CN100575483C - The method for preparing heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase - Google Patents
The method for preparing heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase Download PDFInfo
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Abstract
The invention discloses a kind of method for preparing heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase.This method, be to be carbon source with agricultural wastes, with thermophilic Paecilomyces varioti (Paecilomyces thermophila) J18, at 40-60 ℃, fermentation obtains containing the fermented liquid of heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase, the purified heat resistant xylanase that obtains the electrophoresis level, heat-resisting xylobiase or heat-resisting beta-glucosidase.Method of the present invention, simple, realize suitability for industrialized production easily.The heat resistant xylanase of the inventive method preparation can improve the quality of steamed bun and bread, and heat-resisting xylobiase and zytase synergy can be with the thorough hydrolysis of xylan.Heat-resisting beta-glucosidase is applied to have significant effect in the hydrolysis of amygdalin, daidzin, Genistoside, cell-oligosaccharide etc.
Description
Technical field
The present invention relates to a kind of method for preparing heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase.
Background technology
Zytase (Xylanase, EC 3.2.1.8) is the important lytic enzyme of a class, mainly acts on β-1 on the xylan backbone with internal-cutting way, and 4-glycosidic link, hydrolysate are mainly the oligose and the wood sugar of different polymerization degree.At present, external more relatively to the research of thermophilic fungus zytase, Haltrich et.al. (Enzyme Microb.Technol.15:854-860) is carbon source through fermentation with the Microcrystalline Cellulose with Schizophylum commune BT2115, Xylanase activity reaches the 4839U/mL fermented liquid, is the highest level that present liquid fermenting produces the zytase report.Domestic more existing fungies produce the research report of zytase, but the research of fungi product heat resistant xylanase and thermophilic fungus bacterial strain thereof is reported also seldom.(food and fermentation industries, 2006,32 (1): 23-27) grade has reported that the thermophilic cotton wool bacterium of a strain thermophilic fungus (Thermomyces lanuginosus) CBS288.54-M18 produces heat resistant xylanase to Li Xiuting, and the liquid fermenting enzyme activity is 1834U/ml.
(Aqueous Two-Phase System is to be suggested a kind of " liquid-liquid " extractive technique that is used to separate biochemical substances the sixties in 20th century ATPS) to aqueous two-phase system.Isolated cell, microbial film, virus, protein, nucleic acid and other biomolecules now have been widely used in.But aqueous two-phase system has advantages such as simple and easy, the high carrying capacity of operation, phase system one step generation, the recirculation of phase material, easy mass-producing, becomes a special kind of skill that the industrialization potentiality are arranged most.Moreover, the double water-phase separation system have mild condition, bio-compatibility good, do not have advantage such as toxicity to make it on enzyme, albumen isoreactivity separating substances, have important use to be worth.The research report of more existing abroad in recent years application aqueous two-phase system (ATPS) purifying zytases has carried out separating and extracting as using polyoxyethylene glycol/potassium phosphate salt system to the outer zytase of thermophilic fungus Melanocarpusalbomyces IIS-68 product born of the same parents; Utilize the zytase of polyoxyethylene glycol (PEG)/potassium phosphate salt system purifying from Bacillus pumilus, 12 kinds of ATP systems that comprise different PEG molecular weight, PEG concentration, phosphate concn, sodium chloride concentration have been studied, result's Xylanase activity rate of recovery under optimal conditions reaches more than 90%, has reached quite high purity.The research of present domestic application double water-phase method purifying biological active substance report is more, is not used for the patent and the research report of purifying zytase but see.
Steamed bun and bread are traditional main meal products, but are faced with several similar problems in the suitability for industrialized production of steamed bun and bread, promptly how to delay shelf-lives that wears out, prolongs steamed bun and bread and alternative or minimizing chemical additive consumption in storage process.Therefore, develop improve steamed bun and bread quality the biological enzyme improved formulations just seem extremely important.Have the patent and the research report of many modifier for steamed bread at present both at home and abroad, Yuan Jianguo etc. have invented a kind of method (application number: 200410024487.3) with biological enzyme improvement steamed bread quality, invention is mixed and made into compound enzymic preparation with glucose oxidase, lipase and amylase, improved the whiteness of steamed bun after adding in dough, increased the volume of steamed bun, delayed to wear out, also shortened the production cycle of steamed bun simultaneously.Jia Yingmin etc. have invented a kind of tailored flour compound enzymic preparation (application number is 200510112661.4), have played and above-mentioned close effect after adding in the steamed bun.(oil and foodstuffs science and technology, 2004,12 (1): 4-7) grade has been added prozymes such as commercial amylase, zytase, glucose oxidase to Wang Jinshui in dough, found that the effect that can play certain delaying retrogradation in the steamed bun storage process.The people of Soviet Union and Eastern Europe (Eur Food Res Technol, 2005,220:540-545) grade has been added two kinds of commercial endo-xylanases in dough, makes the specific volume of steamed bun obtain tangible increase, and weave construction also has clear improvement.
The at present domestic patent of also not using in bread about zytase, relevant bibliographical information also seldom.Li Xiuting (food and fermentation industries, 2006,32 (1): 23-27) grade has been studied thermophilic cotton wool bacterium and has been produced heat resistant xylanase to bread quality and aged influence, inquire into heat resistant xylanase and influenced bread aged kinetics, find that an amount of bread quality of adding heat resistant xylanase has obtained obvious improvement, and aging speed slows down.Deng Wei (Chinese grain and oil journal, 2005,20 (1): 1-5) grade has been studied the influence of olive-green streptomycete (Streptomycesolivaceovirdis) E-86 zytase group to the bread quality, test-results shows, the bread specific volume that adds 20ppm zytase group has increased by 38.8%, soft tommy core hardness has reduced by 1/3, and the bread rate of ageing also obviously reduces.Zhang Qinliang (food and fermentation industries, 2004,30 (7): 21-25) grade has been inquired into commercial neutral xylanase to the flour opaque, the influence of degree of aging in bread baking quality and the bread storage process, the result shows, neutral xylanase can be improved the opaque of flour significantly, when addition is 0.3ml/kg (flour), the formation time of dough can reduce about 50%, can improve the bread baking quality effectively, increase the volume and the specific volume of bread significantly, can improve simultaneously the elasticity and the hardness of crumb, reduce the hardness of bread shell, enzyme interpolation scope is 0.05～0.48ml/kg (flour), can increase the anti-aging effect of bread, preserve after 7 days, the consistency and elasticity of bread does not have considerable change, has prolonged shelf-lives.
Xylobiase (β-1,4-Xylosidase, EC 3.2.1.37) is a kind of in the xylan degrading enzyme system, main catalytic hydrolysis β-wood sugar glycosidic bond, thus wood oligose thoroughly is decomposed into wood sugar.The xylan degrading enzyme ties up to industries such as the energy, papermaking and medicine and has wide practical use.In energy industry, xylan in the agricultural wastes generates oligosaccharides after the zytase degraded, degraded fully by xylosidase, the wood sugar of generation can be converted to fuel such as alcohol by bacterium and fungi again, therefore plays an important role in biomass energy is produced.At present, the research of domestic pass xylan degrading enzyme system mainly concentrates on β-1, the 4-zytase, and less relatively to the research of xylobiase, relevant patent is also seldom.Only (China Patent No.: CN96194959.7), the correlative study report also only has the separation and purification of xylosidase in the clonal expression of purifying, xylobiase gene of aspergillus (Aspergillus) xylobiase and a kind of straw mushroom for patent about xylosidase by Denmark company application.Microorganism xylobiase major part is an intracellular enzyme, and the minority fungi can produce the outer xylobiase of born of the same parents.
