CN103865902B - A kind of preparation method of high temperature resistant alkalescent xylanase - Google Patents
A kind of preparation method of high temperature resistant alkalescent xylanase Download PDFInfo
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- CN103865902B CN103865902B CN201310682220.2A CN201310682220A CN103865902B CN 103865902 B CN103865902 B CN 103865902B CN 201310682220 A CN201310682220 A CN 201310682220A CN 103865902 B CN103865902 B CN 103865902B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
- D21C9/10—Bleaching ; Apparatus therefor
Abstract
The invention discloses a kind of preparation method of high temperature resistant alkalescent xylanase, belong to technical field of enzyme preparation.To produce the bacillus acidocldarius (Bacillus thermophilus) 701 of high temperature resistant alkalescent xylanase for starting strain, and by Optimal Medium composition and zymotechnique through liquid submerged fermentation prepare a kind of high temperature resistant, alkali resistance is strong, action condition is wide in range, do not have the high temperature resistant alkalescent xylanase in the field such as applicable papermaking, food and feed of cellulase activity.Specific activity of enzyme is 350-420IU/ml; Optimal reactive temperature is 65 DEG C, and Applicable temperature scope is 40-75 DEG C; Optimal reaction pH value is 10.0, and being suitable for pH value in reaction scope is 5.0-11.0.
Description
Technical field
The invention belongs to technical field of enzyme preparation, particularly a kind of preparation method of high temperature resistant alkalescent xylanase.
Background technology
Mierocrystalline cellulose, hemicellulose and xylogen are the main components of plant hemicellulose, their actings in conjunction constitute the support frame of plant cell wall, among three, hemicellulose occupies more than 35% of plant dry weight, hemicellulose is also the general name of multiple complex plycan, its main component is xylan, and it is a kind of poly five-carbon sugar of complexity.The main chain of xylan by β-1-4 glycosidic link, multiple xylopyranosyl is coupled together, and side chain is connected to different substituting groups, has like this (Collins et al., 2005) such as ethanoyl, glucal acyl group, L-arabinose bases.So the degradable common participation just needing multiple enzyme of xylan.Generally xylan is divided into hardwood xylan and cork xylan.Hardwood xylan is mainly polymerized by 0-acetyl-4-0-methyl glucose alditol acyl wood sugar, is connected with β-1-4 glycosidic link.Cork glycan is polymerized (Beg et al, 2001) by Arabic 4-0-methyl glucose alditol acyl xylose residues.Some sorting techniques are also divided into soluble xylan and insolubility xylan xylan.
Zytase (Isosorbide-5-Nitrae-β-D-xylanase:EC3.2.1.8) is a kind of important industrial enzymes, playing biocatalysis, xylanolitic can be become protein or the RNA of polysaccharide or monose when decomposing xylan.Zytase is extensively present in natural organism.Bacterium, fungi, zytase can be produced in animal body etc.
Due to the using value that zytase is industrially potential, cause more attentions of people within the past ten years, wherein alkalescent xylanase is mainly used in paper pulp and papermaking enterprise.The application of alkalescent xylanase in paper industry mainly comprises slurrying, assists bleaching, changes in fibrous property, deinking etc.
