CN102876590A - Penicillium sp. mutant strain and application of penicillium sp. mutant strain to cellulase preparation - Google Patents

Penicillium sp. mutant strain and application of penicillium sp. mutant strain to cellulase preparation Download PDF

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CN102876590A
CN102876590A CN2012103952271A CN201210395227A CN102876590A CN 102876590 A CN102876590 A CN 102876590A CN 2012103952271 A CN2012103952271 A CN 2012103952271A CN 201210395227 A CN201210395227 A CN 201210395227A CN 102876590 A CN102876590 A CN 102876590A
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cellulase
mutant strain
preparation
penicillium
penicillium oxalicum
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CN102876590B (en
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冯家勋
农清栋
刘君梁
秦秀林
段承杰
张政
黄妹平
蓝健益
卢业飞
吕芳贤
张颖
黄叶萍
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Guangxi Ding Biotechnology Co., Ltd.
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Guangxi University
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Abstract

The invention discloses a penicillium sp. mutant strain and application of the penicillium sp. mutant strain to cellulase preparation. The invention provides a strain of penicillium oxalicum EU2106 with the preservation number being CGMCC (China General Microbiological Center Culture Collection Center) No.6471. The invention also provides a method for producing cellulase or a cellulase preparation. The method comprises the following steps that the penicillium sp. mutant strain EU2106 is cultured for a certain time on a basic or optimized liquid culture medium, culture substances are collected, thalli are removed through centrifugation, and then, obtained supernate can be used as the cellulase preparation. Experiments prove that the penicillium oxalicum EU2106 obtained through the mutation can be used for producing the cellulase preparation, the preparation can be used for effectively hydrolyzing sugarcane slag paper pulp, and in addition, glucose is major ingredients in the final hydrolysis products. The cellulase preparation can be used for simultaneous saccharification and fermentation of sugarcane slag paper pulp for alcohol production.

Description

One strain mould mutant strain and the application in the preparation cellulase thereof
Technical field
The present invention relates to biological technical field, relate in particular to a strain mould mutant strain and the application in the preparation cellulase thereof.
Background technology
Lignocellulose is the renewable organic resource the widest and most abundant that distributes on the earth, and at present, the lignocellulose resource fails to be effectively utilized.Alcohol fuel refers to that dehydrated alcohol is used for a kind of New-type fuel of engine after denaturing agent is processed, and its raw materials for production are common mainly contains W-Gum, tapioca (flour) and sugar cane juice etc.These raw materials are converted into ethanol under the effect of enzyme or microorganism.Utilize microorganism that cellulose conversion is become again producing fuel ethyl alcohol by ferment of glucose, strive grain with the people, strive the problem such as ground with grain and have huge meaning solving take what grain or glucide caused as the raw material production alcohol fuel.
China is since calendar year 2001 comes into effect the alcohol fuel plan, become the third-largest fuel ethanol production state that is only second in the world Brazil and the U.S. (Song Yuanquan is permitted Yun treasure, Liu Dehua. global biofuel general situation of development. the biological industry technology, 2009,5:34-42).The company of granted production alcohol fuel has 5, it is respectively biomass energy company limited of Guangxi China Oil and Food Import and Export Corporation, Jilin Fuel Ethanol Co.,Ltd, sky, Henan hat alcohol fuel company, Fengyuan Biological Chemistry Co., Ltd., Anhui Prov. and Heilongjiang China Resources Alcohol Co., Ltd (make widely known strong, to Weida, Zhou Tao, Deng. China's alcohol fuel current situation and trend analysis. China energy, 2009,31:31-34), above company year produce the alcohol fuel ability and amount to 1,020,000 tons of (Liu Li, Sun Junshe, Kang Liping, etc. Fuel Ethanol Production from Sweet Sorghum Stalk. chemical progress, 2007,19:1109-1115).In addition, COFCO Biochemical Energy (Zhaodong) Co., Ltd. and Chinese Academy Of Sciences Process Engineering Research Institute, adopt and introduce corresponding stalk product fuel alcohol plant, the rapidly industrialized development of propelling cellulose fuel ethanol (Qu Yinbo. lignocellulolytic enzymes and biorefinery. Chemical Industry Press, 2011).Chinese Government promulgated in " planning of renewable energy source Long-and Medium-term Development " in 2007 and points out, greatly develop the raw materials for production take non-grain material as alcohol fuel, the year utilization that expects the year two thousand twenty biofuel ethanol be 1,000 ten thousand tons (Li Yanjun. world fuel ethanol new development and to the enlightenment of China. international economic cooperation, 2008,2:28-34).
Abroad, at present, in the world existing 20 countries nearly promote the use of ethanol petrol (Yue monarch, military National Day, Hao Xiaoming. China's fuel ethanol production Classification and prospect. chemical progress, 2007,19:1084-1089), main country is the U.S. and Brazil.Since 20 century 70s, under the promotion of twice oil crisis, the U.S. and Brazil take W-Gum and sugarcane as raw material, produce alcohol fuel respectively in a large number.Other country has also carried out the production of alcohol fuel in succession.Some tropic countries such as India, European countries such as France etc. all utilize respectively sugarcane and molasses as raw material production alcohol fuel (Cardonaalzate C, Sancheztoro O.Energy consumption analysis of integrated flowsheets for production of fuel ethanol from lignocellulosic biomass.Energy, 2006,31:2447-2459).Canadian Ai Ouji (Iogen) company, Hispanic Abengoa biomass energy company etc. have all built up the biomass energy demonstration plant, the former is first cellulosic ethanol industrialization (Sun Zhimou of company, Jiang Lei, Zhang Junbo, Deng. the process of industrialization of countries in the world Bioconversion of Lignocellulosic Materials for Fuel Ethanol. brewing science and technology, 2007,1:91-94), produce about 2 * 106L alcohol fuel per year, the latter produce per year to surpass 5 * 106L alcohol fuel (Qu Yinbo. lignocellulolytic enzymes and biorefinery. Chemical Industry Press, 2011).In addition, Sweden, France, Japan and other countries also actively utilize different cellulosic materials to produce alcohol fuel.
Polysaccharose substance in the lignocellulose such as Mierocrystalline cellulose and hemicellulose are not easy to be converted to and are the monose molecule, Mierocrystalline cellulose is before quilt acid or enzymolysis processing change into fermentable sugar, must carry out pre-treatment to lignocellulose, pretreated purpose is to remove xylogen and hemicellulose, reduce cellulosic degree of crystallinity, but improve cellulosic contact (Fred AK, Jenny EH, Quang AN.Microbial pretreatment of biomass.Applied Biochemistry and Biotechnology, 105-108:127-141).At present, the cellulose materials pretreatment process has: acid treatment (Chandel AK, Kapoor K, Singh A, et al.Detoxification of sugarcane bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501.Bioresource Technology, 2007,98:1947-1950), alkaline purification (Hernandez-Salas JM, Villa-Ramirez MS, Veloz-Rendon JS, et al.Comparative hydrolysis and fermentation of sugarcane and agave bagasse.Bioresource Technology, 2009,100:1238-1245), steam explosion (steam explosion) is processed, biological treatment (LiX, Ryuichiro K, Kokki S.Biodegradation of sugarcane bagasse with marine fungus Phlebia sp.MG-60.Journal Wood Sci, 2002,48:159-162) and wet oxidation process, ammonia treatment etc.
Mierocrystalline cellulose under the effect of cellulase by saccharification, undergo microbial fermentation again and produce ethanol or other fermentation end product, generally to comprise following four step biocatalytic reaction processes (Qu Yinbo. Industrialization of Cellulosic Ethanol. chemical progress, 2007,19:1098-1108): the production of cellulase, lignocellulose are hydrolyzed into monose, zymohexose and pentose fermentation, finally produce the products such as product such as ethanol.According to these reactors with in various degree the combination, can be divided into different fermentative production flow processs, comprise: fractional hydrolysis fermentation (separate hydrolysis and fermentation, SHF), simultaneous saccharification and fermentation (simultaneous saccharification and fermentation, SSF), the situ production of cellulase (on-site production) and synchronous saccharification (the simultaneous saccharification and co-fermentation that ferments altogether, SSCF) and the comprehensive organism course of processing (consolidated bioprocessing, CBP) etc.