Glucuroide (Glucosidase, EC 3.2.1.21) is the special-shaped lytic enzyme of a class, belongs to a kind of in the cellulose complex enzyme system.Glucuroide has very important hydrolytic action, can be applied to a lot of industries.The glucoside endonuclease capable plays an important role in the process of cellulose degraded Mierocrystalline cellulose production alcohol: cellobiose that generates in by the hydrolytic deterioration process and oligose, reduce the hydrolysis restraining effect of disaccharides, reach the hydrolysis that promotes cellulase, the effect that improves hydrolysis efficiency cellulase; The glucoside endonuclease capable synthesizes some important medicine and precursor substances thereof, has the potential application prospect in pharmaceutical industries; Glucuroide can also the hydrolyzed soy glycosides, and aglycon type soybean isoflavones is hydrolyzed into free glucoside type, and free type has physiological activity more widely; Simultaneously glucuroide can also the hydrolysis amygdalin, plays the effect of the debitterize that takes away the puckery taste in some food.Have many using values just because of glucuroide, more and more be subjected to researchist's attention.The present both at home and abroad correlative study of existing many glucuroides report, wherein major part derives from microorganism, and generally the product enzyme research to mesophilic bacteria is more in the microorganism, and it is less relatively that thermophilic microfungi is produced the research of heat-resisting beta-glucosidase.
Thermophilic microorganism is that a class can be in the metabolic microorganism of normal growth under the comparatively high temps, thermophilic microorganism and the thermostable enzyme that produces and general common micro-organisms produce enzyme and compare and have many remarkable advantages, as the comparatively high temps bottom fermentation is cultivated, be not easy bacteria infection; The enzyme that thermophilic microorganism produced generally all has higher temperature stability and higher temperature of reaction, and higher temperature of reaction can improve the speed of reaction and the hydrolysis efficiency of enzyme; In addition, heat-staple enzyme more helps transportation, storage and uses, and is more suitable in industrial applications etc.At present domestic about thermophilic microorganism and the relevant report of producing enzyme research thereof also seldom, therefore, obtains good thermophilic microorganism bacterial strain and its product enzyme and character is furtherd investigate the attention that more and more is subjected to studying with producers.
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase.
The method for preparing heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase provided by the present invention is to be carbon source with agricultural wastes, with thermophilic Paecilomyces varioti (Paecilomyces thermophila) J18, at 40-60 ℃, fermentation obtains heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase.
The method of described fermentation is solid fermentation or liquid fermenting, described solid fermentation is that described thermophilic Paecilomyces varioti J18 is inoculated into the top fermentation of solid fermentation substratum, with the citrate buffer solution flushing, filter the crude enzyme liquid that obtains containing heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase; Described liquid fermenting is that filtering fermentating liquid obtained containing the crude enzyme liquid of heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase after described thermophilic Paecilomyces varioti J18 was inoculated into the liquid fermentation medium top fermentation; Described citrate buffer solution is the damping fluid that contains citric acid and trisodium citrate, and described citrate buffer solution is preferably the damping fluid that contains 0.05mol/L (50mM) citric acid and 0.05mol/L (50mM) trisodium citrate.
Described solid fermentation substratum is 1 by ratio of weight and number: agricultural wastes powder, yeast extract and the water of 0.01-0.1: 2-6 are formed; It is that 1: 0.04: 5 agricultural wastes powder, yeast extract and water is formed that described solid fermentation substratum is preferably by ratio of weight and number.
Described liquid fermentation medium is for containing agricultural wastes powder 10-60g/L, urea or yeast extract or Tryptones 5-20g/L, CaCl
20.1-1g/L, MgSO
47H
2O 0.1-1g/L, FeSO
40.1-1g/L initial pH value is the substratum of 5-9.Be preferably and contain agricultural wastes powder 40g/L, urea or yeast extract or Tryptones 10g/L, CaCl
20.3g/L, MgSO
47H
2O 0.3g/L, FeSO
40.3g/L initial pH value is 7 substratum.
Described agricultural wastes are wheat straw, wheat bran or corn cob.
The time of described fermentation is 2-10 days.
The used seed of described fermentation is from preserving on the inclined-plane streak inoculation on the PDA inclined-plane or flat board of new preparation, then with the inclined-plane or dull and stereotypedly be positioned over that static the cultivation obtained in 2-8 days in the 40-60 ℃ of incubator; Is that 20% glycerine solution washes as the solid fermentation seed with the spore on the inclined-plane with the quality percentage composition, and the dull and stereotyped thalline of cultivating then is directly used in the liquid fermenting seed.
The crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase according to above-mentioned solid fermentating mode preparation, the vigor of zytase is 5,000-20,000U/g agricultural wastes (butt carbon source), the vigor of xylosidase is 20-100U/g agricultural wastes (butt carbon sources), and the vigor of glucuroide is 20-100U/g agricultural wastes (butt carbon sources).
The crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase for preparing according to the liquid fermenting mode, the vigor of zytase is 200-2, the 000U/ml fermented liquid, the vigor of xylosidase is the 2-4U/ml fermented liquid, and the vigor of glucuroide is at the 1.5-4U/ml fermented liquid.
Described method also comprise the described crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase carried out purifying after, obtain heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase.
The purification process of the heat resistant xylanase of described solid fermentation is the double water-phase method; Described double water-phase method is in the described crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase, adding be described crude enzyme liquid quality 25% polyoxyethylene glycol, be the ammonium sulfate of described crude enzyme liquid quality 50% and be the distilled water of described crude enzyme liquid quality 35%, behind the mixing, left standstill 2 hours or centrifugally treat its complete layering, the upper strata is the zytase of purifying.
The purification process of the heat resistant xylanase of described liquid fermenting comprises the steps:
1) in the crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase that solid fermentation obtains, slowly adds ammonium sulfate powder ammonium sulfate to the enzyme liquid and reach 20% saturation ratio, stirred 30 minutes in the ice-water bath, then 10,000 * g frozen centrifugation 10 minutes, getting supernatant liquor continue to add ammonium sulfate powder ammonium sulfate to the enzyme liquid and reaches 50% saturation ratio, stirred 1 hour in the ice-water bath, 10,000 * g frozen centrifugation 10 minutes, to precipitate with a spot of damping fluid dissolving, solution is the heat resistant xylanase enzyme liquid of preliminary purification.
2) with the heat resistant xylanase enzyme liquid of preliminary purification, cross Sephacry S-200HR gel filtration chromatography, with pH 7.320mM Tris-HCl buffer solution elution, collect target peak.
3) with step 2) elutriant that obtains collects the target peak that obtains and crosses DEAE52 weak anionic chromatography column, with the NaCl solution gradient wash-out of 0.4-0.7mol/L, collects the heat resistant xylanase that the target protein peak is purifying.
Enzymatic activity recovery is more than 90% in the purifying zytase purge process according to the method described above, is 1 than enzyme activity, more than the 000U/mg, compares with crude enzyme liquid, improves 2-5 doubly than enzyme activity.
The step that described purifying obtains heat-resisting xylobiase is:
1) to the described heat resistant xylanase that contains, slowly add ammonium sulfate powder ammonium sulfate to the enzyme liquid in the crude enzyme liquid of heat-resisting xylobiase and heat-resisting beta-glucosidase and reach the 30-50% saturation ratio, stirred 0.5-2 hour in the ice-water bath, then 8,000-14, the centrifugal 10-30 of 000 * g minute, getting supernatant liquor continue to add ammonium sulfate powder ammonium sulfate to the enzyme liquid and reaches the 55-65% saturation ratio, stirred 0.5-2 hour in the ice-water bath, 8,000-14, the centrifugal 10-30 of 000 * g minute, precipitation is obtained the xylosidase liquid of preliminary purification with a spot of phosphoric acid-citrate buffer solution dissolving; Described phosphoric acid-citrate buffer solution is for containing 0.025mol/L citric acid and 0.025mol/L trisodium citrate.