In the industrial production, pulping process is under the condition of alkalescence, delignification is removed by the method for high temperature steaming, 98% is reached for making dissolving pulping fibre element purity, this just needs a large amount of sodium-hydroxide treatment paper pulp, cause serious environmental pollution, many scholars slurrying then trial is carried out a biological disposal upon for this reason, and should be heat-resisting and alkaline-resisting for the zytase of pulping bleaching.Feng Jianliang etc. (Kimura et al.2000) compare test with xylanase pretr eatment wheat straw and conventional chemistry slurrying, and xylanase pretr eatment improves the delignification degree of raw material, can also reduce the Kappa number of slurrying.Zytase has more extensive and deep research in auxiliary bleaching, and achieves very large achievement.Research finds, no matter is softwood pulp, hardwood pulp or bamboo pulp, straw pulp, and zytase all can be degraded xylogen remaining in paper pulp, improves whiteness (AL BALAA et al, 2006; Meek andLipman, 1922).Zytase for association with pulp bleaching must have high temperature resistant feature, Khasin etc. obtain one and derive from alkaline bacterial strain Bacillus sterarothermophilus T26 zytase, this zytase has best bleaching effect (Khasin et al when pH9.0 and 65 DEG C to paper pulp, 1993), a lot of zytase does not possess such feature, limits commercial applications.The waste paper quantity that the whole world produces every year is surprising, and after waste paper reclaims, recycling work just becomes particularly important, can make also to protect environment while resource recycling.1991, ink can remove by application zytase from news paper waste by Korea S scholar reported first.After this, people begin one's study and utilize zytase to carry out deinking, to alleviate or to eliminate the environmental pollution (Cao Junwei etc., 2004) that Chemical Deinking brings.
At present, the green cleaning and bleaching of the paper pulp being representative with element-free chlorine and Totally-chlorine-free bleaching technology has become the inexorable trend of countries in the world paper-making industry association with pulp bleaching development, zytase enzyme process helps drift novel process to be applied in more than 30 large-scale paper plants of family of Europe and North America, becomes biotechnology in the most successful example of paper industry application.Wherein Canada have an appointment 10% sulphate process pulp mill have employed this novel process.Novozymes Company of Denmark and U.S.'s mountain pass this chemical company Deng Duojia zymin manufacturer, be proposed the zytase and cellulase product innovation that are specifically designed to slurrying process one after another, but up to the present, the industrial zytase being applied to association with pulp bleaching is neutral meta-acid mostly, optimal reactive temperature is mostly at about 50 DEG C, as everyone knows, pulp cooking and bleaching are substantially all carry out under the condition of high temperature and highly basic, existing low temperature acid zytase product is made to be subject to great restriction in the application in this field, in addition, at feed and field of food, what zytase was applied in the granulation of feed and food bakes in operation, still require that zytase used can keep higher enzyme to live under the high temperature conditions, therefore, research and develop resistant to elevated temperatures zytase product and will bring good economic benefit and social benefit.
The xylanase bacterium that sifting property is more excellent from natural microbial is the means the most effective effectively obtaining high yield zytase bacterial strain.Since zytase has bio-bleaching function, people have invested alkalescent xylanase more sight, have screened the production bacterial strain much with the alkalescent xylanase of application prospect, have been separated a lot of alkalescent xylanase.1973, the Late Cambrian such as Horikoshi were from the zytase (Horikoshi and Atsukawa, 1973) in alkaline bacterial, and from alkaline bacterial Bacillus sp.No.C25922, purifying obtains zytase, its optimal pH is 6-8.Afterwards, found 2 zytases from alkaline bacterial Bacillus No.C2125: zytase A and zytase N, wherein zytase A also has certain enzyme to live (Honda et al, 1985) when pH12.The basophilic thermophile bacteria Bacillus sp. (NCIM59) that Dey etc. (Dey et al, 1992) are separated, can produce the zytase that 2 do not have cellulase activity, they have good alkali resistance, and optimal pH is 10.0.It is 9.0(Nakamura et al that Nakamura etc. report its optimal pH of Extracellular xylanase J deriving from alkaline bacterial strain Bacillus sp.41M21,1993).Most of mycetogenetic zytase is generally acidic xylanase, but also there is exception, Taneja etc. screen a strain basophilic fungi AspergillusnidulansKK299, the optimal pH of the zytase that it produces is 8(Taneja et al,, and this enzyme has higher affinity to hardwood xylan than cork xylan 2002).