Cellulase is the general name of class polycomponent enzyme system, and their synergies are glucose with cellulose degradation.Cellulase can be divided into 3 large classes by its catalysis: endoglucanase (endo-1,4-β-D-glucanase, E.C3.2.1.4), exoglucanase (exo-1,4-β-D-glucanase) or cellobiohydrolase (cellobiohydrolase) (E.C 3.2.1.91) and beta-glucosidase (β-glucosidase, E.C3.2.1.21) (Zhang PYH, Himmel ME, Mielenz JR.Outlook for cellulase improvement:screening and selection strategies.Biotechnol Advan, 2006,24:452-481).Endoglucanase is the β-1 of the inner noncrystalline domain of cutting fibre element at random, 4 glucoside bonds, staple fiber element long-chain also is translated into the Mierocrystalline cellulose short chain that the polymerization degree reduces, produce small molecules Mierocrystalline cellulose (the Sanchez C.Lignocellulosic residues:biodegradation and bioconversion by fungi.Biotechnol Adv of reduction end or non-reduced end, 2009,27:185-194).The cellulolytic crystallizing field of exoglucanase is attacked Mierocrystalline cellulose reduction end or non-reduced end, discharges cellobiose; Beta-glucosidase hydrolysis fiber disaccharides and cell-oligosaccharide, discharge glucose (Kumar R, Singh S, Singh OV.Bioconversion of lignocellulosic biomass:biochemical and molecular perspectives.J Ind Microbiol Biotechnol, 2008,35:377-391).Yeast or bacterium can utilize glucose as substrate, its fermentation is produced ethanol or other products (Duff SJB, Murray WD.Bioconversion of forest products industry waste cellulosics to fuel ethanol:a review.Bioresour Technol, 1996,55:51-53).
Occurring in nature, the microorganism of degraded cellulose is extremely extensively various, comprise bacterium and fungi (Lynd LR, Weimer PJ, van Zyl WH, et al.Microbial cellulose utilization:fundamentals and biotechnology.Microbiol Mol Biol Rev, 2002,66:506 – 577; Gilbert HJ, Hazelwood GP.Bacterial cellulases and xylanases.Journal of General Microbiology, 1993,139:187-194).Industrial, filamentous fungus is the best producer of cellulase, and the cellulase major part of its generation is extracellular enzyme.Such as: Trichoderma (Trichoderma sp.), Aspergillus (Aspergillus sp.) and Penicillium (Penicillium sp.).At present, in the world many companies to carry out the commercial fibres element zymin of industrial-scale production be (the Gusakov AV.Alternatives to Trichoderma reesei in biofuel production.Trends Biotechnol that uses the mould mutant strain of Rui Shi wood to produce, 2011,29:419-425; Persson I, Tjerneld F,
Figure BDA00002266455200031
BH.Fungal cellulolytic enzyme production:An overview.Proc Biochem, 1991,26:65-74).But, have still that cellulase activity is low, the high problem of production cost.Therefore, be necessary to research and develop the bacterial strain of new High Cellulase Production.
Summary of the invention
First purpose of the present invention provides a strain penicillium oxalicum (Penicillium oxalicum) EU2106.
Penicillium oxalicum provided by the invention (Penicillium oxalicum) EU2106, its deposit number is CGMCC No.6471.
The application of above-mentioned penicillium oxalicum (Penicillium oxalicum) EU2106 in preparation cellulase or cellulase preparation also is the scope of protection of the invention.
Second purpose of the present invention provides the method for a kind of production of cellulose enzyme or cellulase preparation.
Method provided by the invention, above-mentioned penicillium oxalicum (Penicillium oxalicum) EU2106 that comprises the steps: to ferment collects tunning, namely obtains cellulase or cellulase preparation.
Above-mentioned cellulase has filter paper enzyme, CMC enzyme and/or beta-glucosidase enzyme and lives.
In the aforesaid method, the condition of described fermentation is to cultivate 5-7 days at 24-34 ℃, 200rpm; Be specially at 28 ℃, 200rpm and cultivated 6 days.
In the aforesaid method, the component of the fermention medium that described fermentation is adopted is as follows: the described fermention medium of every L is by 4gKH 2PO 4, 4g (NH 4) 2SO 4, 0.6g CaCl 2, 0.6g MgSO 4.7H 2O, 2mL Tween-80,5.0mg FeSO 47H 2O, 1.6mg MnSO 4H 2O, 1.4mg ZnSO 47H 2O, 2.0mg CoCl 2, 40g wheat bran, 10g Avicel and water forms, water is supplied volume;
The pH value of described fermention medium is pH3.5-5.5, and the pH value of described fermention medium is specially 5.5.
In the aforesaid method, described fermentation is for cultivating the spore inoculating of above-mentioned penicillium oxalicum (Penicillium oxalicum) EU2106 to described fermention medium;
The initial kind concentration of spore in fermentation system of described penicillium oxalicum (Penicillium oxalicum) EU2106 is specially 10 6Individual/mL.
In the aforesaid method, behind described collection tunning, also comprise the steps: described tunning centrifugal (the centrifugal 5min of 13,800xg), collect supernatant liquor, obtain cellulase or cellulase preparation.
Above-mentioned penicillium oxalicum (Penicillium oxalicum) EU2106, above-mentioned cellulase or the application of described cellulase preparation in preparation wood sugar, cellobiose and/or glucose also are the scope of protection of the invention; Described preparation wood sugar, cellobiose and/or glucose are for above-mentioned penicillium oxalicum (Penicillium oxalicum) EU2106, above-mentioned cellulase or described cellulase preparation hydrolysis begasse pulp production wood sugar, cellobiose and/or glucose being, concrete hydrolysising condition is: the pH value is 5.0, and temperature is 50 ℃.
Above-mentioned penicillium oxalicum (Penicillium oxalicum) EU2106, above-mentioned cellulase or the application of described cellulase preparation in simultaneous saccharification and fermentation production alcohol also are the scope of protection of the invention; Above-mentioned simultaneous saccharification and fermentation produce alcohol be with in above-mentioned cellulase or the described cellulase preparation adding begasse pulp as fermentation raw material, fermented yeast, thus obtain alcohol (ethanol).
The 3rd purpose of the present invention provides a kind of fermention medium.
Fermention medium provided by the invention, its component is as follows: the described fermention medium of every L is by 4g KH 2PO 4, 4g (NH 4) 2SO 4, 0.6g CaCl 2, 0.6g MgSO 4.7H 2O, 2mL Tween-80,5.0mg FeSO 47H 2O, 1.6mg MnSO 4H 2O, 1.4mg ZnSO 47H 2O, 2.0mg CoCl 2, 40g wheat bran, 10g Avicel and water forms, water is supplied volume.
Above-mentioned mould mutant strain EU2106 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 08 22nd, 2012, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6471, and the Classification And Nomenclature of suggestion is penicillium oxalicum Penicillium oxalicum.
Of the present invention experimental results show that, the present invention has found a strain penicillium oxalicum Penicillium oxalicum through sudden change, it is carried out fermentation culture, fermented liquid is produced wood sugar, cellobiose and/or glucose take begasse pulp as substrate, filter paper enzyme, CMC enzyme and/or beta-glucosidase enzyme with cellulase are lived; And this fermented liquid can also promote the saccharomycetes to make fermentation begasse pulp to produce alcohol, has application potential in the Conversion with the use to bagasse.
Description of drawings
Fig. 1 is the form of mould mutant strain EU2106 on the PDA flat board.
Fig. 2 is the conidium form of mould mutant strain EU2106 under opticmicroscope.
Fig. 3 is the mycelia form of mould mutant strain EU2106 under opticmicroscope.
Fig. 4 is the conidiophore form of mould mutant strain EU2106 under opticmicroscope.
Fig. 5 is total DNA and the ITS sequence amplification fragment thereof of the mould mutant strain EU2106 of extraction.
Fig. 6 is that different initial pH are to the influence curve of mould mutant strain EU2106 cellulase-producing.
Fig. 7 is that different culture temperature are to the influence curve of mould mutant strain EU2106 cellulase-producing.