2) above-mentioned enzyme liquid through preliminary purification is dialysed with 5-50mM pH 5.0-8.0 phosphoric acid buffer, cross DEAE52 weak anionic chromatography column then, with 20-200mM NaCl eluant solution;
3) elutriant that step 2) obtains is crossed QSepharose Fast Flow (QSFF) reinforcing yin essence ion chromatography post then with the phosphoric acid buffer dialysis of 5-50mM pH 5.0-8.0, uses 30-300mM NaCl eluant solution at last, and elutriant is xylosidase.
The ratio enzyme activity of the xylosidase behind the aforesaid method purifying is 37.65U/mg, compares with crude enzyme liquid, has improved 35.64 times than enzyme activity.
The step of the heat-resisting beta-glucosidase of described purifying is:
1) dialysis, with the heat-resisting beta-glucosidase crude enzyme liquid 5-50mM for preparing under the above-mentioned condition, the dialysis of the phosphoric acid buffer of pH 5.2-8.2;
2) step 1) is crossed DEAE52 weak anionic chromatography column, used 5-50mM, the phosphoric acid buffer of pH 5.2-8.2 is used the NaCl eluant solution of 50-500mM then with albumen and other impurity that the flow velocity flush away of 0.2-2ml/min does not adsorb, collects target protein;
3) with step 2) the collection liquid that obtains crosses the Sephacryl-S200 gel chromatography column, uses 5-50mM, and the phosphoric acid buffer wash-out of pH 5.2-8.2, flow velocity are 0.2-2ml/min, collect the glucuroide that target protein is purifying.
Purifying glucuroide according to the method described above, the enzymatic activity recovery of glucuroide is more than 20%, to have brought up to 80U/mg than enzyme activity by 0.38U/mg in the purge process, compares 200 times of purifying with crude enzyme liquid.
Method of the present invention adopts thermophilic Paecilomyces varioti J18 bacterial strain, is carbon source with agricultural wastes such as wheat straw, corn cobs, adopts solid or liquid fermenting mode to produce three kinds of enzymes, greatly reduces production cost.The present invention adopts double water-phase method and chromatography method that three kinds of enzymes of produce are carried out purifying respectively and obtained the pure enzyme of electrophoresis level respectively.Method of the present invention is simple, and the purity and the rate of recovery of the enzyme of preparation are all higher, realize suitability for industrialized production easily.
The heat resistant xylanase of method of the present invention preparation has advantages such as Heat stability is good, hydrolysis property be outstanding; In a lot of industries such as this zytase can be applied to pulping and paper-making, the energy, feed, brewage; Zytase is applied in the course of processing of steamed bun and bread, has played the good effect of improving steamed bun and bread quality, use this zytase processing steamed bun to make the specific volume of steamed bun increase 10-20%; Whiteness is significantly improved; The weave construction of steamed bun improves significantly; Wearing out has obtained delaying to a certain extent, and initial hardness has reduced 10-30%; Use this zytase processing bread to make the specific volume of bread increase 20-45%; The weave construction of bread improves significantly; The aging of bread obtained prolonging to a certain extent, and slow storage is after 7 days, and the hardness of bread core is the 40-80% of contrast.
The heat-resisting xylobiase of method preparation of the present invention, have that hydrolysis substrate is single-minded, advantage such as Heat stability is good, pH stable range are wide, this endonuclease capable and the zytase thorough hydrolyzed xylan that acts synergistically generates wooden monose, this has important effect in the biomass energy performance history, in addition, this enzyme also has application prospect in industries such as food, feed, papermaking.
The heat-resisting beta-glucosidase of method preparation of the present invention, adopt method purifying provided by the present invention to obtain the pure enzyme of electrophoresis level, this enzyme has key properties such as hydrolyzed soy glycosides, amygdalin, Genistoside, saligenin, cell-oligosaccharide, has bigger application potential, when hydrolyzed soy glycosides soybean isoflavones, aglycon type soybean isoflavones is hydrolyzed into free glucoside type, and free type has physiological activity more widely; The characteristic of hydrolysis amygdalin makes it can play the effect of the debitterize that takes away the puckery taste in some food; This enzyme can also play important effect in the biosynthetic process of some new functional sugars and stain remover.
Description of drawings
Fig. 1 is a heat resistant xylanase purifying electrophorogram
Fig. 2 is heat-resisting xylobiase purifying electrophorogram
Fig. 3 is heat-resisting beta-glucoside enzyme purification electrophorogram
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The preparation of embodiment 1, heat resistant xylanase
1, solid fermentation is produced heat resistant xylanase
1) inoculation is with the cultivation of thermophilic Paecilomyces varioti (Paecilomyes thermophila) J18:
Thermophilic Paecilomyces varioti (Paecilomyes thermophila) J18 is (available from DSMZ of Institute of Microorganism, Academia Sinica, preserving number is AS3.6885) seed is from preserving on the PDA inclined-plane streak inoculation on the PDA inclined-plane or flat board of new preparation, then inclined-plane or flat board is positioned in the 40-60 ℃ of incubator static cultivation 2-8 days.With the quality percentage composition is that 20% glycerine solution washes the spore on the inclined-plane, and the spore liquid that washes is used for solid fermentation and produces enzyme, and the flat board of growth then is directly used in liquid fermenting and uses.
2) solid fermentating mode is produced zytase liquid
With thermophilic Paecilomyces varioti J18 is that fermentation strain, natural agricultural wastes wheat straw are that carbon source adopts solid fermentating mode to produce zytase.
The substratum of solid fermentation is: wheat straw 5g/ bottle (250ml triangular flask), and yeast extract 0.04g/g wheat straw powder, adding is 5 times a water of wheat straw powder and yeast extract total mass, sterilizes 20 minutes for 121 ℃.Can inoculate after the cooling.
Is that (spore concentration is 10 to spore to the thermophilic Paecilomyces varioti J18 (available from DSMZ of Institute of Microorganism, Academia Sinica, preserving number is AS3.6885) that washes of 20% glycerine solution with step 1) with the quality percentage composition
6Cfu/ml) be seeded in the 250ml triangular flask that the solid fermentation substratum is housed and (contain the 5g wheat straw in every bottle, the 0.2g yeast extract), every bottle graft kind 10
6Cfu, with spore mixing in substratum, 50 ℃ leave standstill cultivation, after the cultivation, get one bottle every day, extract crude enzyme liquid and detect xylanase activity.
The crude enzyme liquid extracting method is: adding in the substratum of solid fermentation is the citrate buffer solution that contains 0.05mol/L (50mM) phosphoric acid and 0.05mol/L (50mM) trisodium citrate of 10 times of quality of substratum, 200rpm room temperature vibration lixiviate 2 hours, pour damping fluid into centrifuge tube, 10,000 * g frozen centrifugation 10 minutes, the supernatant liquor that obtains is the crude enzyme liquid that contains zytase, heat-resisting xylobiase and heat-resisting beta-glucosidase.
The measuring method of Xylanase activity: adopt the DNS method, promptly get the crude enzyme liquid of the above-mentioned preparation of 0.1ml dilution, join in the 1% birch xylan substrate solution of 0.9ml with the citrate buffer solution preparation of 0.05mol/L, pH 6.20,50 ℃ were reacted 10 minutes; With DNS (3,5, the dinitrosalicylic acid method) the reducing sugar amount that determination of color discharged.The Xylanase activity unit definition is: under 50 ℃ of conditions, it is a unit (U) that per minute generates the needed enzyme amount of 1 μ mol wood sugar.
The result shows that the output that solid fermentation produces zytase began remarkable increase in the time of the 4th day, reach the highest during by the 8th day, was 18,580U/g butt carbon source.Enzyme activity began slow decline in the 9th day.The amount of butt carbon source is meant the dry weight of the configuration used carbon source agricultural wastes of substratum (referring to corn cob meal herein).