Chinese patent CN101701205A discloses a kind of alkali-tolerant xylanase XynE2 and gene thereof and application, produce alkali-tolerant xylanase optimum pH 7.8, optimum temperuture 65 DEG C; Chinese patent CN102586134B discloses ocean green color-producing streptomycete bacterial strain and the application thereof that alkaline-resisting salt-tolerant xylanase is produced in a strain, produce zytase optimum pH 6.0, optimum temperuture 70 DEG C; The high-yield strains that Chinese patent CN102321558A discloses a kind of fire resistant xylanase and the method and the enzyme that obtains that utilize this bacterium to ferment to produce fire resistant xylanase, produce fire resistant xylanase optimum pH 7.0, optimum temperuture 60 DEG C.
In view of this, international and domestic all multi-experts, scholar and Scientific Research Workers are not stinted fund, manpower and time, increase R&D intensity that is high temperature resistant, alkali-tolerant xylanase, be intended to by biotechnology the application in the fields such as papermaking, food, feed push to one higher, upgrade, the stronger New Times, it is no matter the acquisition of novel bacterial, still gene recombination technology is adopted, although consume so big financial resources and energy, but result is unsatisfactory, be difficult to reach high temperature resistant, the R&D target of alkali-tolerant xylanase and requirement, or heatproof is not alkaline-resisting; Alkaline-resisting not heatproof; Alkaline-resisting high temperature resistant scope is not wide in range; Zytase is impure and have cellulase activity; Substrate specificity is uncomfortable etc., and the high temperature resistant alkalescent xylanase prepared higher, the alkaline-resisting high temperature resistant wide scope of stronger, the resistance to temperature of a kind of alkali resistance, do not have cellulase activity and be applicable to the field such as papermaking, food and feed is still the unremitting pursuit of those skilled in the art and R&D direction.
Summary of the invention
Technical problem solved by the invention is the product defects overcoming existing high temperature resistant alkalescent xylanase, to produce the bacillus acidocldarius (Bacillus thermophilus) 701 of high temperature resistant alkalescent xylanase for starting strain, and by Optimal Medium composition and zymotechnique prepare a kind of high temperature resistant, alkali resistance is strong, action condition is wide in range, do not have the high temperature resistant alkalescent xylanase in the field such as applicable papermaking, food and feed of cellulase activity.
The bacillus acidocldarius 701 of product provided by the invention is high temperature resistant alkalescent xylanase gathers soil sample to isolate a strain bacillus acidocldarius HYX0021 from Xue Li paper mill, Hunan Province, through ultraviolet mutagenesis repeatedly and nitrosoguanidine mutagenesis screening obtain, characteristic be high temperature resistant, alkali resistance is strong.
The bacterial strain of product provided by the invention is high temperature resistant alkalescent xylanase is specially bacillus acidocldarius 701.This bacterial strain is preserved in China typical culture collection center on November 3rd, 2013 and (is called for short CCTCC, address is: China. Wuhan. and Wuhan University, postcode: 430072), preserving number is CCTCC NO:M2013537, and Classification And Nomenclature is bacillus acidocldarius 701(Bacillusthermophilus701).
High temperature resistant alkalescent xylanase Rate activity prepared by the present invention is 350-420IU/ml, Applicable temperature scope is 40-75 DEG C, optimal reactive temperature 65 DEG C, at 75 DEG C of enzymes complete stability alive; Being suitable for pH value in reaction scope is 5.0-11.0, and the enzyme complete stability alive when pH value is 11.0, optimal reaction pH value is 10.0.
A preparation method for high temperature resistant alkalescent xylanase, comprises the steps:
(1) actication of culture
The slant strains of intact bacillus acidocldarius 701 is inoculated in slant medium, cultivates 24-36h for 37-45 DEG C and carry out actication of culture, so activation 2-3 time;
Described slant medium consists of: extractum carnis 3-10g, sodium-chlor 5-12g, peptone 10-20g, glucose 2-5g, agar 15-20g, herbal mediciment powder 5-10g, distilled water l000mL, pH value 5-11,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take Radix Astragali 20-30 part, Radix Codonopsis 10-18 part, radix bupleuri 10-15 part, root of large-flowered skullcap 10-15 part; Respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then being cooled to 45-60 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
Described mixed enzyme addition is the 5-10% of mixture gross weight.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10-20 part, outer beta-glucanase 10-20 part, beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta-amylase 10-15 part, neutral protease 10-15 part, aspartic protease 10-15 part, superoxide-dismutase 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part.
