Nghiên cứu phối hợp esterase và hệ enzyme thủy phân từ nấm trong chuyển hóa phụ phẩm công nông nghiệp để thu nhận bioethanol tt tiếng anh

Based on the above reasons, we conducted a research project entitled "Research on the combination of esterases and hydrolases from fungi to convert agro-industrial by-products into bioet

I INTRODUCTION

1 The necesssity of the dissertation

Currently, energy demand is always a problem for any country in the world Among thealternative energy sources currently in use (wind, solar, nuclear energy, etc.), bioenergy is aninevitable development trend, especially in agricultural countries which are importing fuel Based onproduction materials, the bioenergy can be divided into 4 generations: Generation I (from starch such

as corn, cassava, sugarcane), Generation II (from plant biomass such as rice, corn, wheat stalks,bagasse), Generation III (from microalgae species) and Generation IV - advanced fuel (based onbiochemical, chemical, thermal and chemical changes) They are divided into three groups: biodiesel,biogas and bioethanol However, bioethanol is a great concern because the government issued adecision to use E5 bio-fuel (5% bioethanol) to replace RON 92 petrol nationwide on January 1, 2018.Therefore, the demand for bioethanol production and consumption is increasing

The production of bioenergy in general and bioethanol according to Generation I from starchsources (cassava, corn) and sugar (sugarcane) is very popular Besides, bioethanol is also producedfrom lignocellulose according to the second generation This source of materials mainly includes:wood, rice straw, bagasse, corn stalks are the biomass with the most common lignocellulosecomposition among lignocellulose-rich agricultural by-products (ABP) However, this material source

is not used effectively but mainly by traditional methods such as mushroom farming, animal feed,composting for fertilizer, burning Therefore, taking advantage of this substrate sources to producebioethanol is an appropriate solution, especially for countries with agriculture like Vietnam Althoughthe source of lignocellulose is very popular, it is difficult to effectively utilize this biomass source forthe production of higher value products mainly due to the complex structure of lignocellulose, difficult

to convert biochemicals Therefore, the task of finding the right solution to effectively convert thematerial into a useful form of energy is becoming increasingly urgent Traditional methods can usechemical (acid/alkaline) or physicochemical (grinding/explosive steam) However, one of the newways to solve this task is to use biological catalysts (enzymes from fungal species), which are known

to have an efficient catalytic enzyme system that helps them decompose well lignocellulose to releasesugar units needed for bioethanol fermentation However, in general lignocellulose hydrolysingenzymes are weak when used directly on the crude substrate and the efficient conversion of catalystsrequires a combination of enzymes with synergistic effects Based on the above reasons, we conducted

a research project entitled "Research on the

combination of esterases and hydrolases from fungi to convert agro-industrial by-products into bioethanol" using a mixture of suitable enzymes ("enzyme cocktail") includes esterases (acetyl

esterase and feruloyl esterase), hydrolases and/or oxidative enzymes from fungi to decomposecomplex polymer structures to ferment bioethanol The production of bioethanol from biomass ofAIBP on the one hand takes advantage of raw materials to replace traditional raw materials (eg

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cereals, corn, cassava), on the other hand forms a clean energy source to help solve the problem ofenvironmental pollution caused by burning or processing according to traditional methods, anddoes not affect the national food security This is also one of the key stages of generation II infermentation technology of bioethanol.

2 Objectives of the dissertation

Currently, the ability to convert raw materials from lignocellulose-rich biomass bytraditional methods has many limitations Therefore, the objective of the thesis is to exploit avariety of biocatalysts (hydrolases) from fungi to efficiently convert lignocellulose-rich biomassfrom ABP into sugars (C5 and C6) capable of fermenting for bioethanol production The thesisuses "enzyme cocktail" synergistic catalyst to increase the ability of biological transformation.Therein, the study used carbohydrate esterase [feruloyl esterase (FAE), acetyl esterase (AE)] tocoordinate with main circuit attack enzymes (cellulase/xylanase) and branched circuits tohydrolyze lignocellulose structure In order to increase bioavailability, a second objective is tostudy the use of a mixture of enzyme ("enzyme cocktail") and to assess the possibility ofconverting monosaccharide into bioethanol by "enzyme cocktail"

3 Content of the dissertation

To achieve the above objectives, the main research included:

- Isolation, screening and selection of fungal strains which are carbohydrate esterase [feruloyl esterase (FAE), acetyl esterase (AE)] biosynthesis capable

