CN102559511A - Hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of hypocrea - Google Patents

Hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of hypocrea Download PDF

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CN102559511A
CN102559511A CN2011104171049A CN201110417104A CN102559511A CN 102559511 A CN102559511 A CN 102559511A CN 2011104171049 A CN2011104171049 A CN 2011104171049A CN 201110417104 A CN201110417104 A CN 201110417104A CN 102559511 A CN102559511 A CN 102559511A
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beta
glucosidase
hypocrea
ethanol
enzyme activity
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梁翠谊
许敬亮
袁振宏
庄新姝
徐惠娟
张宇
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Guangzhou Institute of Energy Conversion of CAS
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Abstract

The invention discloses hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of the hypocrea. The hypocrea is conserved in China General Microbiological Culture Collection Center (CGMCC) on September 1st, 2011, and the conservation No. is CGMCC No. 5209. The hypocrea can produce novel beta-glucosidase, wherein the enzyme activity of the beta-glucosidase is up to 482.1U/ml, the pH value of the optimum reaction is 4.8, the optimum reaction temperature is 70 DEG C, and the enzyme activity is high in stability at the temperature of 50 DEG C, so the beta-glucosidase is suitable for pyrohydrolysis; ethanol of which the concentration is 10 percent has the maximum promotion effect on the enzyme activity, and the enzyme activity of the beta-glucosidase is improved by nearly 1 time; and the ethanol of which the concentration is less than 30 percent in a reaction system does not inhibit the enzyme activity, and when biomass raw materials are hydrolyzed, the inhibition of cellobiose can be eliminated effectively, so that the yield of the ethanol is improved, and the inhibition of terminal products is eliminated. The production cost of cellulosic ethanol can be reduced, and the industrialized progress of the cellulosic ethanol is accelerated.