3) purifying of heat resistant xylanase
Adopt double water-phase method (ATPS) purifying zytase, choose polyoxyethylene glycol (PEG-4000)/ammonium sulfate as becoming phase system, purification process is: in step 2) described solid fermentation prepares after 8 days and contains heat resistant xylanase, in the crude enzyme liquid of heat-resisting xylobiase and heat-resisting beta-glucosidase, the polyoxyethylene glycol that adds described crude enzyme liquid quality 25%, the ammonium sulfate of described crude enzyme liquid quality 50%, and the distilled water of described crude enzyme liquid quality 35%, behind the mixing, leave standstill and treated its complete layering in 2 hours, the upper strata is the zytase of purifying, enzyme liquid through the electrophoresis purification Identification, the result as shown in Figure 1, the result shows, it is pure that heat resistant xylanase behind the double water-phase method purifying has reached electrophoresis, and the molecular weight of this zytase is 26kDa.Swimming lane 1 is the molecular weight of albumen standard among Fig. 1, and swimming lane 2 produces crude enzyme liquid for solid fermentation, and swimming lane 3 is the zytase of purifying.
According to step 2 in the step 1) measuring method of described Xylanase activity, detect the activity of the heat resistant xylanase behind the purifying, the result is as shown in table 1.The crude enzyme liquid enzymatic activity recovery of purifying is 94.1% with this understanding, and the ratio vigor of zytase is 1, and 335.7U/mg, crude enzyme liquid are 266.3U/mg, compares with crude enzyme liquid, and the ratio vigor of zytase has improved 5 times.
The vitality test of zytase behind table 1 purifying
2, the liquid fermenting mode is produced zytase
1) preparation of crude enzyme liquid
Liquid fermentation medium: corn cob meal carbon source 40g/L, Tryptones 10g/L, CaCl
20.3g/L, MgSO
47H
2O 0.3g/L, FeSO
40.3g/L; Sterilized 20 minutes for 121 ℃, the pH value of substratum is 7.
Liquid fermenting: the inoculation of the step 1) preparation in step 1 is with scraping 1cm on thermophilic Paecilomyces varioti J18 (available from DSMZ of Institute of Microorganism, Academia Sinica, preserving number the is AS3.6885) flat board
2The thermophilic paecilomycerol filament of size, be seeded in the triangular flask that the 60ml liquid fermentation medium is housed, in airbath vibration shaking table, 50 ℃, 200rpm shaking culture, cultivate after 5 days fermented liquid 10,000 * g frozen centrifugation 10 minutes, supernatant liquor is the crude enzyme liquid that contains zytase, heat-resisting xylobiase and heat-resisting beta-glucosidase.
According to step 2 in the step 1) measuring method of described Xylanase activity, the output that zytase is produced in the tracer liquid fermentation, the result shows that the output of zytase in the time of the 5th day reaches maximum 1, the 380U/ml fermented liquid.
2) the liquid fermenting mode is produced the purifying of zytase
1. ammonium sulfate precipitation: in crude enzyme liquid, slowly add ammonium sulfate powder ammonium sulfate to the enzyme liquid and reach 20% saturation ratio, stirred 30 minutes in the ice-water bath, then 10,000 * g frozen centrifugation 10 minutes is got supernatant liquor and continue to be added ammonium sulfate powder ammonium sulfate to the enzyme liquid and reach 50% saturation ratio, stirs 1 hour in the ice-water bath, 10,000 * g frozen centrifugation 10 minutes will precipitate with a spot of damping fluid dissolving, and solution is the heat resistant xylanase enzyme liquid of preliminary purification.
2. gel permeation chromatography: the enzyme liquid behind the ammonium sulfate precipitation purifying is further purified through gel filtration chromatography.The Sephacry S-200HR that selects Pharmacia company for use is the gel column filler, chromatography column length 40cm, diameter 1.0cm.Level pad and elution buffer: pH 7.320mM Tris-HCl, flow velocity 0.3ml/min collects target protein peak (determining the target protein peak by enzyme activity determination).
3. ion exchange chromatography: above-mentioned gained enzyme liquid is through the ion exchange chromatography purifying, the DE52 weak anionic ion-exchanger that adopts the production of Whatman company is as the post material, with 20mM pH 7.3 Tris-HCl damping fluid balances, use the NaCl solution gradient wash-out of 0.4-0.7M behind the last sample, collect target protein peak (determining the target protein peak), check its purity by electrophoresis (SDS-PAGE) at last by enzyme activity determination.
Obtained the pure zytase of electrophoresis through above-mentioned three step purifying, in the purge process, according to step 2 in the step 1) measuring method of described Xylanase activity, reach 1229.4U/mg than enzyme work, compared purifying 2.38 times with crude enzyme liquid, the rate of recovery is 38.3%, concrete outcome such as following table 2.
Table 2 liquid fermenting produces zytase purifying table
The preparation of embodiment 2, heat-resisting xylobiase
1, solid fermentation is produced heat-resisting xylobiase
1) inoculation is with the cultivation of thermophilic Paecilomyces varioti (Paecilomyes thermophila) J18:
Thermophilic Paecilomyces varioti J18 is (available from DSMZ of Institute of Microorganism, Academia Sinica, preserving number is AS3.6885) seed is from preserving on the inclined-plane streak inoculation on the PDA inclined-plane or flat board of new preparation, then inclined-plane or flat board is positioned in the 40-60 ℃ of incubator static cultivation 2-8 days.With the quality percentage composition is that 20% glycerine solution washes the spore on the inclined-plane, and the spore liquid that washes is used for solid fermentation and produces enzyme, and the flat board of growth then is directly used in liquid fermenting and uses.
2) solid fermentating mode is produced heat-resisting xylobiase
With thermophilic Paecilomyces varioti J18 (available from DSMZ of Institute of Microorganism, Academia Sinica, preserving number is AS3.6885) is that fermentation strain, natural agricultural wastes wheat straw are that carbon source adopts solid fermentating mode to produce heat-resisting xylosidase.
The substratum of solid fermentation is: wheat straw powder 5g/ bottle (250ml triangular flask), and yeast extract 0.04g/g wheat straw powder, adding is 4.5 times a water of wheat straw powder and yeast extract total mass, sterilizes 20 minutes for 121 ℃.Can inoculate after the cooling.
(spore concentration is 10 to the thermophilic Paecilomyces varioti J18 spore that step 1) is washed with glycerine solution
6Cfu/ml) be seeded in every bottle graft kind 10 that (contains the 5g wheat straw in every bottle, the 0.2g yeast extract) in the 250ml triangular flask that the solid fermentation substratum is housed
6Cfu, with spore mixing in substratum, 50 ℃ leave standstill cultivation, after the cultivation, get one bottle every day, extract crude enzyme liquid and detect heat-resisting xylosidase activity.
The crude enzyme liquid extracting method is: adding on the fermention medium of the triangular flask of solid fermentation is the citrate buffer solution that contains 0.05mol/L (50mM) phosphoric acid and 0.05mol/L (50mM) trisodium citrate of 10 times of quality of substratum, 200rpm room temperature vibration lixiviate 2 hours, pour damping fluid into centrifuge tube, 10,000 * g frozen centrifugation 10 minutes, the supernatant liquor that obtains is the crude enzyme liquid that contains zytase, heat-resisting xylobiase and heat-resisting beta-glucosidase.
The activity of the heat-resisting xylobiase after the Detection and Extraction, the mensuration of heat-resisting xylobiase enzyme activity is according to Lacke (Methods Enzymol, 1988,160:679-684) method is carried out: the enzyme liquid of 50 μ l dilution joins in 200 μ l 5mM pNP-Xylocopyranoside substrates (preparation of the 50mM pH 6.5 phosphate buffer solutions) solution, 50 ℃ are reacted down after 10 minutes, add Na
2CO
3The solution termination reaction is measured light absorption value at the 410nm place.Heat-resisting xylobiase unit of activity is defined as under 50 ℃ of conditions, and it is a unit (U) that per minute generates the needed enzyme amount of 1 μ mol pNP.The result shows that the vigor of heat-resisting xylobiase is at 40U/g wheat straw powder.
