot represents the optimum region of the enzyme concentration and the hydrolysis time.
13,15) and C-5 6,24) and C-5
), between H-1’’ ) comfirmed the
The HMBC correlations between H-6,89) and C-1’
the structure of B ring and the The structure of C ring was confirmed by
182,1)/ C-
vitexin.
zation of glycosides from Vietnamess plants by BGL-P
galactoside was hydrolysed by different BGL-P enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h,
xperimental
The regression plot represents the optimum region of the
5 5
1’’ ) comfirmed the
’ the structure of B ring and the
The structure of C ring was confirmed by
P enzyme concentrations ( 0.1U / ml; 0.55U / ml and 1.0U / ml), for 2h, 4h,
xperimental
Figue 4.75:
Hydrolyzation of After 5 h of enzymatic reaction catalyzed by BGL
60oC, significant amounts of quercetin (approximately 90%) were obtained.
HydrolyzationNormally, the transformation of rutin catalyzed by mixture of 2
enzymes: catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and catalyzes the cleavage of terminal rutinoside groups from rutin to quercetin and rutinoseto hydrolysis the enzymatic reaction time was 6h.
4.75: The
Hydrolyzation of After 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were
obtained.
H
Figure 4.77 Hydrolyzation quercetin
Normally, the transformation of rutin catalyzed by mixture of 2 enzymes: α-L-rhamnosidase and catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and catalyzes the cleavage of terminal rutinoside groups from rutin to quercetin and rutinoseto hydrolysis and the enzymatic reaction time was 6h.
The effect of
Hydrolyzation of quercetinAfter 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were
OHO
OH O
OHO
OH O
4.77 Hydrolyzation of quercetin
Normally, the transformation of rutin catalyzed by mixture of 2 rhamnosidase and
catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and catalyzes the cleavage of terminal rutinoside groups from rutin to quercetin and rutinose. In this study,
and the obtained maximal yield of quercetin was the enzymatic reaction time was 6h.
of enzyme concentrationrate of the
quercetin-After 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were
O
O
O
OH
O
O
O
O
OH
O
HO
HO
Hydrolyzation of quercetin-3-O-rutinoside
Normally, the transformation of rutin catalyzed by mixture of 2 rhamnosidase and
catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and catalyzes the cleavage of terminal rutinoside groups from rutin to
. In this study, the obtained maximal yield of quercetin was
the enzymatic reaction time was 6h.
16
enzyme concentrationrate of the hydrolysis
-3-O-β-DAfter 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were
OH
OHCOOH
OH
OH
OHOH
OH
5
5h, 60
HiÖu su
HiÖu s
Hydrolyzation of quercetinrutinoside (rutin)
Normally, the transformation of rutin catalyzed by mixture of 2 rhamnosidase and β-D-
catalyzes the cleavage of terminal rhamnoside groups from rutin to isoquercitrin and rhamnose and the same time, catalyzes the cleavage of terminal rutinoside groups from rutin to
. In this study, β-Dthe obtained maximal yield of quercetin was
the enzymatic reaction time was 6h.
enzyme concentration and reaction hydrolysis
D-glucuronideAfter 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were
HO
OH
HO
O
5h, 60oC
60oC
suÊt: 90%
u suÊt: 98%
quercetin glycosides by (rutin)
Normally, the transformation of rutin catalyzed by mixture of 2 -glucosidase
catalyzes the cleavage of terminal rhamnoside groups from rutin to the same time,
catalyzes the cleavage of terminal rutinoside groups from rutin to D-glucosidase
the obtained maximal yield of quercetin was
and reaction
glucuronide After 5 h of enzymatic reaction catalyzed by BGL, significant amounts of quercetin (approximately 90%) were
O
O
OH
O
O
O
OH
OH
O
O
HO
H3HO
8h,6
0oC
HiÖ
usu
Êt:2
5%
glycosides by
Normally, the transformation of rutin catalyzed by mixture of 2 glucosidase. α-L
catalyzes the cleavage of terminal rhamnoside groups from rutin to the same time, β
catalyzes the cleavage of terminal rutinoside groups from rutin to glucosidase (BGL
the obtained maximal yield of quercetin was
and reaction time on the
After 5 h of enzymatic reaction catalyzed by BGL-P, heated at , significant amounts of quercetin (approximately 90%) were
OH
O OH
OH
OH
OH
O
O3CO
HO OH glycosides by BGL
Normally, the transformation of rutin catalyzed by mixture of 2 L-Rhamnosidase
catalyzes the cleavage of terminal rhamnoside groups from rutin to β-D-glucosidase
catalyzes the cleavage of terminal rutinoside groups from rutin to (BGL-P) was used
the obtained maximal yield of quercetin was 25% when
time on the
P, heated at , significant amounts of quercetin (approximately 90%) were
BGL-P
Normally, the transformation of rutin catalyzed by mixture of 2 Rhamnosidase
catalyzes the cleavage of terminal rhamnoside groups from rutin to glucosidase
catalyzes the cleavage of terminal rutinoside groups from rutin to was used % when
P, heated at , significant amounts of quercetin (approximately 90%) were
Normally, the transformation of rutin catalyzed by mixture of 2 Rhamnosidase
catalyzes the cleavage of terminal rhamnoside groups from rutin to glucosidase
catalyzes the cleavage of terminal rutinoside groups from rutin to was used % when
17 4.4.1.2 Hydrolyzation of other glycosides:
Genistin and daidzin were hydrolysed in the condition that the BGL-P enzyme concentration was 0.4U / ml with an incubation time of 5 hours at 60oC. The reaction was stopped by adding 10 ml of MeOH. Hydrolysis results showed high yield to both genistin and daidzin 98%.