Fig. 8 is that the wheat bran of different concns is to the influence curve of mould mutant strain EU2106 cellulase-producing.
Fig. 9 is the (NH of different concns 4) 2SO 4Influence curve to mould mutant strain EU2106 cellulase-producing.
Figure 10 is that the different vaccination amount is to the influence curve of mould mutant strain EU2106 cellulase-producing.
Figure 11 is that different incubation times are to the influence curve of mould mutant strain EU2106 cellulase-producing.
Figure 12 is that different pH are to the influence curve of the cellulase activity of mould mutant strain EU2106 generation.
Figure 13 is that differing temps is to the influence curve of the cellulase activity of mould mutant strain EU2106 generation.
Figure 14 is that the HPLC of the product of the cellulase hydrolysis begasse pulp that produces of mould mutant strain EU2106 analyzes collection of illustrative plates.
Figure 15 is the analysis of product of the begasse pulp of the liquid cellulase preparation hydrolysis different concns that produces of mould mutant strain EU2106.
The as a result figure of Figure 16 for the simultaneous saccharification and fermentation of begasse pulp being produced alcohol with liquid cellulase preparation and the Angel yeast saccharomyces cerevisiae of mould mutant strain EU2106 generation.
Figure 17 produces alcohol for liquid cellulase preparation and Wine brewing yeast strain ZM1-5 with mould mutant strain EU2106 generation to the simultaneous saccharification and fermentation of begasse pulp as a result figure.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
% among the following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
In embodiment 1 and embodiment 2, cellulase activity adopts 3 in measuring, 5-dinitrosalicylic acid (3,5-dinitrosalicylate, DNS) method is measured the reducing sugar (Miller that produces, GL.Use of dinitrosalicyclic acid reagent for determination of reducing sugar.Analytical Chemistry 1959,31:426-428), concrete steps are as follows:
Among the embodiment 1, when the separation screening mutant strain, adopt Congo red-Avicel screening solid medium is (g/L): KH 2PO 42g, (NH 4) 2SO 41.4g, CaCl 20.3g, MgSO 4.7H 2O 0.5g, urea 0.3g, Yeast extract0.8g, Peptone 2g, glucose 2g, Avicel PH-1013g, gelatin 2g, agar 20g, Congo red 0.2g, micro-0.05mL, 5.0,121 ℃ of sterilizations of pH 30min.
When mutant strain is carried out multiple sieve, adopt liquid screening substratum (g/L): KH 2PO 44g, (NH 4) 2SO 42.8g, CaCl 20.6g, urea 0.6g, MgSO 4.7H 2O 0.6g, wheat bran 20g, Avicel 30g, Tween-802mL, the component of micro-0.1mL(trace element and the final concentration in substratum are respectively 5.0mg/LFeSO 47H 2O, 1.6mg/L MnSO 4H 2O, 1.4mg/L ZnSO 47H 2O, 2.0mg/L CoCl 2), 5.0,121 ℃ of sterilizations of pH 30min.
Mutant strain is carried out liquid shaking bottle when sieving again, adopt the method for dull and stereotyped stripping and slicing, mycelia and spore (the related a small amount of substratum of mycelia and spore) are inoculated in the 250mL triangular flask that fills 50mL liquid screening substratum, 28 ℃, 200rpm shaking table shaking culture 5 days are collected crude enzyme liquid and are also measured it to the enzyme activity of filter paper.
When above-mentioned mutant strain sieves again, adopt following system of determination filter paper enzyme activity: the enzyme liquid 100 μ L that will suitably dilute join in Sodium phosphate dibasic-citrate buffer solution (pH 5.0) that 400 μ L contain 1%Whatman I powder filter paper, 50 ℃ of lower insulation reaction 30min, add 1mL DNS reagent, boiling water bath 5min colour developing, be cooled to room temperature, drawing 200 μ L clear liquors adds in the 96 hole enzyme plates, at the light absorption value of wavelength 540nm place working sample, calculate glucose concn in the reaction system according to the glucose concn typical curve of drawing.
In embodiment 2, adopt following methods to measure the enzyme activity of various cellulases.
1), glucose concn typical curve
Glucose reference liquid with deionized water preparation 1mg/mL.By the prescription in the table 1 solution is added in the test tube, the 5min that develops the color in boiling water behind the mixing places cold water to cool off.Draw 200 μ L solution, add in the 96 hole enzyme plates, measure light absorption value under 540nm, draw and obtain the glucose typical curve, its regression equation is Y=3.1803X-0.0417, variance R 2Be 0.9992.
The preparation of table 1 glucose concn standard model
Figure BDA00002266455200061
2), p-NP (p-NP) concentration standard curve
P-NP reference liquid with deionized water preparation 1mg/mL.By the prescription in the table 2 solution is added in the test tube.Behind the mixing, draw 200 μ L solution, in 96 hole enzyme plates, survey its light absorption value under 410nm, drawing standard curve, its regression equation are Y=22.405x+0.0464, variance R 2Be 0.9991.
The preparation of table 2p-NP concentration standard sample
Figure BDA00002266455200071
3), the mensuration of filter paper enzyme activity
The mensuration of filter paper enzyme activity is with reference to Ghose method (Ghose TK.Measurement of cellulase activites.Pure ﹠amp; Appl.Chem., 1987,59:257-268) and slightly revise.It is 1 * 6cm that Whatman I filter paper is cut into size, is involved in the round bottom centrifuge tube of 10mL, adds respectively pH 5.0 Sodium phosphate dibasics-citrate buffer solution 1mL, and filter paper will be cushioned liquid fully and soak, and places the water-bath preheating of 50 ℃ of temperature.Add enzyme liquid 500 μ L, organize in contrast insulation reaction 60min with the enzyme liquid of deactivation.The DNS reagent that adds 3mL, boiling water bath 5min colour developing is placed in the cold water and is cooled to room temperature.Draw 200 μ L reaction supernatant liquor, add in the 96 hole enzyme plates, at the light absorption value of wavelength 540nm place working sample, calculate glucose concn in the reaction system according to the glucose concn typical curve of drafting.
An enzyme activity unit (U) is defined as: under certain condition, the required enzyme amount of reducing sugar (glucose that is equivalent to equivalent) that enzymic hydrolysis filter paper, every 1min produce 1 μ mol is an enzyme activity unit (U).
4), the mensuration of carboxymethylcelluloenzyme enzyme (CMCase) vigor
The mensuration of CMCase enzyme activity adopts people (the Gokhale DV such as Gokhale, Puntambekar US, Deobagkar DN.et al.Production of cellulolytic enzymes by mutant of Aspergillus niger NCIM 1207.Enzyme Microb Technol, 1988, the method for 10:442-445) describing is also revised slightly.
Preparation 1% (w/v) Xylo-Mucine (carboxyl methyl cellulose sodium, CMC-Na) (be dissolved in pH 5.0 Sodium phosphate dibasics-citrate buffer solution) and be stirred to fully dissolving, draw this solution of 1mL in the round bottom centrifuge tube of 10mL, place 50 ℃ water-bath preheating.The enzyme liquid that adds 500 μ L is organized insulation reaction 30min in contrast with the enzyme liquid of deactivation.The DNS reagent that adds 3mL, boiling water bath 5min colour developing is placed in the cold water and is cooled to room temperature.Draw 200 μ L reaction supernatant liquor, add in the 96 hole enzyme plates, at the light absorption value of wavelength 540nm place working sample, calculate glucose concn in the reaction system according to the glucose concn typical curve of drafting.
An enzyme activity unit (U) is defined as: under certain condition, the required enzyme amount of reducing sugar (glucose that is equivalent to equivalent) that enzymic hydrolysis CMC-Na, every 1min produce 1 μ mol is an enzyme activity unit (U).
5), the beta-glucosidase (vitality test of β-glucosidase)
The beta-glucoside enzyme activity determination adopts people (the Gokhale DV such as Gokhale, Puntambekar US, Deobagkar DN.et al.Production of cellulolytic enzymes by mutant of Aspergillus niger NCIM 1207.Enzyme Microb Technol, 1988, the method for 10:442-445.) describing is also revised slightly.