The mass ratio of described ethanol and propyl alcohol is 1:1-1.5.
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: the slant strains 1-2 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 38-43 DEG C, incubation time 10-15h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 38-43 DEG C, incubation time 10-15h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 37-45 DEG C, stirring velocity 200-400rpm, ventilation (V/V) 1:1-2, tank pressure 0.05Mpa, incubation time 10-20h;
Described one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, extractum carnis 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, herbal mediciment powder 1.5-2%, trehalose 1-3%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.1-0.3%, insufficient section pure water is supplied, pH value 5-11,121-123 DEG C of sterilizing 30-40min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 5-15%, yeast powder 0.4-0.8%, herbal mediciment powder 1.5-2%, trehalose 1-3%, peptone 0.1-0.5%, corn steep liquor 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, Trisodium Citrate 0.1-0.5%, birch xylan 0.1-0.8%, insufficient section pure water is supplied, pH value 5-11,121-123 DEG C of sterilizing 30-40min.
Described seeding tank fermented liquid cell concentration is 7.0x10
8-8.0x10
8individual/mL;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 37-45 DEG C, stirring velocity 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 10-15 DEG C, constant temperature culture 15-20h; Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 20-30h; Finally slowly be warming up to 10-15 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Continue slowly to be warming up to 37-45 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 15-30%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 5-11;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/ml-8mg/mL, start to add supplemented medium, feed supplement amount is to maintain fermented liquid reducing sugar content for 2mg/mL-5mg/mL;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, soybean cake powder 15-25g, herbal mediciment powder 30-50g, trehalose 30-40g, birch xylan 5-15g, yeast powder 4-8g, corn steep liquor 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium primary phosphate 1-2g, Trisodium Citrate 1-5g, defoamer 0.1-1g, pure water l000mL, pH value 5-11,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 20-30%, Semen Maydis powder 10-20%, bean powder 15-25%, herbal mediciment powder 5-10%, and insufficient section pure water is supplied, pH value 5-11,121-123 DEG C of sterilizing 30-40min.
The concocting method of described fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 5-11, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 15-30min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 15-30min and liquefy, finally add other raw material, stir, adjust initial pH5-11,121-123 DEG C of sterilizing 30-40min for subsequent use.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry high temperature resistant alkalescent xylanase.
Described allocation process does not add any sanitas, can add concentrated enzyme liquid gross weight 0.5-5% herbal mediciment powder.
Beneficial effect:
1. the method for the high temperature resistant alkalescent xylanase of preparation of the present invention is to have high temperature resistant, that alkali resistance is strong bacillus acidocldarius 701 for bacterial classification, and the high temperature resistant alkalescent xylanase specific activity of enzyme of preparation is 350-420IU/mL; Applicable temperature scope is 40-75 DEG C, optimal reactive temperature 65 DEG C, at 75 DEG C of enzymes complete stability alive; Being suitable for pH value in reaction scope is 5.0-11.0, and the enzyme complete stability alive when pH value is 11.0, optimal reaction pH value is 10.0.Higher than existing high temperature resistant alkaline xylanase activity power, resistance to temperature is high, and alkali resistance is strong, and enzyme effect optimum pH wide scope, is particularly suitable for the industrialization demand in the field such as papermaking, food and feed.
2. the preparation method of the high temperature resistant alkalescent xylanase of the present invention adopts the zymotechnique of gradient cooling and gradient increased temperature to significantly improve the anti-stress ability of bacillus acidocldarius 701, causes strain enzyme-producing ability to manifest to greatest extent.And the present invention implements full optimization to bacillus acidocldarius 701 substratum composition, with the addition of the root of large-flowered skullcap with anti-heat stress original work, radix bupleuri, with the addition of and there is adjustment and repair body function, the immunologic function of enhancing body, there is the Radix Astragali of Tiny ecosystem regulatory function, Radix Codonopsis etc., further enhancing the body function of microorganism under same yeasting, adaptation of virus and collaborate, and then strengthen the metabolic function of bacillus acidocldarius 701, the present invention is made to produce high temperature resistant alkaline xylanase activity power high, action condition is wide in range, stability is strong, be suitable for suitability for industrialized production.