Description

High yield is had a liking for the meat seat bacterium and the application thereof of the warm beta-glucosidase of anti-ethanol
Technical field
The invention belongs to microbial fermentation and enzyme engineering Application Areas thereof; Be specifically related to a kind of high yield and have a liking for meat seat bacterium (Hypocrea sp.) W63 of the warm beta-glucosidase of anti-ethanol and the beta-glucosidase that is produced, and the application of this beta-glucosidase on simultaneous saccharification and fermentation.
Background technology
Beta-glucosidase, English name β-glucosidase is called for short BG, is the glycosidic link between a kind of ability catalytic hydrolysis aryl or alkyl and the glycosyl atomic group, thereby discharges the enzyme of glucose.Beta-glucosidase is extensive in distributed in nature, almost in all organisms, has, and is different because of its source difference character, has the important physical function aspect the sugar metabolism of the mankind's glycogen degraded and animal, plant, mikrobe.
In the cellulosic process of cellulase hydrolysis, cellulose substances becomes the glucose synergy of three kinds of enzymes of needs at least, endoglucanase, VISOSE excision enzyme and beta-glucosidase through enzymatic action.NCE5 becomes cellobiose to cellulose degradation with exocellobiohydrolase, and it is resolved into glucose by beta-glucosidase again.It is a crucial rate-limiting step that beta-glucosidase hydrolysis fiber disaccharides discharges glucose.At present; The beta-glucosidase of using on the market is in hydrolytic process; Activity of beta-glucosidase is subject to the inhibition of hydrolysate glucose, and cellobiose is the suppressor factor of other enzymes in the cellulase system, thereby can cause whole cellulosic hydrolytic process to be suppressed; Secondly, in the bacterial strain of cellulase-producing, beta-glucosidase content all very low (except that black mold) does not far reach the level of practical application; Once more, in a large amount of reaction process, the thermal treatment meeting makes most beta-glucosidase enzymic activity forfeiture.In general, about 40 ℃, the optimum temperature of the beta-glucosidase in most of microbe source is about 50 ℃ from the beta-glucosidase optimum temperuture of plant.At present, a critical bottleneck of restriction cellulosic ethanol industrialization is exactly cellulase use cost is higher.Wherein lacking effective commercialization beta-glucosidase is a key factor.Therefore, exploitation is sought and is had a liking for temperature, anti-alcohol, highly active beta-glucosidase for the industrialization process of accelerating cellulosic ethanol, and expection will have very big promoter action.
Except the application in cellulose degradation, beta-glucosidase also has multiple use.In the fruit juice industry, people utilize beta-glucosidase the effect of glycosidic link to be increased the fragrance of fruit juice; In food service industry, people utilize it that some are given birth to the cyanogen mass degradation to remove the toxicity of raw materials of food processing.Therefore, through the efficient bacterium producing multi enzyme preparation of high flux screening technology screening, use significant for the variation of exploring beta-glucosidase.
Summary of the invention
First purpose of the present invention provides meat seat bacterium (Hypocrea sp.) W63 that a kind of high yield is had a liking for the warm beta-glucosidase of anti-ethanol; This bacterium is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 1st, 2011; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, its deposit number is: CGMCC No.5209.
Meat seat bacterium of the present invention (Hypocrea sp.) W63 is that screening and culturing is come out from materials such as deadwood and rotten leaf, soil, fungus sporophore.Its classification of fungi is learned and shown: this bacterium mycelium is white in color, and bacterium colony is little and fine and close, is flocculence, can produce green spores.The PDA culture medium culturing began long white hypha in 1 day, cultivated to begin long green spores more than 3 days.This bacterium mycelium and spore are as shown in Figure 1.
The taxonomy status of meat seat bacterium of the present invention (Hypocrea sp.) W63 is confirmed according to following method:
Ordinary method is extracted the 18s rDNA of this fungi; Its sequence is shown in SEQ ID NO.1, and this sequence has been submitted to GenBank, and its sequence registration number is JN935058; Known array in this sequence and the GenBank DB is carried out the BLAST comparative analysis; And obtaining the 18S rDNA sequence of relevant kind from DB, constructing system is grown tree, and is as shown in Figure 2.Through comparative analysis; Meat seat bacterium of the present invention (Hypocrea sp.) W63 belongs to meat seat Pseudomonas; With its called after meat seat bacterium (Hypocrea sp.) W63, this bacterium was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on September 1st, 2011; Institute of Microorganism, Academia Sinica, its deposit number is: CGMCC No.5209.