The solution (being dissolved in pH 5.0 Sodium phosphate dibasics-citrate buffer solution) of p-NPG(p-nitrophenyl-β-D-glucopyranoside) of preparation 1mg/mL, draw this solution of 0.9mL in the round bottom centrifuge tube of 10mL, it is constant to place 50 ℃ water-bath to be preheated to water-bath temperature temperature.Add the enzyme liquid of 100 μ L and organize in contrast insulation reaction 30min with the enzyme liquid of deactivation.The 2%Na that adds 2mL 2CO 3Termination reaction is placed in the cold water and is cooled to room temperature.Draw 200 μ L reaction supernatant liquor, in 96 hole enzyme plates, survey the light absorption value under the 410nm, calculate the p-NP concentration that produces in the reaction system according to the p-NP concentration standard curve.
An enzyme activity unit (U) is defined as: under certain condition, it is an enzyme activity unit (U) that enzymic hydrolysis p-NPG, every 1min discharge the required enzyme amount of 1 μ molp-NP.
Citric acid-Sodium phosphate dibasic damping fluid preparation:
Prepare respectively the citric acid of 0.1M and the Sodium phosphate dibasic of 0.2M, the citric acid high-temperature sterilization of 0.1M is mixed with the damping fluid of different pH values according to the experiment needs according to the volume in the following table:
Figure BDA00002266455200081
Separation screening and the evaluation of embodiment 1, mould (Penicillium sp.) mutant strain EU2106
One, the separation screening of mutant strain
The present invention is from the original fungal bacterial strain in laboratory, obtains the higher bacterial strain of cellulase-producing vigor through screening after the mutagenesis.Take mould (Penicillium sp.) wild type strain HP7-1 as starting strain, adopt 3 to take turns Co 60-gamma-ray irradiation has obtained the mutant strain TCO7-4 that a strain filter paper enzyme activity improves than starting strain HP7-1.Again TCO7-4 and mutant strain EU122 thereof are carried out ethylmethane sulfonate (Ethylmethylsulfone, EMS) and ultraviolet (UV) complex mutation and screening, finally obtain mutant strain EU2106.
Bacterial strain is carried out Co 60During-gamma-ray irradiation, the selection lethality rate is that the spore suspension under 85% left and right sides matched doses carries out extensive isolation and screening to mutant strain; And the order of EMS and UV multiple mutated is, first mutant strain TCO7-4 is carried out respectively EMS and processes 24h, shines 5min behind 28h and the 32h under ultraviolet ray, 6min, 7min.Found that no matter be any assembled scheme, lethality rate all reaches 99%.Therefore, choose that 24h EMS processes and 7min shines spore liquid and carries out extensive isolation and screening mutant strain, obtain a mutant strain EU122.Again mutant strain EU122 is carried out EMS and UV complex mutation with above-mentioned same method.Through separating, screening, obtain at last mutant strain EU2106.
⑴ for the first time 60Co-gamma-ray irradiation: wild type strain HP7-1(filter paper enzyme activity 1.61U/mL, liquid screening substratum) at 0.6kGy, 1.0kGy, 1.4kGy, 1.8kGy, 2.2kGy, 2.6kGy, 3.0kGy 3.4kGy and 3.8kGy etc. carries out mutagenic treatment under totally 10 dosage.When mutagenesis dosage is 2.4kGy, lethality rate is 86%, under this dosage, mutant strain is carried out extensive isolation and screening, selecting 17 strains from 235 single bacterium colonies carries out shake-flask culture and sieves again, obtaining a strain filter paper enzyme activity is 2.39U/mL(liquid screening substratum) mutant strain, called after CO7-5.
⑵ for the second time 60The Co-gamma-ray irradiation: be 0.6kGy with CO7-5 at mutagenesis dosage, 1.0kGy, 1.4kGy, 1.8kGy, 2.0kGy, 2.2kGy, 2.6kGy and 3.0kGy process under 8 dosage totally.When mutagenesis dosage is 2.0kGy, be 88% to the lethality rate of this bacterial strain, under this dosage, mutant strain is carried out extensive isolation and screening.Select 109 strains and carry out shake-flask culture and sieve again from 716 single bacterium colonies, obtaining a strain filter paper enzyme activity is 4.03U/mL(liquid screening substratum) mutant strain SCO7-113.
⑶ for the third time 60The Co-gamma-ray irradiation: with mutant strain SCO7-113 at 1.4kGy, 1.6kGy, 1.8kGy, 2.0kGy and 2.2kGy carry out mutagenic treatment under totally 5 dosage.Lethality rate is 86% when dosage is 1.4kGy, and the mutant strain under this dosage is carried out extensive isolation and screening, selects 175 strains and carry out shake-flask culture and sieve again from 834 strains, obtains the mutant strain TCO7-4 that a strain filter paper enzyme activity is 4.09U/mL.
⑷ EMS-UV complex mutation: take TCO7-4 as starting strain, through and then using uviolizing after the EMS processing, the bacterial strain of processing through EMS does not shine 7min under ultraviolet ray, and when lethality rate was 88%, twice EMS and UV complex mutation, lethality rate all reached 99%.For the first time complex mutation is selected 178 strains and is carried out shake-flask culture and sieve again from 2380 strains, obtains the mutant strain EU122 that a strain filter paper enzyme activity is 4.14U/mL.Complex mutation is take EU122 as starting strain for the second time, selecting 137 bacterium colonies from 1566 single bacterium colonies carries out shake-flask culture and sieves again, obtain at last the mutant strain EU2106 that a strain filter paper enzyme activity is 5.26U/mL, its filter paper enzyme activity is 3.3 times of filter paper enzyme activity (1.61U/mL) of wild type strain HP7-1.
Two, the evaluation of mould mutant strain EU2106
When mould mutant strain EU2106 is carried out morphological observation, it is inoculated on the PDA flat board 28 ℃ of constant temperature culture.After 3 days, this bacterial strain produces the cyan spore, and there is the white hypha circle of a projection in bacterium colony central authorities, diameter 0.5cm, and mycelia is for white and measure few fine hair shape.
Be cultured to the 5th day, the product spore district of mould mutant strain EU2106 is that central mycelia circle begins to the about 0.5cm of colony edge place, and the spore of close mycelia circle is intensive and color is darker, darkcyan, and spore rareness and color around it are more shallow; More smooth and the pros and cons of bacterium colony is radial, and spore is form of powdery particles, produces the spore district and is circular concentric (Fig. 1).Under opticmicroscope, the spore shape of mould mutant strain EU2106 has a small amount of circle (Fig. 2) concurrently all take ellipse as main; Mycelia is forked (Fig. 3); Conidiophore all expands and is the broom shape, has two and takes turns conidium stigma (Fig. 4).
Extract total DNA of mould mutant strain EU2106, with λ DNA ladder as standard model, take total DNA of the bacterial strain EU2106 that extracts as template, take universal primer ITS1(5 ' TCCGTAGGTGAACCTGCGG 3 ') and ITS4(5 ' TCCTCCGCTTATTGATATG 3 ') be primer, pcr amplification.
The result as shown in Figure 5, Fig. 5 A is total DNA of mould mutant strain EU2106, wherein, M is molecular weight standard λ DNA, the 1 total DNA for the mould mutant strain EU2106 that extracts; Fig. 5 B is the ITS sequence of the mould mutant strain EU2106 of pcr amplification, and wherein, M is molecular weight standard 1kb DNA ladder, and 1 is the ITS sequence of the mould mutant strain EU2106 of pcr amplification; Find out, obtain the ITS of the bacterial strain EU2106 of about 600bp size.
Through order-checking, the PCR product of this 600bp size has the Nucleotide shown in the sequence 1 in the sequence table.
Sequence analysis the analysis showed that: the homogeny of the ITS sequence of mould mutant strain EU2106 and wild type strain mould (Penicillium sp.) HP7-1, penicillium oxalicum (Penicillium oxalicum) bacterial strain B3-11 (2), mould (Penicillium sp.) bacterial strain P18E1, mould (Penicillium sp.) bacterial strain LH33, mould (Penicillium sp.) bacterial strain 07, mould (Penicillium sp.) bacterial strain JSC94 etc. is 100%.Confirm that mould mutant strain EU2106 suddenlys change from wild type strain mould (Penicillium sp.) HP7-1.