-Biosynthesis and purification of FAE and AE from the culture medium and characterization

of purified enzymes

- Transformation of lignocellulosic material into monosaccharide (hexose and pentose) capable

of fermentation using single enzymes and enzyme cocktails cellulase/xylanase and esterase

- Optimizing the process of transformation and research on the production of bioethanol from transformation process in laboratory-scale

4 New contributions of the dissertation

- 44 species belong to Basidiomycota and Ascomycota isolated in Vietnam were screened for their ability of carbohydrate esterase (feruloyl esterase, acetyl esterase) production

- Feruloyl esterase from Alternaria tenuissima and acetyl esterase from Xylaria polymorpha were firstly purified from lignocellulose-rich cultures Of which Alt.tenuissima feruloyl esterase (AltFAE) has molecular weight (Mw) of 30.3 kDa and specific activity of 11.2 U/mg protein While X.polymorpha (XpoAE) acetyl esterase has Mw of 44 kDa and specific activity of 13.1 U/mg.

These two enzymes are highly stable at temperatures of 40 to 45ºC and pH 5.0-5.5

- Above purified carbohydrate-esterases in combination with the commercial cellulases (“enzymecocktail” with cellulase/xylanse activities) were compositionly optimized and used for theconversion of the raw plant-biomass (bagasse cane, rice straw, meals wood, seaweed …) into

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fermentable monosaccharides sugar Of which bagasse cane was chose as the most suitable

substrate for C5/C6 monosaccharides production (glucose, xylose) with the efficiency of 49.8%

- Evaluated the ability of fermentation of enzymatically hydrolyed C5/C6-sugars to produce

bioethanol by Saccharomyces cerevisiae with the efficiency of 79.8% according to the theory.

5 The scientification of the dissertation

In addition to the production of bioethanol from starch (cassava, corn) and sugar (sugarcane),bioethanol can be produced from lignocellulose Lignocellulose is the most common biomass inthe biosphere Bioethanol production from lignocellulose is a good solution especially forcountries with agriculture like Vietnam Annual domestic agro-industry production of hundreds ofmillions of tons of lignocellulose-rich by-products is an extremely abundant source of rawmaterials for clean energy production, on the other hand addressing the problem of environmentalpollution caused by not being treated or exclusion by traditional methods

Published literatures and our experience show that lignocellulase are generally weak whenused directly (not processed) on raw substrates This can be overcome by using “enzymescocktail” that include hydrolase and/or oxidative enzymes so that they play an important role infinding new enzymes with high bioactivity of complex polymeric structures In addition to,screening of enzymes by micro-organism culture and useful properties

The thesis is not intended to address the whole process of converting crude biomass intobiofuel whose main objective is to exploit and use bio-diversity of fungus and optimize it forconverting biomass into sugar capable of fermenting This is also one of the key stages ofgeneration II in the generation of bioethanol

6 Dissertation frame

The dissertation consists of 163 pages with 21 tables, 58 images,143 references Thestructure of thesis: Introduction (4 pages), Chapter 1: Overview (39 pages), Chapter 2: Materialsand methods (26 pages), Chapter 3: Results and discussion: (67 pages), Conclusion (2 pages), List

of published literatures (1 page), References (13 pages), Appendix (39 pages)

II CONTENT OF THE THESIS

The introduction discusses the scientification, novelty, mission and content of thesis

Chapter 1 Overview

This section presents:

- An overview of lignocellulose-rich agricultural by-products (ABP), which describes the origins,current status of ABP use in Vietnam in general and the origin, current status, environmentalissues from bagasse in Vietnam in particular

- Transformation of lignocellulose-rich materials by biocatalysis It highlights the mechanism ofenzyme metabolism in lignocellulose hydrolysis, enzymatic source from diverse fungi in Vietnam,

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lignocellulose enzyme from fungus and the role of carbohydrate esterase in lignocellulose

- Commercial enzymes that purified from Trichoderma reesei (carboxymethyl cellulase and

glucuronone xylanase; 1-1.6 U/mg, Cell/Xyl, AB Enzyme, Darmstadt, Germany) optimized for

pH 5.0-5.5 and temperature is 40-45ºC

- Saccharomyces cerevisiae SH1 is supplied by the Department of environmental microbiology,

Institute of Environmental Technology

2.2.3 Screening for esterase enzyme activity and selection of fungal strains

2.2.4 Evaluation of acetyl esterase and feruloyl esterase assay

2.2.5 Optimization of esterase biosynthesis

2.2.6 Purification of enzyme, SDS-PAGE and In-Gel Digestion

2.2.7 Biochemistry method: TLC; High performance liquid chromatography (HPLC); Identification

of peptides by mass spectrometry (MS); Reducing sugar by dinitrosalicylic acid method (DNS);processing of materials (by alkaline, acid and heat); Determination of bioethanol content