It is fermentation strain with meat seat bacterium (Hypocrea sp.) W63 that second purpose of the present invention provided a kind of, the beta-glucosidase that makes through liquid fermenting.
Assaying reaction according to beta-glucosidase specific substrate 4-nitrophenyl β-D-glucopyranoside (pNPG); Find meat seat bacterium Hypocrea sp.W63 ability high-yield beta-glucosidase of the present invention; The highest enzyme work of the liquid fermenting of this enzyme is up to 482.1U/mL (amount that catalysis produces 1 μ mol/L p-nitrophenol in 1min is defined as a unit of enzyme); Optimal reaction pH value is 4.8, and optimal reactive temperature is 65 ℃, and the glucose feedback inhibition is obvious; Alcohol concn is that 10% pair of enzyme work has maximum promoter action, the beta-glucosidase enzyme is lived improve 1 times nearly, and the ethanol tolerance is up to 30%.The beta-glucosidase of having reported at present; Enzyme is lived and generally is no more than 300U; Its optimal reactive temperature is generally at 50 ℃; Can work have promoter action to the beta-glucosidase enzyme below 8% at alcohol concn, but, will give birth to restraining effect to the enzyme life birth of beta-glucosidase along with the increase of alcohol concn.Therefore, the beta-glucosidase that meat seat bacterium Hypocrea sp.W63 of the present invention is produced has tangible difference with beta-glucosidase in the existing report, is a kind of novel have a liking for temperature, beta-glucosidase of anti-the alcoholic acid.
The 3rd purpose of the present invention provides the application of meat seat bacterium of the present invention (Hypocrea sp.) W63 on the preparation beta-glucosidase.
The beta-glucosidase of meat seat bacterium of the present invention (Hypocrea sp.) W63 preparation can be applied in the production of cellulosic ethanol.The stalk gas explosion is pulverized, add yeast, cellulase and beta-glucoside zymin then and ferment, help the Mierocrystalline cellulose simultaneous saccharification and fermentation and use, can effectively eliminate cellobiose and suppress, improve ethanol production, eliminate end product and suppress.
The 4th purpose of the present invention provides the application of beta-glucosidase on the lignocellulosic material simultaneous saccharification and fermentation that meat seat bacterium of the present invention (Hypocrea sp.) W63 produces.
Meat seat bacterium (Hypocrea sp.) W63 of the present invention's screening; This bacterium can produce a kind of new beta-glucosidase; The enzyme work of this enzyme is up to 482.1U/mL (amount that catalysis produces 1 μ mol/L p-nitrophenol in 1min is defined as a unit of enzyme U), and optimal reaction pH value is 4.8, and optimal reactive temperature is 70 ℃; Enzyme work can show high stability in the time of 50 ℃, is suitable for pyrohydrolysis; Alcohol concn is that 10% pair of enzyme work has maximum promoter action; Improve 1 times nearly to the beta-glucosidase enzyme is alive; Alcohol concn 30% can not cause inhibition to enzyme work with interior in the reaction system, and when the hydrolyzing biomass raw material, can effectively eliminate cellobiose and suppress; Improve ethanol production, eliminate end product and suppress.Expection can reduce production costs of cellulosic ethanol, accelerates its industrialization process.
Meat seat bacterium of the present invention (Hypocrea sp.) W63 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 1st, 2011; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, its deposit number is: CGMCC No.5209.
Description of drawings
Fig. 1 is the electron-microscope scanning figure of meat seat bacterium of the present invention (Hypocrea sp.) W63, and wherein, the magnification of A is 7500, and the magnification of B is 12500, and the magnification of C is 15000, and the magnification of D is 30000;
Fig. 2 is a 18S rDNA phylogenetic tree of using meat seat bacterium of the present invention (Hypocrea sp.) W63;
Fig. 3 is the pH value and the pH stability of the beta-glucosidase of meat seat bacterium of the present invention (Hypocrea sp.) W63 product;
Fig. 4 is the influence of temperature to the activity of beta-glucosidase of meat seat bacterium of the present invention (Hypocrea sp.) W63 product;
Fig. 5 is that the beta-glucosidase that produces of meat seat bacterium of the present invention (Hypocrea sp.) W63 is to the alcoholic acid tolerance;
Fig. 6 adds the influence of the beta-glucosidase of meat seat bacterium of the present invention (Hypocrea sp.) W63 product to the simultaneous saccharification and fermentation effect, and wherein solid-line curve is represented total sugar content, and hollow curve is represented alcohol concn.
Embodiment:
Following examples is to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1: the screening of bacterial strain