According to " form of fungi and the classification " of wearing fragrant billows (Dai Fanglan. the form of fungi and classification. Beijing: Science Press, 1987,92-96) and Wei Jingchao " fungi identification handbook " (Wei Jingchao. the fungi identification handbook. Shanghai: Shanghai science tech publishing house, 1982.) in form describe, and to the analytical results of ITS sequence, preliminary evaluation mould mutant strain EU2106 is penicillium oxalicum (Penicillium oxalicum).
Above-mentioned bacterial strains EU2106 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 08 22nd, 2012, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.6471, and the Classification And Nomenclature of suggestion is penicillium oxalicum Penicillium oxalicum.
The optimization of the culture condition of embodiment 2, mould (Penicillium sp.) mutant strain EU2106 production of cellulose enzyme
When the culture condition that carries out mould (Penicillium sp.) mutant strain EU2106 production of cellulose enzyme was optimized, used medium was liquid-based basal culture medium (g/L): KH 2PO 44g, (NH 4) 2SO 42.8g, CaCl 20.6g, MgSO 4.7H 2(component of trace element and the final concentration in substratum are respectively 5.0mg/L FeSO for O 0.6g, Tween-802mL, trace element 47H 2O, 1.6mg/L MnSO 4H 2O, 1.4mg/L ZnSO 47H 2O, 2.0mg/LCoCl 2) 0.1mL, wheat bran 50g, pH value regulate 5.0,121 ℃ of lower sterilization 30min with the 2M HCl aqueous solution or the 2M NaOH aqueous solution.
One, the preparation of spore suspension
1, with 112 ℃ of sterilizations of PDA substratum 20min.
2, make spore suspension after the spore of 7 days mould (Penicillium sp.) the mutant strain EU2106 that is obtained by embodiment 1 of activation that goes down to posterity on the PDA flat board is washed with sterilized water, spore concentration is 1 * 10 8Individual/mL.
Two, the optimization of pH value
1, the basic fermention medium of liquid of the different pH values of preparation (4.0,4.5,5.0,5.5 or 6.0).
2, the spore suspension of mutant strain EU2106 is seeded in the aforesaid liquid minimum medium by 1% inoculum size (volumn concentration), 28 ℃, 200rpm were cultivated 5 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.
The results are shown in Figure 6, the suitableeest initial pH that mutant strain EU2106 produces filter paper enzyme (Fig. 6 A), CMC enzyme (Fig. 6 B) and beta-glucosidase (Fig. 6 C) is 5.5.
Three, the optimization of culture temperature
1, the liquid-based basal culture medium of preparation pH 5.5.
2, the spore suspension of mutant strain EU2106 is seeded in the liquid-based basal culture medium of pH 5.5 by 1% inoculum size (volumn concentration), place respectively the shaking table 200rpm of differing temps (24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃ or 34 ℃) to cultivate 5 days, centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.
The results are shown in Figure 7, the result shows that the suitableeest culture temperature that mutant strain EU2106 produces filter paper enzyme (Fig. 7 A) and CMC enzyme (Fig. 7 B) is 28 ℃, and the suitableeest culture temperature of producing beta-glucosidase (Fig. 7 C) is 30 ℃.
Four, substratum determining of the suitableeest carbon source
1, the various carbon source substratum of preparation
The substratum (pH5.5) that contains various carbon sources: other carbon source (corn cob, bagasse, Tapioca Starch, glucose, glycerine, lactose or lactose+lactobionic acid) of quality such as use to replace wheat bran in the liquid-based basal culture medium, obtain containing the substratum of various carbon sources.
2, the spore liquid of mutant strain EU2106 is seeded to respectively by 1% inoculum size (volumn concentration) contains in the substratum of various carbon sources, 28 ℃, 200rpm were cultivated 5 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.
The results are shown in Table 3, can find out, the suitableeest carbon source that mutant strain EU2106 produces filter paper enzyme, CMC enzyme, beta-glucosidase all is wheat bran.
Determining of the suitableeest carbon source of table 3 mutant strain EU2106 production of cellulose enzyme
Figure BDA00002266455200111
Figure BDA00002266455200121
ND: expression can't detect enzyme activity
Five, the wheat bran optimal concentration determines
1, prepares various substratum
Preparation contains the liquid-based basal culture medium (pH 5.5) of various concentration wheat bran:
KH 2PO 44g, (NH 4) 2SO 42.8g, CaCl 20.6g, MgSO 4.7H 2O 0.6g, Tween-802mL, micro-0.1mL, wheat bran (10g, 15g, 20g, 25g, 30g, 35g, 40g, 45g or 50g), pH value regulate 5.0 with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; Be settled to 1L with distilled water; 121 ℃ of sterilization 30min.
2, the spore suspension of mutant strain EU2106 is seeded to respectively in the above-mentioned liquid-based basal culture medium that contains various concentration wheat bran by 1% inoculum size (volumn concentration), 28 ℃, 200rpm were cultivated 5 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.
The results are shown in Figure 8, the result shows that when the concentration of adding wheat bran was 40g/L, mutant strain EU2106 produced the amount maximum of filter paper enzyme (Fig. 8 A), CMC enzyme (Fig. 8 B), and 30g/L wheat bran is the optimal concentration that mutant strain EU2106 produces beta-glucosidase (Fig. 8 C).
Six, substratum the most proper combination carbon source determines
1, prepares various substratum
Preparation contains the substratum (pH 5.5) of various combination carbon sources: other combination carbon source (wheat bran adds Tapioca Starch, Avicel(Microcrystalline Cellulose), potato, paper pulp or the corn cob of the quality such as using) replace the wheat bran in the liquid-based basal culture medium, obtain containing the substratum of various combination carbon sources.
2, the spore suspension of mutant strain EU2106 is seeded to respectively in the above-mentioned substratum that contains various combination carbon sources by 1% inoculum size (volumn concentration), 28 ℃, 200rpm were cultivated 5 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.
The results are shown in Table 4, show that wheat bran and Avicel are the most proper combination carbon sources that mutant strain EU2106 produces enzyme.
Determining of the most proper combination carbon source of table 4 mutant strain EU2106 production of cellulose enzyme
Figure BDA00002266455200131
Annotate: the standard deviation of mean value, different combination carbon source groups are compared with the Avicel group with wheat bran and carried out the T-test statistical analysis in SPSS, " * * ": difference is (p＜0.01) extremely significantly; " * ": significant difference (p＜0.05).
Seven, combination carbon source optimal concentration determines
1, prepares various substratum
Preparation contains the substratum (pH 5.5) of various concentration wheat bran and Avicel:
KH 2PO 44g, (NH 4) 2SO 42.8g, CaCl 20.6g, MgSO 4.7H 2O 0.6g, Tween-802mL, micro-0.1mL, wheat bran add Avicel(10g add 20g, 10g add 30g, 10g add 40g, 10g add 10g, 20g add 20g, 20g add 30g, 30g add 10g, 30g add 20g or 40g add 10g), the pH value regulates with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; Be settled to 1L with distilled water; 121 ℃ of sterilization 30min.
2, the spore suspension of mutant strain EU2106 is seeded to respectively in the above-mentioned substratum that contains various concentration wheat bran and Avicel by 1% inoculum size (volumn concentration), cultivated 5 days, centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.
The results are shown in Table 5, the result shows that when the ratio of wheat bran and Avicel was 4:1 (w:w), mutant strain EU2106 can produce the highest filter paper enzyme and CMC enzyme activity.
Determining of the most proper combination carbon source concentration of table 5 mutant strain EU2106 production of cellulose enzyme
Figure BDA00002266455200141
Annotate: the standard deviation of mean value, the combination carbon source of different ratios and ratio are that the combination carbon source of 4:1 is compared and carried out the T-test statistical analysis in SPSS, " * * ": difference is (p＜0.01) extremely significantly; " * ": significant difference (p＜0.05).