3) solid fermentating mode is produced the purifying of heat-resisting xylobiase
1. ammonium sulfate precipitation: in crude enzyme liquid, slowly add ammonium sulfate powder ammonium sulfate to the enzyme liquid and reach 45% saturation ratio, stirred 1 hour in the ice-water bath, then 10,000 * g frozen centrifugation 10 minutes is got supernatant liquor and continue to be added ammonium sulfate powder ammonium sulfate to the enzyme liquid and reach 60% saturation ratio, stirs 1 hour in the ice-water bath, 10,000 * g frozen centrifugation 10 minutes will precipitate with a spot of damping fluid dissolving, and solution is the heat-resisting xylobiase liquid of preliminary purification.
2. with step 1. in through the xylosidase liquid of preliminary purification with the dialysis of 25mM pH 5.2 phosphoric acid buffers, cross DEAE52 weak anionic exchange column (Whatman company 4057200) then, to go up sample after the ion column usefulness 25mM pH 5.2 phosphoric acid buffer balances, then wash with same buffer, treat steadily back 0-150mM NaCl solution gradient wash-out of baseline, elutriant is heat-resisting xylobiase.
3. at last the xylosidase component of above-mentioned collection is dialysed with the phosphoric acid buffer of 25mM pH 5.2, (medium is Q-Sepharose Fast Flow to cross QSFF reinforcing yin essence ion column then, Amersham Bioscience company, 17-0510-01), with going up sample after the phosphoric acid buffer balance of 25mM pH 6.5, then with the same buffer flushing, treat steadily back 0-150mM NaCl solution gradient wash-out of baseline at last, elutriant is heat-resisting xylobiase liquid.
According to step 2) method, the ratio vigor of the heat-resisting xylobiase behind the detection purifying, the result is as shown in table 3, the result shows that the ratio enzyme activity of the heat-resisting xylobiase behind the purifying is 31.68U/mg (crude enzyme liquid is 0.97U/mg), compare with crude enzyme liquid, improved 32.66 times than enzyme activity, enzymatic activity recovery is 7.5%.
Table 3 solid fermentating mode is produced the purifying table of heat-resisting xylobiase
2, liquid fermenting is produced heat-resisting xylobiase
1) preparation of liquid fermenting crude enzyme liquid
Liquid fermentation medium: corn cob meal carbon source 50g/L, urea 10g/L, CaCl
20.3g/L, MgSO
47H
2O 0.3g/L, FeSO
40.3g/L; Sterilized 20 minutes for 121 ℃, the pH value of substratum is 6.5.
Liquid fermenting: the inoculation of the step 1) preparation in embodiment 1 step 1 is with scraping 1cm on the thermophilic Paecilomyces varioti J18 flat board
2The thermophilic paecilomycerol filament of size, be seeded in the triangular flask that the 50ml liquid fermentation medium is housed, in airbath vibration shaking table, 45 ℃, 170rpm shaking culture, cultivate after 5 days, with fermented liquid 10,000 * g frozen centrifugation 10 minutes, supernatant liquor was the crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase.
In the aforesaid liquid fermenting process, measure the output of xylosidase every day, the mensuration of heat-resisting xylobiase enzyme activity is according in the step 1 2) described method measures.
The result shows, begins remarkable increase in the time of the 3rd day, reaches the highest during by the 5th day, and enzyme activity is the 3.15U/ml fermented liquid, is 2.43U/mg albumen than enzyme activity, and enzyme activity began slow decline in the 6th day.
2) purifying of heat-resisting xylobiase
After 5 days, the crude enzyme liquid that contains zytase, heat-resisting xylobiase and heat-resisting beta-glucosidase that obtains carries out purifying, obtains heat-resisting xylobiase with the aforesaid liquid fermentation, and concrete grammar is as described below:
1. ammonium sulfate precipitation: in crude enzyme liquid, slowly add ammonium sulfate powder ammonium sulfate to the enzyme liquid and reach 45% saturation ratio, stirred 1 hour in the ice-water bath, 10,000 * g frozen centrifugation is 10 minutes then, gets supernatant liquor and continue to add ammonium sulfate powder ammonium sulfate to the enzyme liquid and reach 60% saturation ratio, stirred 1 hour in the ice-water bath, 10,000 * g frozen centrifugation 10 minutes will precipitate with a spot of damping fluid dissolving, solution is the heat-resisting xylobiase liquid of preliminary purification, waits until being further purified.
2. with step 1. in through the xylosidase liquid of preliminary purification with the dialysis of 25mM pH 5.2 phosphoric acid buffers, cross DEAE52 weak anionic exchange column (Whatman company 4057200) then, to go up sample after the ion column usefulness 25mMpH 5.2 phosphoric acid buffer balances, then wash with same buffer, treat steadily back 50mM NaCl eluant solution of baseline, elutriant is heat-resisting xylobiase component.
3. with above-mentioned phosphoric acid buffer dialysis of crossing the xylosidase component of weak anionic exchange column collection with 25mM pH 5.2, (medium is Q-Sepharose Fast Flow to cross QSFF reinforcing yin essence ion column then, Amersham Bioscience company, 17-0510-01), go up sample after the phosphoric acid buffer balance with ion column usefulness 25mM pH 6.5, follow with the same buffer flushing, treat steadily back, back 30-120mM NaCl solution gradient wash-out of baseline, elutriant is heat-resisting xylobiase liquid.
The xylosidase liquid of getting this purifying carries out gel electrophoresis, and it is pure that the result shows that the xylosidase behind the purifying reaches electrophoresis, and the proteic molecular weight size of this xylosidase is 53.5kDa.Its electrophoretogram as shown in Figure 2, swimming lane 1 is the molecular weight of albumen standard among Fig. 2, swimming lane 2 is the heat-resisting xylobiase of purifying.
According in the step 1 2) described method detects the ratio vigor of the heat-resisting xylobiase behind the purifying, the result is as shown in table 4, the result shows that the ratio enzyme activity of the heat-resisting xylobiase behind the purifying is 37.65U/mg (crude enzyme liquid is 1.06U/mg), compare with crude enzyme liquid, improved 35.64 times than enzyme activity, enzymatic activity recovery is 3.03%.
Table 4 liquid fermenting produces the purifying table of heat-resisting xylobiase
3) heat-resisting xylobiase character detects
Respectively under differing temps (0-100 ℃, 5 ℃ gradient) according to step 2 in the step 1) described method measures the vigor of heat-resisting xylobiase, the temperature during with the enzyme activity vertex is as the optimum temperuture of heat-resisting xylobiase; Enzyme liquid was handled 30 minutes under optimum temperuture, and residual enzyme activity is measured in the ice-water bath cooling then, measures the temperature stability of heat-resisting xylobiase in contrast with undressed enzyme liquid.The result shows that the optimum temperuture of heat-resisting xylobiase is 55 ℃, has certain thermotolerance, and with the insulation of this enzyme after 30 minutes, residual enzyme activity is 71.2% of a control value under optimum temperuture.
(use citrate buffer solution pH 2-6 respectively at condition of different pH respectively, phosphoric acid buffer pH 5-7, Tris-HCl pH of buffer 7-9, the pH value that glycosides propylhomoserin pH of buffer 9-11 regulates heat-resisting xylobiase liquid) under, according to step 2 in the step 1) described method measures under the condition of different pH enzyme activity of heat-resisting xylobiase that thermophilic Paecilomyces varioti produces.The result shows that this enzyme enzyme activity when pH 6.5 is the highest.When pH was lower than 5.0, enzyme was lived and almost completely is suppressed, and when pH 9.0, still has 35.3% remnant enzyme activity.
With buffered soln (the citrate buffer solution pH 2-6 of heat-resisting xylobiase with different pH, phosphoric acid buffer pH 5-7, Tris-HCl pH of buffer 7-9, glycosides propylhomoserin pH of buffer 9-11) after the mixing, 50 ℃ are incubated 30 minutes down, measure enzyme activity according to the described method of step 1 then, it is residual enzyme activity, with not treated enzyme liquid (vigor is 100%) in contrast, the result shows that this enzyme all has stability preferably in pH 6.5-9.0 scope, enzyme activity loss about 20% illustrates that this enzyme is comparatively stable under the condition of the inclined to one side alkali of neutrality.