Materials such as sampling deadwood and rotten leaf, soil, fungus sporophore.Directly place Congo red primary dcreening operation substratum, the prescription of this substratum is: contain KH in every liter of substratum 2PO 40.5g, (NH 4) 2SO 42.0g, MgSO 47H 2O 0.25g, cellulose powder (microcrystalline cellulose) 2.0g, Congo red 0.2g, agar 18～20g, surplus is a water, natural pH value.Cultivate 2～5d for 30 ℃, single bacterium colony purifying on the PDA substratum that picking hydrolysis transparent circle is bigger, the bacterial strain of purifying carry out the PDA inclined-plane and preserve.
Bacterial strain produces the beta-glucoside enzyme activity and sieves again: the purifying bacterial strain that the PDA inclined-plane is preserved is inoculated into respectively in the multiple sieve fermention medium of equivalent, and 30 ℃, 120rpm cultivates 48h.Frozen centrifugation is got supernatant, is crude enzyme liquid, measures the beta-glucoside enzyme activity of the crude enzyme liquid of each bacterial strain product respectively with standard pNPG method.Concrete grammar is: draw the crude enzyme liquid of 0.1mL appropriateness dilution with liquid-transfering gun, join the 0.2mol/LNa of 0.9mL pH5.0 2HPO 4In-0.1mol/L the citrate buffer solution,, add the pNPG solution of the 1mL 5mmol/L of preheating 5～10min more rapidly,, add the Na of 3mL 1mmol/L immediately with the accurate clock reaction 10min of stopwatch in 50 ℃ of water bath with thermostatic control 5min 2CO 3The solution termination reaction, room temperature is placed 5min, in 400nm place photometry absorption value (OD).Select the higher bacterial strain of beta-glucoside enzyme activity to use the PDA inclined-plane to preserve then.The described fermention medium that sieves again is that every liter of substratum contains KH 2PO 42g, (NH 4) 2SO 42.5g, MgSO 47H 2O 0.3g, CaCl 20.3g, rice straw 50g, surplus is a water.
Obtain the highest bacterial strain of 1 strain beta-glucoside enzyme activity through screening.Adopt modified CTAB method to extract the total DNA of this bacterial strain, the universal primer NS1 of selection amplification fungi 18S rDNA sequence (5 '-GTATCATATGCTTGTCTC-3 ') and NS8 (5 '-TCCGCAGGTTCACCTACGGA-3 ') the total DNA of meat seat bacterium (Hypocrea sp.) W63 is carried out the amplification of 18SrDNA sequence.In the PCR reaction system of 20 μ L, contain 10 * Buffer, 2 μ L and (contain MgCl 2, 2.5mmol/L), dNTP (10mmol/L) 0.4 μ L, primer (NS1 and NS8) amount respectively is 20pmol, rTag (5U/ μ L) 0.2 μ L, the template DNA of about 50ng, all the other volumes are supplied with aseptic ultrapure water.The pcr amplification condition is: 95 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 1min, 52 ℃ of annealing 50s, 72 ℃ are extended 50s, 35 circulations, 72 ℃ are extended 10min.Pcr amplification product adopts dna gel to reclaim the test kit recovery of tapping rubber.Purified product is connected to pMD18-T-Vecter, connects product and is transformed into Escherichia coli JM109 competent cell, carries out the screening of ampicillin selection and blue hickie.Picking mono-clonal bacterium liquid checks order.Through order-checking; Its sequence is shown in SEQ ID NO.1, and this sequence has been submitted to GenBank, and its sequence registration number is JN935058; Known array in this sequence and the GenBank DB is carried out the BLAST comparative analysis; And obtaining the 18S rDNA sequence of relevant kind from DB, constructing system is grown tree, and is as shown in Figure 2.Through comparative analysis; This Pseudomonas is in meat seat Pseudomonas; With its called after meat seat bacterium (Hypocrea sp.) W63, this bacterium was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on September 1st, 2011; Institute of Microorganism, Academia Sinica, its deposit number is: CGMCC No.5209.
Embodiment 2: the preparation of beta-glucoside zymin and enzyme activity thereof, zymologic property are measured
1. the preparation of beta-glucoside zymin: with meat seat bacterium (Hypocrea sp.) W63 activation, through seed culture, seed culture medium is that every liter of substratum contains potato and soaks juice 200g, glucose 20g, and surplus is a water, 115 ℃ of sterilization 30min.30 ℃, 120rpm cultivated 3 days, obtained seed culture fluid.The inoculum size of seed culture fluid with 5% (v/v) is inoculated in the fermention medium, and the prescription of this fermention medium is that every liter of substratum contains KH 2PO 42g, (NH 4) 2SO 42.5g, MgSO 47H 2O 0.3g, CaCl 20.3g, rice straw 50g, liquid microelement 200 μ L (CuSO 40.1g, MnSO 40.1g, FeSO 47H 2O 0.1g, CoCl 20.1g, be settled to 100mL, water 100mL).30 ℃, 120rpm, fermentation 5d collects fermented liquid then, and 4 ℃ of frozen centrifugation 5min of 8000rpm get supernatant and are the beta-glucoside zymin, and 4 ℃ of preservations are subsequent use.
2. the enzyme activity determination of beta-glucosidase: as substrate, reaction system is 2mL with 4-nitrophenyl β-D-glucopyranoside (pNPG), earlier with 1mL pNPG (5mmol/L) and 0.9mL pH 5.0Na 2HPO 4-citrate buffer solution mixing adds the beta-glucoside zymin that the suitable last step of diluting of 0.1mL obtains again, and 50 ℃ are reacted 10min, add the Na of 3mL1mol/L immediately 2CO 3The solution termination reaction, room temperature is placed 5min, in 400nm place photometry absorption value (OD).Through measuring, the vigor of the beta-glucoside zymin that meat seat bacterium (Hypocrea sp.) W63 that obtains through step 1 produces reaches 482.1U/mL, has higher enzyme activity.