Eight, substratum determining of the suitableeest nitrogenous source
1, prepares various substratum
Preparation contains the substratum (pH 5.5) of various nitrogenous sources:
Ammonium sulfate: KH 2PO 44g, ammonium sulfate 2.8g, CaCl 20.6g, MgSO 4.7H 2O 0.6g, Tween-802mL, micro-0.1mL, 40g wheat bran, 10g Avicel, pH value regulate 5.5 with the 2M HCl aqueous solution or the 2MNaOH aqueous solution; Be settled to 1L with distilled water; 121 ℃ of sterilization 30min
With etc. other nitrogenous source (SODIUMNITRATE, peptone, soybean cake powder, ammonium nitrate or urea) of quality replace ammonium sulfate in the liquid-based basal culture medium, obtain containing the substratum of various nitrogenous sources,
2, the spore suspension of mutant strain EU2106 is seeded to respectively in the above-mentioned substratum that contains various nitrogenous sources by 1% inoculum size (volumn concentration), 28 ℃, 200rpm were cultivated 5 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.The results are shown in Table 6.The suitableeest nitrogenous source that mutant strain EU2106 produces filter paper enzyme, CMC enzyme all is ammonium sulfate.
Table 6 is the determining of the suitableeest nitrogenous source of mutant strain EU2106 production of cellulose enzyme
Figure BDA00002266455200142
Annotate: the standard deviation of mean value, all nitrogenous sources are compared with ammonium sulfate and carried out the T-test statistical analysis in SPSS, " * * ": difference is (p＜0.01) extremely significantly; " * ": significant difference (p＜0.05).
Nine, the ammonium sulfate optimal concentration determines
1, prepares various substratum
Preparation contains the substratum (pH 5.5) of various concentration ammonium sulfate:
KH 2PO 44g, (NH 4) 2SO 4(1g, 2g, 3g, 4g, 5g, 6g, 7g or 8g), CaCl 20.6g, MgSO 4.7H 2O 0.6g, Tween-802mL, micro-0.1mL, wheat bran 40g, Avicel 10g, pH value are regulated with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; Be settled to 1L with distilled water; 121 ℃ of sterilization 30min.
2, the spore suspension of mutant strain EU2106 is inoculated respectively in the above-mentioned substratum that contains various concentration ammonium sulfate by 1% inoculum size (volumn concentration), 28 ℃, 200rpm were cultivated 5 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.
The results are shown in Figure 9, the result shows, is 4g/L when adding ammonium sulfate concentrations, and the filter paper enzyme activity of mutant strain EU2106 (Fig. 9 A), CMC enzyme activity (Fig. 9 B) and beta-glucoside enzyme activity (Fig. 9 C) reach the highest.
Ten, determining of the suitableeest inoculum size
1, preparation best medium:
KH 2PO 44g, (NH 4) 2SO 44g, CaCl 20.6g, MgSO 4.7H 2O 0.6g, Tween-802mL, micro-0.1mL, wheat bran 40g, Avicel 10g, pH value are regulated pH 5.5 with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; Be settled to 1L with distilled water; 121 ℃ of sterilization 30min.
2, the spore suspension with mutant strain EU2106 is seeded in the above-mentioned best medium, and the final concentration of spore liquid is respectively 10 3Individual/mL, 10 4Individual/mL, 10 5Individual/mL, 10 6Individual/mL or 10 7Individual/mL, 28 ℃, 200rpm were cultivated 5 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme and CMC enzyme activity.
The results are shown in Figure 10, the spore final concentration of inoculation is 10 6Individual/mL is the optimal concentration that this mutant strain produces enzyme.
11, determining of the suitableeest incubation time
1, preparation best medium:
KH 2PO 44g, (NH 4) 2SO 44g, CaCl 20.6g, MgSO 4.7H 2O 0.6g, Tween-802mL, micro-0.1mL, wheat bran 40g, Avicel 10g, pH value are regulated pH 5.5 with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; Be settled to 1L with distilled water; 121 ℃ of sterilization 30min.
2, the spore suspension with mutant strain EU2106 is seeded in the above-mentioned best medium, and the final concentration of spore liquid is 10 6Individual/mL, 28 ℃, 200rpm were cultivated 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or 9 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme and CMC enzyme activity.
The results are shown in Figure 11, the suitableeest incubation time of the product enzyme of mutant strain EU2106 is the 6th day.
12, the comparison of mould (Penicillium sp.) mutant strain EU2106 production of cellulose enzyme situation in the substratum of liquid-based basal culture medium and optimization
1, prepares various substratum
(1) the liquid-based basal culture medium of preparation pH 5.0.
(2) substratum of the optimization of preparation pH 5.5: KH 2PO 44g, (NH 4) 2SO 44g, CaCl 20.6g, MgSO 4.7H 2O0.6g, Tween-802mL, micro-0.1mL, wheat bran 40g, Avicel 10g, pH value are regulated with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; Be settled to 1L with distilled water; 121 ℃ of moist heat sterilization 30min.
2, the spore suspension of mutant strain EU2106 is seeded to respectively in the substratum of optimization of the liquid-based basal culture medium of pH 5.0 and pH 5.5, the final concentration of spore liquid is 10 6Individual/mL, 28 ℃, 200rpm were cultivated 6 days, and centrifugal collection supernatant liquor is crude enzyme liquid.
3, collect crude enzyme liquid as solution to be measured, carry out the mensuration of filter paper enzyme, CMC enzyme and beta-glucoside enzyme activity.
The results are shown in Table 7, filter paper enzyme, the CMC enzyme that mutant strain EU2106 produces in optimizing liquid nutrient medium all is significantly higher than enzyme corresponding in the liquid-based basal culture medium and lives, and changes not obvious before and after the optimization of beta-glucosidase liquid medium within.Liquid nutrient medium filter paper enzyme activity after optimizing is that 6.87U/mL, CMC enzyme activity are 12.77U/mL, and they are respectively filter paper enzyme activity (2.74U/mL) before optimizing, CMC enzyme activity (6.23U/mL) 2.5 times, 2.0 times.
The product enzyme of table 7 mutant strain EU2106 in the liquid nutrient medium of liquid-based basal culture medium and optimization relatively
Figure BDA00002266455200161
Annotate: the standard deviation of mean value, the optimization liquid nutrient medium is compared with basic liquid nutrient medium and carried out the T-test statistical analysis in SPSS, " * * ": difference is (p＜0.01) extremely significantly; " * ": significant difference (p＜0.05).
Embodiment 3, utilize mould (Penicillium sp.) mutant strain EU2106 to prepare cellulase preparation (cellulase)
1, preparation fermention medium
The Optimal Medium of preparation pH 5.5: KH 2PO 44g, (NH 4) 2SO 44g, CaCl 20.6g, MgSO 4.7H 2The component of O0.6g, Tween-802mL, micro-0.1mL(trace element and the final concentration in substratum are respectively 5.0mg/L FeSO 47H 2O, 1.6mg/L MnSO 4H 2O, 1.4mg/L ZnSO 47H 2O, 2.0mg/LCoCl 2), wheat bran 40g, Avicel 10g, pH value regulate with the 2M HCl aqueous solution or the 2M NaOH aqueous solution; Be settled to 1L with distilled water; 121 ℃ of moist heat sterilization 30min.
2, the preparation of spore liquid
(1) preparation PDA solid medium and with its 112 ℃ the sterilization 20min.
(2) mould (Penicillium sp.) mutant strain EU2106 is inoculated on the PDA solid medium flat board, in 28 ℃ of constant incubators, cultivated 5 days.0.9%NaCl with certain volume washes spore, move into to be equipped with in the 50mL centrifuge tube of an amount of granulated glass sphere, with forced oscillation to break up spore, through 4 layers of filtered through gauze.At microscopically, the blood counting chamber counting is also regulated spore concentration to 10 8Individual/mL, the inoculum size by 1% is inoculated in the Optimal Medium (being contained in the 250mL triangular flask) of the pH 5.5 of 50mL, and the final concentration that makes spore is 10 6Individual/mL.
3, the preparation of cellulase preparation
(1) the above-mentioned 250mL triangular flask of having inoculated mutant strain EU2106 spore suspension substratum be will fill and 28 ℃ of constant-temperature tables, 200rpm shaking culture placed 6 days.
(2) 13, the centrifugal 5min culture of 800 * g is removed thalline, collects supernatant liquor as solution to be measured, carries out the mensuration of cellulase activity.