2.2.8 Bio-metabolism of lignocellulose-rich materials

2.2.9 Experimental planning method

2.3 Develop research diagrams

2.3.1 Diagram of fermentation conditions for experimental optimal models

2.3.2 Diagram of hydrolysis sugarcane bagasse by using physicochemical method combining

"enzyme cocktail"

2.3.3 Diagram of bioethanol fermentation conditions

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Chapter 3: Results and discussion

3.1 Isolation and screening of fungi

44 fungi, which included 13 families, were identified and species names, fungal taxa isolatedfrom fungal samples collected from Cuc Phuong (Ninh Binh)

3.1.1 The Basidiomycota

Polyporaceae family: Coriolus unicolor, Tyromyces lacteus, Trametes insularis, Tr cons, Tr gibbosa, Hexagonia apiaria, Nigroporus aratus

Ganoderma family: Ganoderma komingshegii, G applanatum, Ganoderma sp.

Coriolaceae family: Poria versipora

Marasmiaceae family: Campanella junghuhnii, Marasmius maximus

Family of Agaricaceae: Coprinus disseminates

Mycenaceae family: Mycena galericulata

Auriculariaceae family: Auricularia delicate

Hymernochaetaceae family: Inonotus substygius, Polystictus didrichsenii

Hygrophoropsidaceae: Hygrophoropsis aurantiaca

3.1.2 The Ascomycota

Xylariaceae family: Xylaria polymorpha, X carpophila, Hypoxylon monticulosum (CP629), Rosellinia sp (CP564), Nodulisporium sp (CP582).

Helotiaceae family: Bisporella citrine

Pleosporaceae family: Alternaria tenuissima (SP66)

Bionectriaceae family: Bionectria sp (CP587)

3.2 Esterase biosynthesis and the selected fungal strains

3.2.1 Screening of Feruloyl esterase activity

The feruloyl esterase synthesis of the 44 fungal species selected was evaluated by the ethyl

ester (ethyl 4-hydroxy-3-methoxycinnamate) cleavage on agar plates Alt.tenuissima SP66 for

high feruloyl esterase activity should be selected for subsequent studies to determine cultureconditions such as cultivation-time, substrate/carbon source, nitrogen source, temperature and pH

3.2.2 Screening of acetyl esterase activity

After fermentation of 44 fungal species, centrifugation used to remove biomass and othercomponents and then determine acetyl esterase activity (AE) by ability of p-nitrophenyl acetate

hydrolysis X.polymorpha A35 should be selected for further studies due to high acetyl esterase

activity After screening activity of feruloyl esterase and acetyl esterase, two highly active fungal

species Alt.tenuissima SP66 and X.polymorpha A35 were selected Then studies on optimum

fermentation conditions for enzyme synthesis, purification and characterization of AE and FAE aswell as the use of enzymes for lignocellulose catalytic conversion will be carried out

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3.3 Identification of fungi by molecular biology method

Figure 3.1 (A) Total DNA electrophoresis on 1% agarose gel and (B) PCR

product of SP66 and A35From the classification results by molecular biology method and morphological analysis

method, it can be concluded that the fungal isolate SP66 is the Alternaria tenuissima SP66 in the

family Pleosporaceae and the A35 fungal strain is Xylaria Polymorpha A35 in the family

Xylariaceae

3.4 Kinetics of biosynthesis of esterase

Optimization of the acetyl esterase and feruloyl esterase biosynthesis of the two strains of X.

polymorpha A35 and Alt.tenuissima Sp66 as follows:

Acetyl esterase enzyme activity: X.polymorpha A35 cultured on a basic medium supplemented

with straw substrate, nitrogen source is pepton, culture time is 10 days, temperature is 25ºC, pH 7,

stirring rate 200 rpm, then the highest acetyl esterase activity was 135.4 U/l

Feruloyl esterase enzyme activity: Alt.tenuissima was cultured on a basic medium

supplemented with straw substitute, culture time is 12 days, temperature is 25ºC, pH 7, stirring

rate 200 rpm, then the highest acetyl esterase activity was 1154.4 U/l

3.5 Purification of enzyme from culture medium

3.5.1 Purification and properties of esterase from X polymorpha A35 (XpoAE)

Crude enzyme extraction from culture medium after 3 weeks was concentrated by ultrafiltration

(10 kDa cut-off filter) Then, the enzyme protein which expresses the XpoAE activity for the

p-nitrophenyl acetate substrate is purified by liquid chromatography The first step of the protein elution

was carried out via an anaerobic DEAE Sepharose anion exchange chromatograph and the results

were obtained by three active fractions of XpoAE (Sections I, II and III) The eluent volume of the

highest enzyme activity fraction (III) was added to the SuperdexTM 75 column After the gel filtration

chromatography step, XpoAE enzyme activity fraction was collected (Figure 3.2).