The enzyme definition of living: the amount that catalysis produces 1 μ mol/L p-nitrophenol in 1min is defined as a unit of enzyme.
3. the mensuration of the zymologic property of beta-glucosidase: beta-glucoside zymin; Press the pNPG measuring method; Under the constant situation of other conditions; Regulating different pH damping fluids, differing temps, different ethanol concentration and carry out the enzyme activity determination reaction, is 100% to record the highest enzyme work, and this condition is for recording the beta-glucosidase optimum reaction conditions; Stability study: regulate under different pH damping fluids, differing temps, the different ethanol concentration condition, zymin is carried out enzyme activity determination after preserving 1h, is 100% to record the highest enzyme work, is pH, the temperature stability result who records beta-glucosidase.
The result shows that the optimum response pH of the beta-glucosidase that meat seat bacterium of the present invention (Hypocrea sp.) W63 produces is 4.8, and under the condition of pH4.8, keeps enzyme to live stable (as shown in Figure 3), and optimal reactive temperature is 65 ℃ (as shown in Figure 4); Anti-alcohol concn is up to 30%, and wherein alcohol concn is that 10% pair of enzyme activity has significantly facilitation effect (as shown in Figure 5).The beta-glucosidase of having reported at present; Enzyme is lived and generally is no more than 300U; Its optimal reactive temperature is generally at 50 ℃; Can work have promoter action to the beta-glucosidase enzyme below 8% at alcohol concn, but, will give birth to restraining effect to the enzyme life birth of beta-glucosidase along with the increase of alcohol concn.Therefore, the beta-glucosidase that meat seat bacterium Hypocrea sp.W63 of the present invention is produced has tangible difference with beta-glucosidase in the existing report, is a kind of novel have a liking for temperature, beta-glucosidase of anti-the alcoholic acid.This beta-glucosidase has stronger have a liking for temperature, heatproof, anti-alcohol performance, during the simultaneous saccharification and fermentation that especially is fit to be applied to lignocellulosic material is used.
Embodiment 3: beta-glucosidase is applied to simultaneous saccharification and fermentation
1. substrate: the gas explosion straw is crushed to 60 orders, dries to constant weight for 105 ℃.
2. yeast is cultivated the 24h activation for 30 ℃ with the YPD liquid nutrient medium.
3. add cellulase 30FPU/g substrates enzymes amount, 50 ℃ of prehydrolysis 24h to reaction system.
4. the beta-glucoside zymin 30FPU/g substrate that adds embodiment 2 again is in reaction system, and to reaction system, 0h is counted in 37 ℃ of fermentations during with inoculation yeast by the yeast after the inoculum size inoculation activation of 5% (v/v); In 0,4,8; 12,24,48; 96, the 120h spot sampling, HPLC detects ethanol, reducing sugar content.
Above-mentioned reaction system is: 500mL shakes reaction cartridge liquid 200mL in the bottle, contains gas explosion stalk 20g, inorganic salt composition ((NH in this reaction solution 4) 2HPO 40.5g/L, MgSO 47H 2O 0.025g/L), yeast extract paste 1.0g/L, surplus is the Na of pH 4.8 2HPO 4-citrate buffer solution.
Not add beta-glucoside zymin of the present invention as negative control; With the beta-glucosidase (available from sigma company) that adds commercial prodn as positive control, research meat seat bacterium Hypocrea sp.W63 beta-glucosidase that bacterial strain produces is applied to the effect of simultaneous saccharification and fermentation.
The result is as shown in Figure 6; Screening gained meat seat bacterium Hypocrea sp.W63 bacterial strain produces beta-glucosidase and is applied in the simultaneous saccharification and fermentation, ferments to 120h to get the ethanol production peak, and institute's producing and ethanol content is up to 41.25g/L; Compare with negative, positive control, ethanol production improves nearly 2 times.This shows that the beta-glucoside enzymatic property that meat seat bacterium Hypocrea sp.W63 bacterial strain of the present invention is produced all helps the application of Mierocrystalline cellulose simultaneous saccharification and fermentation, can effectively eliminate end product and suppress.And the beta-glucosidase enzyme activity that this bacterial strain produced is higher, is applied to the simultaneous saccharification and fermentation process and has tangible facilitation effect, has vast potential for future development for the industrialization process that promotes cellulosic ethanol.
Above-mentioned detailed description is to the specifying of possible embodiments of the present invention, and this embodiment is not in order to limiting claim of the present invention, does not allly break away from equivalence of the present invention and implements or change, all should be contained in the claim of the present invention.
Figure IDA0000118975920000011
Figure IDA0000118975920000021