Method is the same, the result is as follows: the filter paper enzyme activity of solution to be measured is that 6.79U/mL, CMC enzyme activity are that 12.81U/mL and beta-glucoside enzyme activity are 1.51U/mL, and the fermented supernatant fluid liquid of this mutant strain EU2106 is liquid cellulase preparation (namely obtaining cellulase).
The enzymatic property of embodiment 4, liquid cellulase preparation
1, the Optimun pH of liquid cellulase preparation
Detect the difference of liquid cellulase preparation enzyme activity under the condition of different pH: prepare different pH(3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0) citric acid-Sodium phosphate dibasic damping fluid, measure liquid cellulase preparation that above-described embodiments 3 obtain to the enzyme activity of substrate filter paper, CMC and p-NPG down at 50 ℃.Concerning the same substrate, take high enzymatic activity as 100%, the enzyme activity of other pH value is enzyme activity with the ratio of high enzymatic activity, and take the pH value as X-coordinate, enzyme activity is the ordinate zou mapping.
The results are shown in Figure 12, A: filter paper enzyme activity; B: carboxymethylcelluloenzyme enzyme activity; C: beta-glucoside enzyme activity; Show that mould mutant strain EU2106 liquid cellulase preparation is 4.5(Figure 12 A to the suitableeest action pH of filter paper), be 3.5(Figure 12 B to the suitableeest action pH of CMC), be 5.0(Figure 12 C to the suitableeest action pH of p-NPG).
2, the optimum temperature of liquid cellulase preparation
Detect the difference of liquid cellulase preparation enzyme activity under the condition of different temperatures: under the suitableeest action pH condition, measure above-described embodiment 3 obtains under the different temperature (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ or 70 ℃) liquid cellulase preparation to the enzyme activity of filter paper, CMC and p-NPG.Concerning the same substrate, take high enzymatic activity as 100%, the enzyme activity under other temperature is enzyme activity with the ratio of high enzymatic activity, and take temperature as X-coordinate, enzyme activity is the ordinate zou mapping.
The results are shown in Figure 13, A: filter paper enzyme activity; B: carboxymethylcelluloenzyme enzyme activity; C: the beta-glucoside enzyme activity shows: mould mutant strain EU2106 crude enzyme liquid is to filter paper (Figure 13 A), CMC(Figure 13 B) and p-NPG(Figure 13 C) optimum temperature be respectively 55 ℃, 45 ℃ and 70 ℃.
3, the substrate specificity of liquid cellulase preparation
Detect the difference of liquid cellulase preparation enzyme activity under the different substrate conditions: use respectively the different substrates (Avicel, steam explosion are processed bagasse, Peracetic Acid pre-treatment bagasse, dilute hydrochloric acid pre-treatment bagasse, begasse pulp and steam explosion and processed corn cob) of equal in quality to replace filter paper, the filter paper enzyme activity measuring method of the other the same as in Example 3.
Begasse pulp is taken from Pu Miao paper mill, Nanning, becomes clarification with distilled water immersion certain hour to water, transfers pH to 7.0 with 2M NaOH or 2M HCl, oven dry, 80 order sieving for standby after smashing to pieces.Peracetic Acid pre-treatment bagasse: bagasse is taken from Pu Miao paper mill, Nanning, bagasse is dried and smashed to pieces, after sieving, 80 orders add 10% NaOH by 1:3 (w:v), 100 ℃ of water-bath 1.5h wash with deionized water, filter, press again the peracetic acid soln of 1:1 (w:v) adding 15%, 75 ℃ of water-bath 2.5h transfer pH to 5.0, dry for standby.The pretreated bagasse of steam explosion and corn cob are taken from Guangxi Plant Inst..Hydrochloric acid pre-treatment bagasse: bagasse is taken from Pu Miao paper mill, Nanning, dilute hydrochloric acid with 1.2% with smash into granular bagasse to pieces and mix with the ratio of 15:1 (v/w), in 121 ℃ of sterilization 4h, transfer pH to 5.0 with 0.1MNaOH or 0.1M HCl, again sterilized water is diluted to 30:1 (v/w) with it, filtered through gauze and oven dry, powdered through smashing to pieces, 80 order sieving for standby.
The enzyme activity definition: it is an enzyme activity unit (U) that 4.5,55 ℃ of Water Under solutions of pH substrate, every min discharge the required enzyme amount of 1 μ mol reducing sugar (glucose that is equivalent to equivalent).
The protein concn of the liquid cellulase preparation that use Micro BCA analysis of protein reagent (Pierce, Rockford, IL) detection above-described embodiment 3 obtains.
The enzyme activity determination of liquid cellulase preparation the results are shown in Table 8 under the different substrate conditions, show, the cellulase preparation of above-mentioned preparation is different to the hydrolysis ability of various substrates, hydrolysis ability by by force to a little less than be followed successively by: begasse pulp, Peracetic Acid pre-treatment bagasse, steam explosion pre-treatment bagasse, CMC, filter paper, Avicel, steam explosion pre-treatment corn cob, dilute hydrochloric acid pre-treatment bagasse.
The substrate specificity of table 8 mutant strain EU2106 cellulase preparation
Figure BDA00002266455200181
4, the product analysis of liquid cellulase preparation hydrolysis begasse pulp
(1), preparation begasse pulp suspension
The 1g begasse pulp is suspended from the citric acid of 100mL pH4.5-Sodium phosphate dibasic damping fluid, obtains the begasse pulp suspension.
(2), product analysis
1% of the above-mentioned preparation of adding 20mL begasse pulp suspension in the 50mL triangular flask, add simultaneously the liquid cellulase preparation that above-described embodiment 3 of 10mL obtains, be positioned over 50 ℃, the insulation of 150rpm shaking table, take out sample 4mL during respectively at 3h and 6h, boiling water boiling 5min cools off with the deactivation cellulase, 13, the centrifugal 10min of 800xg gets supernatant liquor, and HPLC detects the sugar in the supernatant liquor.Standard model is the mixed solution of glucose, cellobiose and the wood sugar of 1mg/mL.All samples filters with the biofilter of 0.22 μ m.
Chromatographic apparatus is Shimadzu CBM-10A system, and the chromatographic column model is BP-800pb ++, detector models is RID-10A, and column temperature remains on 80 ℃, and moving phase is deionized water, and sampling volume is 20 μ L, flow velocity is 0.8mL/mim.
The results are shown in Figure 14, the A. standard model; B. hydrolytic action time 3h, hydrolysis temperature is 50 ℃; C. hydrolytic action time 6h, hydrolysis temperature is 50 ℃, wherein, 1: cellobiose; 2: glucose; 3: wood sugar shows that the primary product of liquid cellulase preparation hydrolysis begasse pulp is glucose, but a small amount of cellobiose and wood sugar are arranged.
Infer that thus mould (Penicillium sp.) mutant strain EU2106 liquid cellulase preparation has each component of complete cellulase system.
Embodiment 5, utilize liquid cellulase preparation hydrolysis begasse pulp
1,4 groups of processing is set, every group of processing arranges 3 repetitions: the citric acid of liquid cellulase preparation, aseptic deionized water and the pH 5.0 that add begasse pulp in the triangular flask of 150mL, is obtained by embodiment 3-Sodium phosphate dibasic damping fluid (each group process in add-on of each component see Table 9) obtains reaction system; The reaction system mixing is placed in 50 ℃, 150rpm shaking table; Enzyme digestion reaction 96h; Interval sampling in 12 hours before the reaction 24h, interval sampling in 24 hours after the reaction 24h, the amount of the reducing sugar that produces in the detection reaction system such as cellobiose and glucose.
The add-on of each component during each group of table 9 is processed
Figure BDA00002266455200191
2, take the time as X-coordinate, the sugared concentration of generation is ordinate zou mapping, the results are shown in Figure 15, the analysis of the reducing sugar that A. produces hydrolysis; B. to being hydrolyzed the analysis of the cellobiose that produces; C. to being hydrolyzed the analysis of the glucose that produces; The result shows that mould (Penicillium sp.) mutant strain EU2106 liquid cellulase preparation can be hydrolyzed begasse pulp production reducing sugar such as cellobiose and the glucose of different concns effectively.