Hình 4.78 Hydrolyzation of genistin and daidzin Maximum yield of picied hydrolisis was 99% when the enzymatic
reaction time was 5h at 60oC.
Hình 4.79 Thủy phân hợp chất picied
Under the same conditions, isorhamnetin-3-O-β-D-glucuronide was hydrolysed with the yield 85% after 5 hour.
Figue 4.80 Hydrolyzation of isorhamnetin-3-O-β-D-glucuronide BGL-P hydrolysed isoapigenin-6-C-glucoside and apigenin-8-C-
glucoside, with similar yield.
Figue 4.81 Hydrolyzation of isoapigenin-6-C-glucoside and apigenin-8-
C-glucoside
4.4.1.3
The results of hydrolysis of eand physcionhydrolysed glycosides.
4.4.2 Hydrolyzation by Quercetin
immobilization at the appropriate conditions.
Figue
Enzyme BGLtimes before lost 50% activity. This is a potential
4.4.1.3 Hydrolyzation ofThe results of hydrolysis of ephyscion-8
hydrolysed anthraquinone glycosides with the same yeild of flavonoid glycosides.
HHO
H3C
HOHO
OH
Figue Hydrolyzation by Quercetin
immobilization at the appropriate conditions.
Figue 4.84:
Enzyme BGLtimes before lost 50% activity. This is a potential
Hydrolyzation ofThe results of hydrolysis of e
8-O-β-Danthraquinone glycosides with the same yeild of flavonoid
HO
OHOO
OH
OH
O
CO
O
O
O
H
OH
(20)
Figue 4.82: Hydrolyzation by BGLQuercetin -3-O-beta
immobilization at the appropriate conditions.
4.84: Reusable of
Enzyme BGL-P immobilized onto alginate can be reused in 5 times before lost 50% activity. This is a potential
Hydrolyzation of anthraquinone glycosidesThe results of hydrolysis of e
D-glucopyranoside (20) anthraquinone glycosides with the same yeild of flavonoid
O OHO
O
(19)
OH
CH
O
O
Hydrolyzation of BGL-P immobilizationbeta-galactoside was hydrolized by
immobilization at the appropriate conditions.
Reusable of BGLspent
P immobilized onto alginate can be reused in 5 times before lost 50% activity. This is a potential
18
anthraquinone glycosidesThe results of hydrolysis of emodin
glucopyranoside (20) anthraquinone glycosides with the same yeild of flavonoid
OH
CH3
4h(96%
H3
95%
Hydrolyzation of immobilization
galactoside was hydrolized by immobilization at the appropriate conditions.
BGL-P immobilized spent coffee grounds
P immobilized onto alginate can be reused in 5 times before lost 50% activity. This is a potential
anthraquinone glycosidesmodin-8-O-β-
glucopyranoside (20) showed thatanthraquinone glycosides with the same yeild of flavonoid
HO
h, 60oC%)
4h, 60oC%
Hydrolyzation of anthraquinone glycosidesimmobilization:
galactoside was hydrolized by immobilization at the appropriate conditions.