Claims (4)

1. a meat seat bacterium (Hypocrea sp.) W63, its preserving number is: CGMCC No.5209.
2. a beta-glucosidase is characterized in that, is to be fermentation strain with the described meat seat of claim 1 bacterium (Hypocrea sp.) W63, makes through liquid state fermentation.
3. the described meat seat of claim 1 bacterium (Hypocrea sp.) W63 is in the application of producing on the beta-glucosidase.
4. the application of the described beta-glucosidase of claim 2 on the lignocellulosic material simultaneous saccharification and fermentation.
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CN104046605A (en) * 2014-05-29 2014-09-17 中国科学院广州能源研究所 Mesophile ethanol-tolerant beta-glucosidase, and coding gene and application thereof
CN105037479A (en) * 2015-06-10 2015-11-11 中国科学院微生物研究所 Antimicrobial compound based on fungus transformation, and preparation method and application thereof

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CN101705213A (en) * 2009-11-16 2010-05-12 浙江大学 Preparation method of beta-glucosaccharase
CN101792717A (en) * 2009-12-30 2010-08-04 吉林大学 Thermophilic acid-resisting beta-glucosaccharase producing strain and preparation method thereof
CN102080050A (en) * 2010-12-07 2011-06-01 中国科学院广州能源研究所 Trichoderma viride W2 capable of producing thermophilic ethanol-resistant beta-glucosidase and application thereof

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CN101705213A (en) * 2009-11-16 2010-05-12 浙江大学 Preparation method of beta-glucosaccharase
CN101792717A (en) * 2009-12-30 2010-08-04 吉林大学 Thermophilic acid-resisting beta-glucosaccharase producing strain and preparation method thereof
CN102080050A (en) * 2010-12-07 2011-06-01 中国科学院广州能源研究所 Trichoderma viride W2 capable of producing thermophilic ethanol-resistant beta-glucosidase and application thereof

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Publication number Priority date Publication date Assignee Title
CN104046605A (en) * 2014-05-29 2014-09-17 中国科学院广州能源研究所 Mesophile ethanol-tolerant beta-glucosidase, and coding gene and application thereof
US9890371B2 (en) * 2014-05-29 2018-02-13 Guangzhou Institute Of Energy Conversion, Chinese Academy Of Sciences Thermophilic ethanol-resistant β-glucosidase and encoding gene and application thereof
CN105037479A (en) * 2015-06-10 2015-11-11 中国科学院微生物研究所 Antimicrobial compound based on fungus transformation, and preparation method and application thereof

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