Embodiment 6, utilize liquid cellulase preparation simultaneous saccharification and fermentation begasse pulp to produce alcohol
1, preparation fermention medium
(1) first order seed substratum YPD
Yeast powder 10g/L, peptone 20g/L, glucose 20g/L prepares with distilled water; 121 ℃ of sterilization 20min.
(2) secondary seed medium
Yeast powder 10g/L, peptone 20g/L, liquid cellulase preparation are hydrolyzed the enzymolysis solution (containing glucose 10g/L) of 4% begasse pulp (20FPU/g) 12h; 121 ℃ of sterilization 20min.
(3) simultaneous saccharification and fermentation substratum
(NH) 4SO 42g/L, KH 2PO 45g/L, Yeast Extract 10g/L, MgSO 41g/L, CaCl 20.2g/L, 121 ℃ of sterilization 20min; Add warp before the fermentation beginning 60The begasse pulp of Co-gamma-ray irradiation sterilization is to final concentration 20g/L(2%), 40g/L(4%), 60g/L(6%) or 80g/L(8%), add the liquid cellulase preparation that obtained by embodiment 3 to final concentration 20FPU/g begasse pulp.
2, yeast saccharomyces cerevisiae dry yeast in Angel is available from Hubei Angel Yeast Co.,Ltd; Activate according to packing instruction.
3, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ZM1-5CGMCC No.3761.
4, will be seeded to secondary seed medium by 10% inoculum size (volumn concentration) at the two Yeasts liquid that 30 ℃, 150rpm are cultivated among the first order seed substratum YPD of 24h respectively, cultivate 24h in 30 ℃, 150rpm; Secondary seed being cultivated bacterium liquid is seeded to respectively in the simultaneous saccharification and fermentation substratum of different begasse pulp concentration by 10% inoculum size (volumn concentration) again, with bottle sealing, 40 ℃, the 150rpm 96h that ferments, glucose amount, alcohol output are measured in 24h sampling in interval.
5, glucose is measured: detect with HPLC.Standard model is the glucose of 1mg/mL.Chromatographic apparatus is Shimadzu CBM-10A system, and the chromatographic column model is BP-800pb ++, detector models is RID-10A, and column temperature remains on 80 ℃, and moving phase is deionized water, and sampling volume is 20 μ L, flow velocity is 0.8mL/mim.
6, ethanol concn is measured: detect with HPLC.The standard alcohol concn is 1mg/mL.Chromatographic column is TransgenomicIC Sep ICE-Corcgel 87H3 (300mm * 7.8 organic acid posts; Spectra-Physics 6040XR differential refraction monitor; Column temperature remains on 60 ℃, and moving phase is 5mM H 2SO 4, flow velocity 0.5mL/min.
The results are shown in Figure 16 and 17, Figure 16 produces the as a result figure of alcohol for the liquid cellulase preparation that produces with mould mutant strain EU2106 and Angel yeast saccharomyces cerevisiae to the simultaneous saccharification and fermentation of begasse pulp, Figure 17 produces alcohol for the liquid cellulase preparation that produces with mould mutant strain EU2106 and Wine brewing yeast strain ZM1-5 to the simultaneous saccharification and fermentation of begasse pulp as a result figure; The result shows, after having added mould (Penicillium sp.) mutant strain EU2106 liquid cellulase preparation, begasse pulp is degraded to glucose, yeast becomes alcohol with glucose fermentation simultaneously, and enzymic hydrolysis begasse pulp and saccharomycetes to make fermentation glucose carry out in same container simultaneously.Figure 16 is as showing, the Angel yeast saccharomyces cerevisiae is that along with the increase of fermentation time, glucose consumes gradually under 60g/L and the 80g/L condition at begasse pulp content, and alcohol is gradually accumulation then.Figure 17 is as showing, yeast saccharomyces cerevisiae ZM1-5 is under 60g/L, 80g/L and the 100g/L condition at begasse pulp content, increase along with fermentation time, glucose consumes gradually, alcohol is gradually accumulation then, and yeast saccharomyces cerevisiae ZM1-5 liquor output behind synchronous fermentation 48h can reach 22g/L, and glucose content is very low by (＜0.1%, g/100mL), illustrate the fermentation carry out better.These data show that all the simultaneous saccharification and fermentation that mould (Penicillium sp.) mutant strain EU2106 liquid cellulase preparation can be applied to begasse pulp produces in the technique of alcohol, have application potential.

Claims (10)

1. penicillium oxalicum (Penicillium oxalicum) EU2106, its deposit number is CGMCC No.6471.
2. the application of penicillium oxalicum claimed in claim 1 (Penicillium oxalicum) EU2106 in preparation cellulase or cellulase preparation.
3. the method for a production of cellulose enzyme or cellulase preparation, the penicillium oxalicum claimed in claim 1 that comprises the steps: to ferment (Penicillium oxalicum) EU2106 collects tunning, namely obtains cellulase or cellulase preparation.
4. method as claimed in claim 3 is characterized in that: the condition of described fermentation is for cultivating 5-7 days at 24-34 ℃, 200rpm; The condition of described fermentation is specially at 28 ℃, 200rpm and cultivated 6 days.
5. method as claimed in claim 4 is characterized in that: the component of the fermention medium that described fermentation is adopted is as follows: the described fermention medium of every L is by 4g KH 2PO 4, 4g (NH 4) 2SO 4, 0.6g CaCl 2, 0.6g MgSO 4.7H 2O, 2mL Tween-80,5.0mg FeSO 47H 2O, 1.6mg MnSO 4H 2O, 1.4mg ZnSO 47H 2O, 2.0mgCoCl 2, 40g wheat bran, 10g Avicel and water forms, water is supplied volume;
The pH value of described fermention medium is pH3.5-5.5, and the pH value of described fermention medium is specially 5.5.
6. such as arbitrary described method among the claim 3-5, it is characterized in that:
Described fermentation is for cultivating the spore inoculating of penicillium oxalicum claimed in claim 1 (Penicillium oxalieum) EU2106 to described fermention medium;
The initial kind concentration of spore in fermentation system of described penicillium oxalicum (Penicillium oxalicum) EU2106 is specially 10 6Individual/mL.
7. such as arbitrary described method among the claim 3-6, it is characterized in that: behind described collection tunning, also comprise the steps: described tunning centrifugally, collect supernatant liquor, obtain cellulase or cellulase preparation.
8. the cellulase or the cellulase preparation that are prepared by arbitrary described method among the claim 3-7.
9. penicillium oxalicum claimed in claim 1 (Penicillium oxalicum) EU2106, cellulase claimed in claim 8 or the described cellulase preparation application in preparation wood sugar, cellobiose and/or glucose;
Or penicillium oxalicum claimed in claim 1 (Penicillium oxalicum) EU2106, cellulase claimed in claim 8 or the described cellulase preparation application in simultaneous saccharification and fermentation production alcohol.
10. fermention medium, its component is as follows: the described fermention medium of every L is by 4g KH 2PO 4, 4g (NH 4) 2SO 4, 0.6g CaCl 2, 0.6g MgSO 4.7H 2O, 2mL Tween-80,5.0mg FeSO 47H 2O, 1.6mg MnSO 4H 2O, 1.4mg ZnSO 47H 2O, 2.0mg CoCl 2, 40g wheat bran, 10g Avicel and water forms, water is supplied volume.
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CN103740597A (en) * 2013-11-21 2014-04-23 上海海洋大学 Penicillium oxalicum FH6 strain, screening method and application thereof
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CN105925594A (en) * 2016-06-13 2016-09-07 广西大学 Raw starch-digesting glucoamylase, preparation method thereof and application of raw starch-digesting glucoamylase to raw starch hydrolysis and preparation of ethanol by simultaneous saccharification and fermentation of raw starch
CN106047730A (en) * 2016-08-19 2016-10-26 广西大学 Application of Penicillium oxalicum EU2101 in preparation of cellulase preparation and degradation of cellulose
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CN113604522A (en) * 2021-08-02 2021-11-05 广西大学 Penicillium D306 strain capable of producing extracellular polysaccharide and application thereof in preparation of bile acid binder

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