After purification, the amount of purified enzyme protein was 20.6 mg, equivalent to 27 U

and 26.8% efficiency with purity of 18.3 times This purified enzyme fraction is used for further

studies of enzymatic protein properties as well as the in vitro transformation of lignocellulose-rich

material (Table 3.1).

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2700

Figure 3.2 Purified X.polymorpha A35 acetyl esterase via liquid chromatography steps

(A) DEAE Sepharose anion exchange chromatography and (B) SuperdexTM 75 gel filtration

chromatography; (─) absorption at λ = 280 nm and (●) XpoAE activity on p-nitrophenyl acetate

Table 3.1 Purification of the acetyl esterase activity

Physical-chemical properties of AltFAE: The SDS-PAGE of faction III via the purification

steps above shows a protein band corresponding to XpoAE with MW = 44 kDa after staining with

a colloidal blue Staining Kit IEF electrodes showed two adjacent protein bands with pI values of

3.5 and 3.6 respectively (Figure 3.3) The physical and chemical characteristics of XpoAE

correspond to the characteristics of published AE (34-56 kDa)

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Figure 3.3 Purified protein (1,3) expresses acetyl esterase activity (XpoAE) on

SDS-PAGE (A) and IEF (B) gel cartilage; (2,4) protein maker

Optimal temperature and pH: To determine the optimal reaction temperature, the reaction between the purified XpoAE enzyme and the p-nitrophenyl acetate substrate is carried out at a temperature of 35-70°C Results showed that the XpoAE activity increased from 40% at 35°C to

the maximum at 42°C (100%) Then, as the temperature increased, the activity of the enzyme

decreased to 51% at 61 ° C (Fig 3.4-A) The pH value is in the range of 5.0-5.5 Relative activity decreases from 100% at pH 6.5 to 57% at pH 7.0 (Figure 3.4-B)

A

Figure 3.4 Effects of temperature (B) and pH (A) on the activity of XpoAE

from X.polymorpha A35 Thermal stability and pH of enzyme XpoAE: Enzymes are relatively stable at 400C after 3 hours

of incubation After that, the activity decreased by more than 50% when incubated for 4 hours andlonger Enzyme activity decreased rapidly at 600C and lost most of the activity after 1 hour ofincubation at this temperature Purified enzymes show active stability at pH 5.0 but lost over 90% of

the activity during 1 hour incubation under strong acid conditions (pH 3; Figure 3.5) Purified XpoAE

enzyme from X.polymorpha A35 is relatively stable at a temperature of 40-42°C at pH 5.

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Figure 3.5 The purity of acetyl esterase from X.polymorpha A35 (XpoAE) at different temperature

conditions (A) and pH (B): (A): ♦ 40 o C, ▲60 o C; (B): ▲ pH 3, ● pH 5, ♦ pH 6

3.5.2 Purification and properties of feruloyl esterase from Alt.tenuissima SP66 (AltFAE)

Crude enzyme from the culture medium after 2 weeks was cut-off 5 kDa, through whichsmall molecular weight proteins and impurities were also removed Then, the enzyme proteinexpresses the feruloyl esterase activity for methyl ferulate substrate by purification by liquidchromatography The first step in the protein elution process is carried out via the DEAESepharose anion exchange chromatograph After the first step of purification, the purity increasedslightly (from 1.4 times) but most of the pigments (possibly polyphenolic compounds) wereremoved from the target FAE active protein The total elution volume of the highest enzymeactivity fraction was added to the SuperdexTM 75 column The highest purification efficiency atthe gel filtration step, expressed in purity, increased from ~ 12 to ~ 30 times with performance ~

44%) Meanwhile, using strong anionic chromatography (Q Sepharose; Figure 3.6) in the next

step the purity increased to 35.8 times, but the amount of protein and total activity corresponding

to the purified efficiency decreased to nearly ½ (last performance was 28.9%; Thus, the final

purification step is required for basic research (requiring maximum purity), in the AltFAE enzyme study of Alt.tenuissima SP66 can be used immediately after gel filter chromatography to ensure

high recovery efficiency and save time and costs

Figure 3.6 Purification of feruloyl esterase enzyme from Alt.tenuissima SP66 (AltFAE) via Q