P immobilized coffee grounds
P immobilized onto alginate can be reused in 5 times before lost 50% activity. This is a potential
anthraquinone glycosides: -D-glucopyranoside (19)
showed thatanthraquinone glycosides with the same yeild of flavonoid
OH O
O(
OH
H3CO
anthraquinone glycosides
galactoside was hydrolized by
P immobilized onto Ca
P immobilized onto alginate can be reused in 5 times before lost 50% activity. This is a potential results for applied to
glucopyranoside (19) showed that: BGL
anthraquinone glycosides with the same yeild of flavonoid
OH
CH3
(18)
OHO
O(23)
anthraquinone glycosides
galactoside was hydrolized by
Ca-alginate and
P immobilized onto alginate can be reused in 5 results for applied to
glucopyranoside (19) BGL-P can
anthraquinone glycosides with the same yeild of flavonoid
CH3
anthraquinone glycosides
galactoside was hydrolized by BGL-P
alginate and
P immobilized onto alginate can be reused in 5 results for applied to
glucopyranoside (19) P can
anthraquinone glycosides with the same yeild of flavonoid
P
P immobilized onto alginate can be reused in 5 results for applied to
19 hydrolysis of glycoside compounds with the higher performance and the lower cost. 4.5 Disinfection of microorganisms by electro-chemical method: In microbiological studies, keeping the environment safe, controlling the spread of microorganisms during and after the study is of utmost importance. Therefore, after each microbial experiment, it should be carefully disinfected before disposal. Bacillus spores are resistant to disinfection methods and they represent a potential threat that requires improved methods to ensure water safety. In this study, Bacillus cereus spores were used to investigate the effectiveness of the electrochemical (EC) disinfection process. The results of study show that the optimum conditions of the electro-chemical disinfection method is: electric potential 2A, water contain 50mg/L Cl-, pH 6,8 with 0,01M phosphate buffer. Applied this results on the disinfection of P. citrinum, after 30 min 100% P.citrinium was killed in direct experiment and after 60 min in indirect experiment.
Figure 4.88: Effect of storage time to the disinfection ability of spore of
P. citrinum by electro-disinfection 4.6. Biological activities of isolated compounds and their aglycone: 4.6.1 Antioxidant activity by DPPH assay
22 compounds were evaluated for their antioxidant activity by DPPH assay:
Table 4.14 : Antioxidant activity by DPPH assay of aglycone and glycosides
No Name of compound EC50
-5
-4
-3
-2
-1
00 15 30 45 60
Log
N/N
o
thời gian (phút)
trực tiếp
Gián tiếp
20
(mM) (µg/ml) 1 Quercetin 0,027 8,2 2 quercetin-3-O-β-galactoside 0,055 25,6 3 quercetin-3-O-rutinoside 0,143 87,4 4 quercetin-3-O-β-D-glucuronide 0,047 22,5 5 Luteolin 0,015 4,3 6 Kaemferol 0,060 17,2 7 Isorhamnetin 0,032 10,1 8 isorhamnetin-3-O-β-D-glucuronide 0,133 65,5 9 Apigenin 0,047 12, 6 10 apigenin-6-C-glucoside 0,055 23,8 11 apigenin-8-C-glucoside 0,055 23,8 12 Genistein >0,948 >256 13 Genistein 7-O-beta-D-glucoside 0,256 110,7 14 Daidzein 0,532 135,3 15 Daidzein7-O-beta-D-glucoside >0,615 >256 16 Resveratrol 0,036 8,2 17 Resveratrol 3 –beta-mono-D-glucoside 0,043 16,8 18 Emodin 0,205 55,4 19 emodin-8-O-β-D-glucopyranoside >0,592 >256 20 Physcion 0, 731 207,8 21 physcion-8-O-β-D-glucopyranoside >0,573 >256 22 Catechin 0,038 11,0 As results, almost compounds had antioxidant activity and the
aglycone usually had higher activity than their glycosides such as EC50 of quercetin and quercetin-3-O-rutinoside were 0,027 μM and 0,143 μM, respectively or isorhamnetin and isorhamnetin-3-O-β-D-glucuronide were 0,032 μM and 0,133 μM, respectively. DPPH scavenging activity of compounds is given in descending order as follows: quercetin > quercetin-3-O-β-D-glucuronide > quercetin-3-O-β-galactoside > rutin; hay resveratrol > resveratrol 3 –beta-mono-D-glucoside; isorhamnetin > isorhamnetin-3-O-β-D-glucuronide.
So the hydrolysis of glycosides onto aglycone help to create
21 compounds with higher antioxidant capacity to enhance application.
4.6.2 α-Glucosidase inhibition: To assess the applicability of the treatment of diabetes, compounds
were evaluated for their α-Glucosidase inhibition activity. Table 4.15 : Results of α-Glucosidase inhibition activity
No Name of compound IC50
(µg/ml) (mM) 1 quercetin 6,3 0,021 2 quercetin-3-O-β-galactoside 44,1 0,095 3 quercetin-3-O-rutinoside 131,9 0,216 4 quercetin-3-O-β-D-glucuronide 68,9 0,144 5 apigenin 53,46 0,198 6 apigenin-6-C-glucoside 117,2 0,271 7 apigenin-8-C-glucoside 107,2 0,248 8 genistein 13,5 0,050 9 genistein 7-O-beta-D-glucoside >256 >0,592 10 daidzein 26,9 0,106 11 daidzein7-O-beta-D-glucoside >256 >0,615 12 acarbose 192,1 0,297
Flavonoids group isolated from vietnamess plants had great potential for use in treatment of diabetes, many compounds are able to inhibit enzyme α- glucosidase higher than acarbose.