With different synthetic substrate (pNP-β-Xylocopyranoside, pNP-β-Arabinocopyranoside, xylo-bioses, xylotriose, Xylotetrose, wooden pentasaccharides) be configured to the solution of 5mM concentration respectively, measure the enzyme activity of heat-resisting xylobiase then according to the described method of step 1; Xylo-oligosaccharide is configured to the solution of 5mM concentration respectively, adds enzymic hydrolysis then, analyze results of hydrolysis with HPLC and TLC.The result shows that this enzyme mainly acts on pNP-β-Xylocopyranoside, can hydrolysis pNP-β-Arabinocopyranoside (only be equivalent to the former 10.9%), synthetic substrate to other does not almost have hydrolytic action, illustrates that this enzyme has stronger Substratspezifitaet; Simultaneously, xylo-oligosaccharides such as this endonuclease capable hydrolysis xylo-bioses, xylotriose, Xylotetrose, wooden pentasaccharides.
The preparation of embodiment 3, heat-resisting beta-glucosidase liquid
1, solid fermentation is produced heat-resisting beta-glucosidase
1) inoculation is with the cultivation of thermophilic Paecilomyces varioti (Paecilomyes thermophila) J18:
Thermophilic Paecilomyces varioti J18 seed is from preserving on the inclined-plane streak inoculation on the PDA inclined-plane or flat board of new preparation, then inclined-plane or flat board is positioned in the 40-60 ℃ of incubator static cultivation 2-8 days.With the quality percentage composition is that 20% glycerine solution washes the spore on the inclined-plane, and the spore liquid that washes is used for solid fermentation and produces enzyme, and the flat board of growth then is directly used in liquid fermenting and uses.
2) solid fermentating mode is produced heat-resisting beta-glucosidase
With thermophilic Paecilomyces varioti J18 is that fermentation strain, natural agricultural wastes wheat straw are that carbon source adopts solid fermentating mode to produce heat-resisting glucuroide.
The substratum of solid fermentation is: wheat straw powder 5g/ bottle (250ml triangular flask), and yeast extract 0.04g/g wheat straw powder, adding is 4.5 times a water of wheat straw powder and yeast extract total mass, sterilizes 20 minutes for 121 ℃.Can inoculate after the cooling.
Is that (spore concentration is 10 to the thermophilic Paecilomyces varioti J18 spore that washes of 20% glycerine solution with step 1) with the quality percentage composition
6Cfu/ml) be seeded in the 250ml triangular flask that the solid fermentation substratum is housed and (contain the 5g wheat straw in every bottle, the 0.2g yeast extract), every bottle graft kind 10
6Cfu, with spore mixing in substratum, 50 ℃ leave standstill cultivation, after the cultivation, get one bottle every day, extract crude enzyme liquid and detect glucosidase activity.
The crude enzyme liquid extracting method is: adding on the fermention medium of the triangular flask of solid fermentation is the citrate buffer solution that contains 0.05mol/L (50mM) phosphoric acid and 0.05mol/L (50mM) trisodium citrate of 10 times of quality of nutrient solution, 200rpm room temperature vibration lixiviate 2 hours, pour damping fluid into centrifuge tube, 10,000 * g frozen centrifugation 10 minutes, the supernatant liquor that obtains is the crude enzyme liquid that contains zytase, heat-resisting xylobiase and heat-resisting beta-glucosidase.
The mensuration of glucoside enzyme activity is with reference to Rosane et al. (FEMS Microbiology Letters, 1977,146:291-295.) measuring method and a little the change, be specially: with the heat resistant xylanase that contains of the above-mentioned preparation of 25 μ l dilution, the crude enzyme liquid of heat-resisting xylobiase and heat-resisting beta-glucosidase joins in 225 μ l (preparation of 50mM pH 6.5 phosphate buffer solutions) the 5mM pNP-Glucopyranoside substrate solution, 50 ℃ are reacted the saturated sodium tetraborate solution of adding 750 μ l termination reaction after 10 minutes down, measure light absorption value at the 405nm place.The glucoside enzyme activity unit is defined as under 50 ℃ of conditions, and it is a unit (U) that per minute generates the needed enzyme amount of 1 μ mol pNP.
The activity of the heat-resisting beta-glucosidase after the Detection and Extraction shows that the vigor of heat-resisting beta-glucosidase is a 50U/g wheat straw powder.
3) solid fermentating mode is produced the purifying of heat-resisting beta-glucoside carbohydrase
1. with the crude enzyme liquid 20mM that contains zytase, heat-resisting xylobiase and heat-resisting beta-glucosidase of step 1) preparation, the phosphoric acid buffer of pH 7.2 is dialysed, and the final pH that makes enzyme liquid is 7.2.
2. the good enzyme liquid of will dialysing is then crossed with phosphoric acid buffer (20mM, pH 7.2) DEAE52 weak anionic post that balance is good, the albumen and other impurity that do not adsorb with the flow velocity flush away of 1.0ml/min with identical damping fluid, use the NaCl solution gradient wash-out of 100-200mM then, collect target protein peak (determining the target protein peak) by enzyme activity determination.
3. cross the S-200 gel column behind the glucuroide part ultrafiltration and concentration of at last previous step being collected, elution flow rate is 0.3ml/min, collect target protein peak (determining the target protein peak), through obtaining the pure enzyme of glucuroide behind the above-mentioned three step purifying by enzyme activity determination.
Show that by gel detection the molecular weight subunit of this glucuroide is 116kDa; According to step 2) described method measures the vigor of the glucuroide of above-mentioned purifying, the result shows, the rate of recovery of the glucuroide of purifying is 29.1%, glucuroide behind the purifying is 84.2U/mg (crude enzyme liquid is 1.3U/mg) than enzyme activity, compare with crude enzyme liquid, improved 64.8 times than enzyme activity.
Table 5 solid fermentating mode is produced the purifying table of heat-resisting beta-glucoside carbohydrase
2, liquid fermenting is produced heat-resisting beta-glucosidase
Liquid fermentation medium: corn cob meal carbon source 50g/L, yeast extract 10g/L, CaCl
20.3g/L, MgSO
47H
2O 0.3g/L, FeSO
40.3g/L; Sterilized 20 minutes for 121 ℃, the pH value of substratum is 7.0.
Liquid fermenting: the inoculation of the step 1) preparation in the step 1 of embodiment 1 is with scraping 1cm on the thermophilic Paecilomyces varioti J18 flat board
2The thermophilic paecilomycerol filament of size, be seeded in the triangular flask that the 80ml liquid fermentation medium is housed, in airbath vibration shaking table, 50 ℃, 200rpm shaking culture, cultivate after 5 days, with fermented liquid 10,000 * g frozen centrifugation 10 minutes, supernatant liquor was the crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase.
In the aforesaid liquid fermenting process, measure the glucoside enzyme activity every day, the mensuration of glucoside enzyme activity is with reference to the step 2 in the step 1).
The result shows that the high yield enzyme level of liquid fermenting malaga Glycosylase reached at the 5th day, was 2.75U/ml, and protein content is 1.9mg/ml, is 1.44U/mg albumen than enzyme activity, and enzyme activity began slow decline in the 6th day.
2) purifying of heat-resisting beta-glucosidase
At first with the crude enzyme liquid 20mM that contains zytase, heat-resisting xylobiase and heat-resisting beta-glucosidase of step 1) preparation, the phosphoric acid buffer of pH 7.2 is dialysed, and the final pH that makes enzyme liquid is 7.2.The good enzyme liquid of will dialysing is then crossed with phosphoric acid buffer (20mM, pH 7.2) DEAE52 weak anionic post that balance is good, the albumen and other impurity that do not adsorb with the flow velocity flush away of 1.0ml/min with identical damping fluid, use the NaCl solution gradient wash-out of 100-200mM then, collect target protein peak (determining the target protein peak) by enzyme activity determination.