Sepharose® anion exchange chromatography (●) AltFAE activity for methyl ferulate substrate,

(─) absorption protein at λ = 280 nm

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Table 3.2 Purification of feruloyl esterase from Alt.tenuissima SP66 (AltFAE)

Physical and chemical properties of the AltFAE: The SDS-PAGE electrode after the final

purification step through the Q Sepharose® column shows a protein band that expresses FAE

activity (methyl ferulate) after staining with Colloidal Blue Staining Kit MW = 30.3 kDa (Figure 3.7).

Figure 3.7 SDS-PAGE of the AltFAE under denaturing conditions

Lane 1 - AltFAE after Q Sepharose elution; lane 2 - protein markers Optimal pH and temperature: The temperature and pH of the AltFAE are tested from 30-80°C

and pH 4.0-9.0 Relative activity decreases from 100% at pH 7.0 to 84% at pH 9.0 (Fig 3.8-A).

The enzyme was purified for 2 hours in culture, the remaining 97% at pH 6.0 and 89% at pH 8.0.Conversely, the reaction at pH 4.0 resulted in a loss of 57% activity after 2 hours The temperature

of 60°C causes the operation to lose more than 50% within 2 hours, when the temperature rises to70°C, the enzyme activity decreases to only 30% Thus, the optimum temperature and pH with thecorresponding purified enzyme are 42-45°C, pH 6-6.5

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Figure 3.8 Effect of temperature (A) and pH (B) on the AltFAE

Residual activities were determined at temperature intervals of 20–80°C and pH values of 4.0– 9.0 All experiments were performed in triplicates, standard deviation (SD) < 5% Thermal stability and pH of enzyme AltFAE: Enzymes are relatively stable at 25°C and 40°C

after 2 hours of incubation, followed by a reduction of more than 10% in 4 hours at 25°C and35% by incubation in 2 hours at 40ºC Enzyme activity decreases rapidly at 60ºC and loses most

of its activity after 1 hour of incubation at this temperature The enzyme was stable at neutral (pH5) but lost more than 75% of its activity during the 2 hours incubation under strong acid

Figure 3.9 Stability of the AltFAE at different temperatures (A) and pH-values (B) in

dependence of incubation time

(A): 25°C (diamond), 40°C (circle), 60°C (triangle) and (B): pH 10 (diamond), PH 8.0 (circle),

pH 7.0 (star), pH 5.0 (triangle) Peptide analysis results of AltFAE: The peptide sequence of the protein expresses FAE activity from Alt tenuissima SP66 (AltFAE) was successfully determined by ESI-MS mass

spectra (Figure 3.10 & Table 3.3) as the basis for initial classification and could use data for primer determination of the coding gene for this protein.

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Figure 3.10 ESI-MS/MS spectrum of peptide fragments of purified enzyme protein

from Alt.tenuissima SP66 (AltFAE)

Table 3.3 Peptides of purified enzyme protein from Alt.tenuissima SP66 (AltFAE) determined by

hydrolysis with trypsin and LC-ESI-MS/MS

* Symbols for amino acids in accordance with IUPAC (International Union of Pure and Applied Chemistry)

3.5.3 Fermentation, extraction and purification of esterase from fungi

The procedure of fermentation for biosynthesis and purification of esterase consists of thefollowing main steps:

Cultivation of fungi and enzyme biosynthesis on the medium (5 L/batch): Xylaria polymorpha A35 strain is fermented 5 liters/batch on a basic medium (for 1 liter) with the

following composition: MgSO4: 0.5 g; KH2 PO4: 1.5 g; High yeast: 2.0 g; Micro-trace elements(trace): 1 mL Base medium supplemented with straw substrate, nitrogen source is pepton,temperature 25ºC, pH 7 under culture conditions of 200 rpm for 10 days Simultaneously withliquid fermentation, the fungus was fermented using 2-3 kg of dry straw, soaked in waterovernight and then placed in heat-resistant plastic bags and sterilized at 121ºC for 30 minutes Use2-3 petri of peptic algae-malt agar with homogenized mycelium Next, transfer the whole broth toeach plastic bag with a straw substrate, and the entire culture is carried out under sterileconditions The mycelium was incubated at 23ºC for 10-14 days, then the surface fermentationmedium was extracted with distilled water (d.H2O) overnight on a shaker

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