3. in the preparation method of the high temperature resistant alkalescent xylanase of the present invention, the important component part Chinese herbal medicine powder of substratum adopts enzymolysis process and aqueous extraction-alcohol precipitation technology to combine, not only significantly improve various medium-height grass the effective elements of the medicine, enhance the action potency of herbal medicine, but also provide rare nutritive substance (as herbal polysaccharide etc.) for fermentable.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Embodiment 1
The bacillus acidocldarius 701 of product provided by the invention is high temperature resistant alkalescent xylanase gathers soil sample to isolate a strain bacillus acidocldarius HYX0021 from Xue Li paper mill, Hunan Province, through ultraviolet mutagenesis repeatedly and nitrosoguanidine mutagenesis screening obtain, characteristic be high temperature resistant, alkali resistance is strong.
The bacterial strain of product provided by the invention is high temperature resistant alkalescent xylanase is specially bacillus acidocldarius 701.This bacterial strain is preserved in China typical culture collection center on October 12nd, 2013, and (be called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College postcode: 430072), preserving number is CCTCC M2013537.
Described slant medium consists of: extractum carnis 3-10g, sodium-chlor 5-12g, peptone 10-20g, glucose 2-5g, agar 15-20g, herbal mediciment powder 5-10g, distilled water l000mL, pH value 5-11,121 DEG C of sterilizing 20min;
Institute's one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, extractum carnis 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, herbal mediciment powder 1.5-2%, trehalose 1-3%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.1-0.3%, insufficient section pure water is supplied, pH value 5-11,121-123 DEG C of sterilizing 30-40min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 5-15%, yeast powder 0.4-0.8%, herbal mediciment powder 1.5-2%, trehalose 1-3%, peptone 0.1-0.5%, corn steep liquor 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, Trisodium Citrate 0.1-0.5%, birch xylan 0.1-0.8%, insufficient section pure water is supplied, pH value 5-11,121-123 DEG C of sterilizing 30-40min.
Described fermention medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, soybean cake powder 15-25g, herbal mediciment powder 30-50g, trehalose 30-40g, birch xylan 5-15g, yeast powder 4-8g, corn steep liquor 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium primary phosphate 1-2g, Trisodium Citrate 1-5g, defoamer 0.1-1g, pure water l000mL, pH value 5-11,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 20-30%, Semen Maydis powder 10-20%, bean powder 15-25%, herbal mediciment powder 5-10%, and insufficient section pure water is supplied, pH value 5-11,121-123 DEG C of sterilizing 30-40min.
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take Radix Astragali 20-30 part, Radix Codonopsis 10-18 part; Radix bupleuri 10-15 part, root of large-flowered skullcap 10-15 part, respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then being cooled to 45-60 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
Described mixed enzyme addition is the 5-10% of mixture gross weight.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10-20 part, outer beta-glucanase 10-20 part, beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta-amylase 10-15 part, neutral protease 10-15 part, aspartic protease 10-15 part, superoxide-dismutase 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part.
The mass ratio of described ethanol and propyl alcohol is 1:1-1.5.