Cross the S-200 gel column behind the glucuroide part ultrafiltration and concentration of at last previous step being collected, use 5-50mM, the phosphoric acid buffer wash-out of pH 5.2-8.2, flow velocity is 0.3ml/min, collect target protein peak (determining the target protein peak), through obtaining the pure enzyme of glucuroide behind the above-mentioned three step purifying by enzyme activity determination.
Show that by gel detection the molecular weight subunit of this glucuroide is 116kDa; Gel detection result as shown in Figure 3, swimming lane 1 is the pure enzyme of glucuroide among Fig. 3, swimming lane 2 is the molecular weight of albumen standard.
Measure the vigor of the glucuroide of above-mentioned purifying according to the described method of step 1), the result shows, the rate of recovery of the glucuroide of purifying is 23.3%, glucuroide behind the purifying is 80.83U/mg (crude enzyme liquid is 0.38U/mg) than enzyme activity, compare with crude enzyme liquid, improved 212.5 times than enzyme activity.
The purifying table of the heat-resisting beta-glucosidase of table 6
The optimum temperuture of heat-resisting beta-glucosidase: the vigor of measuring glucuroide respectively under differing temps is to determine the optimal reactive temperature of heat-resisting glucuroide, and the temperature of reaction during with the enzyme activity vertex is an optimum temperuture, with this enzyme activity as 100%.
Measure the stability under the differing temps of heat-resisting beta-glucosidase: enzyme liquid is handled 30min respectively under different temperature, residual enzyme activity is measured in the ice-water bath cooling then, with undressed enzyme liquid (100%) in contrast.
The mensuration of glucoside enzyme activity is according to the step 2 in the step 1) carry out.
Measurement result shows that the optimal reactive temperature of heat-resisting beta-glucosidase is 75 ℃, and enzyme activity descends rapidly after temperature is higher than 75 ℃, and 80 ℃ of enzyme activities are reduced to 60% of maximum.When temperature of reaction reduced, enzyme activity also sharply descended, and the enzyme activity of measuring in the time of 60 ℃ has only about 50% of maximum, illustrates that this enzyme is a thermostable enzyme, has stronger temperature tolerance.The temperature stability result of enzyme liquid shows, enzyme liquid is very stable when being lower than 60 ℃, handle and almost do not have the enzyme activity loss after 30 minutes, handling after 30 minutes enzyme activity for 65 ℃ still preserves about 96%, enzyme activity sharply descends after 70 ℃ of processing, lost about 50%, illustrated that this enzyme has temperature stability preferably.
The optimal pH of heat-resisting beta-glucosidase and pH stability: the damping fluid that damping fluid is changed to different pH values and system respectively joins in the substrate measures this enzyme optimal pH under 50 ℃ of conditions, with the enzyme activity vertex as 100%,
The result shows that the optimum pH of this heat-resisting beta-glucosidase is pH 6.2, and when pH was between the 5-7, this enzyme was more stable, and enzyme activity can keep about 80%, and when the pH of reaction system meta-acid or inclined to one side alkali, enzymic activity is suppressed, and enzyme activity descends very fast.The pH stability measurement result of glucuroide shows, when pH is between 5.0-8.2, enzyme liquid is more stable down at 60 ℃, handle that enzyme activity all can be kept at more than 70% after 2 hours, when pH 6.2,6.7 and 7.2 the time, enzyme activity can be kept at more than 80%, illustrates that this enzyme has good stability under the condition of neutral meta-acid.
Embodiment 4, the application of heat resistant xylanase in steamed bun processing
Add 50g water, 1g salt respectively in 6 parts of 100g flour the inside, add 0 (contrast), 2.5,5,10,15 or 20ppm (10 then respectively
-6G/g) behind embodiment 1 solid fermentation and the zytase that obtains of purifying, stirred 5 minutes in the dough mixing machine middling speed respectively, cut apart (50g/) then, rub circle with the hands, the room temperature steamed bun of moulding is left standstill after 15 minutes in 38 ℃, proof 30 minutes under 85% humidity condition, last boiling water steams 25 minutes, treats steamed bun cooling back its volume of mensuration and quality, calculate specific volume, concrete method of calculation are: specific volume=steamed bun volume (ml)/quality of steamed bread (g).
The result is as shown in table 7, has significantly improved the specific volume of steamed bun in dough behind the interpolation zytase, and not enzyme-added control group steamed bun specific volume is 2.67, and enzyme-added back steamed bun specific volume has just begun to increase along with the raising of enzyme concentration, when enzyme concentration is 5-10ppm (10
-6G/g) time, the specific volume of steamed bun reaches maximum value 3.05, and the specific volume of steamed bun begins to descend along with the rising of enzyme concentration thereafter, when enzyme concentration is 20ppm (10
-6G/g) time, specific volume reduces to 2.87.Add that the whiteness of steamed bun is significantly improved behind a certain amount of zytase; The weave construction of steamed bun improves significantly; Wearing out has obtained delaying to a certain extent, and initial hardness has reduced 10-30% (measuring steamed bun core STRESS VARIATION by matter structure instrument reflects).
The different zytase additions of table 7 are to producing the influence of steamed bun specific volume
Embodiment 5, the application of heat resistant xylanase in bread processing
Add 60g water respectively 7 parts of 100g bread powder the insides and (contain 0 (contrast), 5,10,20,40,60,80ppm (10 respectively
-6G/g) behind embodiment 1 liquid fermenting and the zytase that obtains of purifying), in dough mixing machine according to bread manufacture method and face, add a certain amount of salt and shortening midway, leave standstill after question handler is become reconciled, cut apart, weighing, stranding circle, fermentation, moulding, then the bread of moulding is proofed 60 minutes under 38 ℃, 85% humidity condition, last baking 20 minutes treats to measure behind the bread naturally cooling specific volume of bread, and concrete method of calculation are: specific volume=loaf volume (ml)/Bread Quality (g)
The result is as shown in table 8, and the result shows, has significantly improved the specific volume of bread in dough behind the interpolation zytase, and not enzyme-added control group bread specific volume is 5.8, and enzyme-added back bread specific volume has just begun to increase along with the raising of enzyme concentration, when enzyme concentration is 60ppm (10
-6G/g) time, the specific volume of bread reaches maximum value 8.0, has improved 38% compared with the control, and the specific volume of bread begins to descend along with the rising of enzyme concentration thereafter.Add that the weave construction of bread improves significantly behind a certain amount of zytase; The aging of bread obtained prolonging to a certain extent, and slow storage is after 7 days, and the hardness of bread core is the 40-80% (measuring bread core STRESS VARIATION by matter structure instrument reflects) of contrast.
The different zytase additions of table 8 are to the influence of bread specific volume:
The application of embodiment 6, heat-resisting xylobiase and heat resistant xylanase synergetic hydrolysis xylan
With behind embodiment 1 solid fermentation and behind the zytase 50U that obtains of purifying and embodiment 2 liquid fermentings and the xylosidase 2.5U that obtains of purifying make the mixed enzyme solution of zytase and xylosidase after mixing, add the 100ml mass concentration respectively and be 1% corn cob xylan substrate solution or mass concentration and be in 1% the birch xylan substrate solution, the reaction that under 50 ℃ of conditions, is hydrolyzed, with behind independent adding 50U embodiment 1 solid fermentation and the zytase that obtains of purifying be contrast.After reaction, gathered hydrolyzation sample respectively in 1,2,4,6,12 and 24 hour, adopt Somogyi method (Somogyi then, M.A new reagent for the determination of sugars.J.Biol.Chem., 1945,160:61-68.) measure the reducing sugar amount that generates.