In this test, quercetin, apigenin, genistein and daidzein had an IC50 value at lower concentrations than their glycosides. 4.6.3 An angiotensin converting enzyme inhibitor
Angiotensin-converting enzyme (ACE) inhibitors is widely used in the treatment of hypertension, chronic kidney disease, and heart failure. In addition to efficacy, these agents have the additional advantage of being particularly well tolerated since they produce few idiosyncratic side effects and do not have the adverse effects on lipid and glucose metabolism seen with higher doses of diuretics or beta blockers. To compare Angiotensin-converting enzyme (ACE) inhibitors of aglycone and their glycosides, we evaluted the bioactivity of them.
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Table 4.16: Results of an angiotensin converting enzyme inhibitor
Name of compound IC50
(µg/ml) (mM)
Quercetin 23,6 0,078
quercetin-3-O-rutinoside 70,34 0,115
quercetin-3-O-β-D-glucuronide >256 >0,535
Captopril 0,000326 0,000015
Therefore, ACE inhibitors of compounds sample were much lower than the control, however the results showed that: effects of quercetin was higher than their glycosides.
CONCLUSION
1. Isolated and identified of Penicillium citrinum which hight produced β-glucosidase from roots of Clerodendron cyrtophyllum Turcz:
- Fermentation and evaluation of kinetic parameters of free and immobilized β-glucosidase from P. citrinum
- P. citrinum cultured on Pd medium at 6 days, 27oC, 200 rpm. Free BGL-P showed Michaelis–Menten kinetics for pNPG substrates tested with Km values 0,011 µmol, Vmax 13,91 µmol/min, to 60o
- BGP-L immobilized on Ca-alginate: 50oC, pH: 5,5-6,2, Km= 0,034 µmol, Vmax = 13,09 µmol/min. Reused from 5 to 7 times dependent on substances
- BGP-L immobilized on spent coffee grounds: 40oC, pH: 6,0, Km= 0,022 µmol, Vmax= 14,45 µmol/min.
2. Seventeen flavonoide glycoside and aglycone compounds were isolated: Genistein, daidzein, genistin, daidzin, catechin, hyperoside, quercetin, kaempferol, isorhamnetin-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide, vitexin, isovitexin, luteolin, taxifolin, rutin, resveratrol, picied, three anthraquinone glycoside and aglycone
23 compounds were isolated: emodin, emodin-8-O-β-D-glucopyranoside, physcion-8-O-β-D-glucopyranoside
3. The optimum condition of hydrolysis by BGL-P process: reaction at 60oC in 5h, 0,4U/ml BGL-P in citrate buffer pH 6,0.
4. Hydrolyzed ten glycoside compounds in the optimum condition: the yield of reactions from 85 to 98%, except rutin (25%).
5. The optimum conditions of the electro-chemical disinfection method is: electric potential 2A, water contain 50mg/L Cl-, pH 6,8 with 0,01M phosphate buffer.
6. The bioactivity of most aglycone were higher than their glycosides.
- DPPH scavenging activity of compounds is given in descending order as follows: quercetin > quercetin-3-O-β-D-glucuronide > quercetin-3-O-β-galactoside > rutin; hay resveratrol > resveratrol 3 –beta-mono-D-glucoside; isorhamnetin > isorhamnetin-3-O-β-D-glucuronide.
- α-Glucosidase inhibition: quercetin, apigenin, genistein and daidzein had an IC50 value at lower concentrations than their glycosides.
- ACE inhibitors of compounds sample were much lower than the control, however the results showed that: effects of quercetin was higher than their glycosides.
RECOMMENDATIONS
1. BGL-P isolated from P. citrinum had high activity and it can hydrolysis many kind of substances. Therefore, there should be more research is needed to expand the scope of the application 2. Further research is needed on the applicability of aglycone compounds in practice.
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NEW FINDINGS OF THE THESIS Isolated and identified of Penicillium citrinum which hight
produced β-glucosidase Purificated enzyme beta-glucosidase from Penicilium citrinum to
hydrolysis glycosides with high yield. This is an interdisciplinary study with the combination of chemistry,
biology, electrochemistry ... solving the whole problem from isolation to application and finally treatment without affecting the environment.