Embodiment 2
A preparation method for high temperature resistant alkalescent xylanase, comprises the steps:
(1) actication of culture
The slant strains of intact bacillus acidocldarius 701 is inoculated in slant medium, cultivates 24h for 37 DEG C and carry out actication of culture, so activation 2 times;
Described slant medium consists of: extractum carnis 3g, sodium-chlor 5g, peptone 10g, glucose 2g, agar 15g, herbal mediciment powder 5g, distilled water 1000mL, pH value 5,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take the Radix Astragali 20 parts, Radix Codonopsis 10 parts, radix bupleuri 10 parts, the root of large-flowered skullcap 10 parts, respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3 times of weight in container, control temperature 70 DEG C keeps 2h, is then cooled to 45 DEG C, add mixing enzyme preparation and carry out enzymolysis, be 5.5 by lactic acid adjust ph, enzymolysis 2h, finally adds the mixture of mixture 0.5 times of w ethanol and propyl alcohol, control temperature to 60 DEG C keeps 3h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
Described mixed enzyme addition is 5% of mixture gross weight.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10 parts, outer beta-glucanase 10 parts, beta-glucosidase 10 parts, zytase 15 parts, pentosanase 15 parts, Pullulanase 20 parts, beta-amylase 10 parts, neutral protease 10 parts, aspartic protease 10 parts, superoxide-dismutase 5 parts, glucose oxidase 5 parts, acid phosphatase 5 parts.
The mass ratio of described ethanol and propyl alcohol is 1:1.
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: slant strains 2 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 38 DEG C, incubation time 10h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 38 DEG C, incubation time 10h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 37 DEG C, stirring velocity 200rpm, ventilation (V/V) 1:1, tank pressure 0.05Mpa, incubation time 10h;
Institute's one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.3%, glucose 1%, peptone 0.3%, extractum carnis 0.5%, dipotassium hydrogen phosphate 0.8%, herbal mediciment powder 1.5%, trehalose 1%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.1%, insufficient section pure water is supplied, pH value 5,121 DEG C of sterilizing 30min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 5%, yeast powder 0.4%, herbal mediciment powder 1.5%, trehalose 1%, peptone 0.1%, corn steep liquor 0.1%, dipotassium hydrogen phosphate 0.8%, magnesium sulfate 0.05%, Trisodium Citrate 0.1%, birch xylan 0.1%, insufficient section pure water is supplied, pH value 5,121 DEG C of sterilizing 30min.
Described seeding tank fermented liquid cell concentration is 7.0x10
8individual/mL;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 37 DEG C, stirring velocity 200rpm, ventilation (V/V) 1:1, incubation time 10h; Then with 1 DEG C/h rate of temperature fall slow cooling to 10 DEG C, constant temperature culture 15h; Continue with 1 DEG C/h rate of temperature fall slow cooling to 2 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 20h; Finally slowly be warming up to 10 DEG C, constant temperature culture 15h with 1 DEG C/h temperature rise rate; Continue slowly to be warming up to 37 DEG C, constant temperature culture 15h with 1 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 15%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 5;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/mL-8mg/mL, start to add supplemented medium, feed supplement amount is to maintain fermented liquid reducing sugar content for 2mg/mL-5mg/mL;
Put tank standard: the old and feeble self-dissolving of 60% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 50g, Semen Maydis powder 50g, soybean cake powder 15g, herbal mediciment powder 30g, trehalose 30g, birch xylan 5g, yeast powder 4g, corn steep liquor 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium primary phosphate 1g, Trisodium Citrate 1g, defoamer 0.1g, pure water 1000mL, pH value 5,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 20%, Semen Maydis powder 10%, bean powder 15%, herbal mediciment powder 5%, and insufficient section pure water is supplied, pH value 5,121 DEG C of sterilizing 30min.
The concocting method of described fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 5, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 15min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 15min and liquefy, finally add other raw material, stir, adjust initial pH5,121 DEG C of sterilizing 30min are for subsequent use.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry high temperature resistant alkalescent xylanase.
Described allocation process does not add any sanitas, can add concentrated enzyme liquid gross weight 0.5% herbal mediciment powder.
The high temperature resistant alkalescent xylanase liquid enzymes Rate activity obtained through above-mentioned preparation method is 350IU/mL.