The result shows: when being substrate with the birch xylan, in initial reaction stage, the xylan hydrolysis rate of mixed enzyme solution is with little with the percent hydrolysis difference of zytase contrast separately, reacted 1 hour, the amount of reducing sugar is respectively 1.31mg/ml and 0.93mg/ml in the hydrolyzed solution of mixed enzyme and independent zytase, prolongation along with the reaction times, both speed of reaction difference strengthen gradually, hydrolysis 12 hours, the amount of reducing sugar is respectively 2.76mg/ml and 1.72mg/ml in both hydrolyzed solutions, after this reducing sugar amount does not almost change in the hydrolyzed solution, and the content of reducing sugar exceeds 64% than xylanase hydrolysis liquid in final 24 hours synergy hydrolyzed solutions; Results of hydrolysis and the birch xylan that with the corn cob xylan is substrate is that the results of hydrolysis of substrate is similar, and hydrolysis is after 24 hours, and the reducing sugar amount exceeds 40% than xylanase hydrolysis liquid in the thick enzyme corn cob hydrolyzed solution.
Get birch xylan hydrolyzed solution sample and carry out the TLC analysis, found that zytase can only be hydrolyzed to birch xylan xylo-bioses and xylotriose, it can not be degraded into wood sugar fully, birch xylan then all is degraded to wood sugar behind the adding xylosidase.The above results shows that the synergy of heat resistant xylanase and heat-resisting xylobiase can improve the hydrolysis efficiency of birch xylan greatly, and heat-resisting xylobiase is degraded to wood sugar to realize aspect the bio-transformation very big application prospect being arranged fully at xylan.
The hydrolysis properties of embodiment 7, heat-resisting beta-glucosidase
Be substrate (specifically as shown in table 9) with different disaccharides, glucosides and glycan respectively, the heat-resisting glucuroide behind adding embodiment 3 liquid fermentings and the purifying, the reaction that is hydrolyzed under 50 ℃ of conditions by measuring glucose content, is measured its activity.Unit of activity is defined as under 50 ℃ of conditions, the reaction per minute generates 1 μ mol pNP or the needed enzyme amount of glucose is a unit (U), glucose content adopts oxidation enzyme process test kit (the safe clinical reagent of Beijing northization company limited, catalog number: 001017) measure, measurement result is as shown in table 9, the result shows that the heat-resisting beta-glucosidase behind embodiment 3 liquid fermentings and the purifying has very strong specificity to synthetic substrate, hydrolysis effect to pNP-β-Glucopyranoside (p-nitrophenyl-β-glucopyranoside) is best, and other substrate is shown very low enzyme activity.Simultaneously, this enzyme can also the hydrolysis fiber disaccharides, gentiobiose, saligenin, Genistoside, amygdalin, Cyclosiversioside F and daidzin, wherein best to the hydrolysis effect of cellobiose, the enzyme activity that hydrolysis reaches is 70.12U/g, glycan such as this enzyme can not hydrolyzed xylan, Mierocrystalline cellulose, dextran, starch.
The hydrolysis specificity of table 9 glucuroide
Claims (8)
1, a kind of method for preparing heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase, be to be carbon source with agricultural wastes, with thermophilic Paecilomyces varioti (Paecilomyces thermophila) J18, at 40-60 ℃, after being inoculated into the liquid fermentation medium top fermentation, filtering fermentating liquid obtains the crude enzyme liquid of heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase;
Described liquid fermentation medium is for containing agricultural wastes powder 10-60g/L, urea or yeast extract or Tryptones 5-20g/L, CaCl
20.1-1g/L, MgSO
47H
2O 0.1-1g/L, FeSO
40.1-1g/L initial pH value is the substratum of 5-9;
Described agricultural wastes are wheat straw, wheat bran or corn cob.
2, method according to claim 1 is characterized in that: described liquid fermentation medium is for containing agricultural wastes powder 40g/L, urea or yeast extract or Tryptones 10g/L, CaCl
20.3g/L, MgSO
47H
2O 0.3g/L, FeSO
40.3g/L initial pH value is 7 substratum;
Described agricultural wastes are wheat straw, wheat bran or corn cob.
3, method according to claim 2 is characterized in that: the time of described fermentation is 2-10 days.
4, according to any described method among the claim 1-3, it is characterized in that: described method also comprise the described crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase carried out purifying after, obtain heat resistant xylanase, heat-resisting xylobiase or heat-resisting beta-glucosidase.
5, method according to claim 4 is characterized in that: the purification process of the heat resistant xylanase of described liquid fermenting comprises the steps:
1) in the crude enzyme liquid that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase that liquid fermenting obtains, slowly adds ammonium sulfate powder ammonium sulfate to the enzyme liquid and reach 20% saturation ratio, stirred 30-60 minute in the ice-water bath, frozen centrifugation then, getting supernatant liquor continue to add ammonium sulfate powder ammonium sulfate to the enzyme liquid and reaches 50% saturation ratio, stirred 0.5-1.5 hour in the ice-water bath, frozen centrifugation, to precipitate with a spot of damping fluid dissolving, solution is the heat resistant xylanase enzyme liquid of preliminary purification;
2) with the heat resistant xylanase enzyme liquid of preliminary purification, cross Sephacry S-200 HR gel filtration chromatography, with pH 7.320mM Tris-HCl buffer solution elution, collect target peak;
3) with step 2) elutriant that obtains collects the target peak that obtains and crosses DEAE52 weak anionic chromatography column, with the NaCl solution gradient wash-out of 0.4-0.7mol/L, collects the heat resistant xylanase that the target protein peak is purifying.
6, method according to claim 4 is characterized in that: the purification process of described heat-resisting xylobiase or heat-resisting beta-glucosidase is a chromatography.
7, method according to claim 6 is characterized in that: the step that described purifying obtains heat-resisting xylobiase is:
1) contains heat resistant xylanase to what described liquid fermenting obtained, slowly add ammonium sulfate powder ammonium sulfate to the enzyme liquid in the crude enzyme liquid of heat-resisting xylobiase and heat-resisting beta-glucosidase and reach the 30-50% saturation ratio, stirred 0.5-2 hour in the ice-water bath, the centrifugal 10-30 of 8000-14000 * g minute then, get supernatant liquor continuation interpolation ammonium sulfate powder to enzyme liquid and reach the 55-65% saturation ratio, stirred 0.5-2 hour in the ice-water bath, 8,000-14, the centrifugal 10-30 of 000 * g minute, precipitation is dissolved the heat-resisting xylobiase liquid that obtains preliminary purification with the phosphoric acid citrate buffer solution;
2) above-mentioned enzyme liquid through preliminary purification is dialysed with 5-50mM pH 5.0-8.0 phosphoric acid buffer, cross DEAE52 weak anionic chromatography column then, use the 20-200mMNaCl eluant solution;
3) elutriant that step 2) obtains is crossed Q-Sepharose Fast Flow reinforcing yin essence ion chromatography post then with the phosphoric acid buffer dialysis of 5-50mM pH 5.0-8.0, uses 30-300mM NaCl eluant solution at last, and elutriant is heat-resisting xylobiase.
8, method according to claim 6 is characterized in that: the step of the heat-resisting beta-glucosidase of described purifying is:
1) the crude enzyme liquid 5-50mM that contains heat resistant xylanase, heat-resisting xylobiase and heat-resisting beta-glucosidase that described liquid fermenting is obtained, the phosphoric acid buffer dialysis of pH 5.2-8.2;
2) step 1) is crossed DEAE52 weak anionic chromatography column, used 5-50mM, the phosphoric acid buffer of pH 5.2-8.2 is used the NaCl eluant solution of 50-500mM then with albumen and other impurity that the flow velocity flush away of 0.2-2ml/min does not adsorb, collects target protein;
3) with step 2) the collection liquid that obtains crosses Sephacryl-S 200HR chromatography column, uses 5-50mM, and the phosphoric acid buffer wash-out of pH 5.2-8.2, flow velocity are 0.2-2ml/min, collect the heat-resisting beta-glucosidase that target protein is purifying.
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