Study of hydrolysis of natural glycosides by β-Glucosidase enzyme and bioactivities of their products

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: H 2 O (97:3) and EtOAc:H 2 O: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 CH 2 Cl 2 D1.1 (12.8mg) D1.2 (3.4 mg) Kết tinh CH 2 Cl 2 3.3.4 Isolation and Styphnolobium japonicum Characteristic of the compound 1H NMR (500 MHz, DMSO 6’’’); 3,09 J= 7,0 Hz, H H-8); 6,84 (1H, d, (1H, dd, 13C (C-4); 161,2 (C 103,9 (C 116,2 (C 3’’); 70, (CRha-2’’’); 71,3(C 6’’’). 3.3.5 Isolation cuspidati - 5,00 (proton Glc- J=2,0; 8,0 Hz, H -NMR (125 MHz, DMSO -10); 121,1 (C -5’); 121,5(C 3 (CGlc- purification 1’’); 6,19 (1H, d, J= 8,0 Hz, H -5); 100,1 (C -6’); 101,2 (C 4’’); 75,8 (C Rha-3’’’); 71,8 (C and purification (L.) Schott -d6 s CH-OH ); 5,2 (1H, brs, H - -6’); 12,58 (1H, s, OH -6); 164,1 (C -1’); 115,2 (C Glc-5’’); 66,9 (C 6 of glycosides from : melting point ): =0,99 ppm (3H, d, J= 2,0 Hz, H 5’); 7,52 (1H, -d6):  -2’); 144,7 (C Glc-1’’); 74,1 (C Rha-4’’’); 68,2 (C of glycosides from -6); 6,38 (1H, d, d, -5). 156,5 (C -7); 93,6 (C Glc-6’’); 98,7 (C flower of : 242oC J Rha-1’’’); 5,34 (1H, d, J = 2,0 Hz, -2); 133,3 (C -8); 156,4 (C -3’); 148,4 (C Glc-2’’); 76,4 (C Rha-5’’’); 18,6 (C Rhizoma = 6,5Hz, H J= 2,0 Hz, H-2’); 7,55 -3); 177,4 Rha-1’’’); 70,5 P Rha- -9); -4’); Glc- Rha- 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 (%) = ܳܿ ܯ1 ܯ2 ܳ݋ ݔ100 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: Where 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 fungal glucosidase. 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 , A CHAPTER Isolation of fungal beta : Colonies of fungal were isolated from root of -like appearance (a penicillus). sample and A -pNG method. Analysis of the culture filtrate of C5 fungal beta ITS4 re blank 4. RESULTS AND DISCUSSION cyrtophyllum Turcz -glucosidase gions for fungal identification 9 = (Acontrol were the optical density of the extract at control -glucosidases: and screened them for prodution with the -glucosidases: the fungal. – Asample -glucosidases Turcz C5 gave maximum enzyme activity was We used 5.8S gene and )/(Acontrol Clerodendron was identified – Ablank roots of 33,628U/ml . Constructing ) β- of in next mostly 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 BGL- . Neighbour joining tree with bootstrapping gave us Fig. -Nitrophenyl β P was used at free enzyme an activity was observed. oC. Temperature is an important factor for enzymatic ged to 4.4: Colonies -BGL was carried out by partially purified enzyme (BGL -P: 6.0. BGL 10 Penicillium citrinum , phialides β- , lyophilized. -D -P C5. glucosidase -glucopyranoside (5 mM) as d immobilized enzyme. The best temperature for BGL al errors. Bootstrap It is because more than , thus strongly of C5 from culture ammonium sulphate Activity of the BGL 5 - 0 to 70°C after P) was -P 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 Immobil