Embodiment 3
A preparation method for high temperature resistant alkalescent xylanase, comprises the steps:
(1) actication of culture
The slant strains of intact bacillus acidocldarius 701 is inoculated in slant medium, cultivates 30h for 41 DEG C and carry out actication of culture, so activation 3 times;
Described slant medium consists of: extractum carnis 6g, sodium-chlor 8g, peptone 15g, glucose 5g, agar 20g, herbal mediciment powder 8g, distilled water 1000mL, pH value 8,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take the Radix Astragali 25 parts, Radix Codonopsis 15 parts, radix bupleuri 12 parts, the root of large-flowered skullcap 12 parts, respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 5 times of weight in container, control temperature 80 DEG C keeps 3h, is then cooled to 50 DEG C, add mixing enzyme preparation and carry out enzymolysis, be 6 by lactic acid adjust ph, enzymolysis 3h, finally adds the mixture of mixture 2 times of w ethanol and propyl alcohol, control temperature to 70 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
Described mixed enzyme addition is 8% of mixture gross weight.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 15 parts, outer beta-glucanase 15 parts, beta-glucosidase 12 parts, zytase 18 parts, pentosanase 18 parts, Pullulanase 25 parts, beta-amylase 18 parts, neutral protease 12 parts, aspartic protease 12 parts, superoxide-dismutase 8 parts, glucose oxidase 8 parts, acid phosphatase 8 parts.
The mass ratio of described ethanol and propyl alcohol is 1:1.2.
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: slant strains 2 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 40 DEG C, incubation time 10h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 40 DEG C, incubation time 10-15h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 41 DEG C, stirring velocity 300rpm, ventilation (V/V) 1:1.5, tank pressure 0.05Mpa, incubation time 15h;
Described one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.4%, glucose 1.2%, peptone 0.4%, extractum carnis 0.6%, dipotassium hydrogen phosphate 1.1%, herbal mediciment powder 1.8%, trehalose 2%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.2%, insufficient section pure water is supplied, pH value 8,123 DEG C of sterilizing 40min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 10%, yeast powder 0.6%, herbal mediciment powder 1.8%, trehalose 2%, peptone 0.3%, corn steep liquor 0.3%, dipotassium hydrogen phosphate 1.1%, magnesium sulfate 0.08%, Trisodium Citrate 0.3%, birch xylan 0.4%, insufficient section pure water is supplied, pH value 8,123 DEG C of sterilizing 40min.
Described seeding tank fermented liquid cell concentration is 8.0x10
8individual/mL;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 41 DEG C, stirring velocity 500rpm, ventilation (V/V) 1:2, incubation time 12h; Then with 2 DEG C/h rate of temperature fall slow cooling to 12 DEG C, constant temperature culture 18h; Continue with 2 DEG C/h rate of temperature fall slow cooling to 4 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 25h; Finally slowly be warming up to 12 DEG C, constant temperature culture 18h with 2 DEG C/h temperature rise rate; Continue slowly to be warming up to 41 DEG C, constant temperature culture 18h with 2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 20%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 8;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/mL-8mg/mL, start to add supplemented medium, feed supplement amount is to maintain fermented liquid reducing sugar content for 2mg/mL-5mg/mL;
Put tank standard: the old and feeble self-dissolving of 70% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 100g, Semen Maydis powder 55g, soybean cake powder 20g, herbal mediciment powder 40g, trehalose 35g, birch xylan 10g, yeast powder 6g, corn steep liquor 3g, ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 3g, defoamer 0.5g, pure water 1000mL, pH value 8,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 25%, Semen Maydis powder 15%, bean powder 20%, herbal mediciment powder 8%, and insufficient section pure water is supplied, pH value 8,123 DEG C of sterilizing 40min.
The concocting method of described fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 8, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 20min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 20min and liquefy, finally add other raw material, stir, adjust initial pH8,123 DEG C of sterilizing 40min are for subsequent use.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry high temperature resistant alkalescent xylanase.
Described allocation process does not add any sanitas, can add concentrated enzyme liquid gross weight 3% herbal mediciment powder.
The high temperature resistant alkalescent xylanase liquid enzymes Rate activity obtained through above-mentioned preparation method is 400IU/mL.
Embodiment 4
A preparation method for high temperature resistant alkalescent xylanase, comprises the steps:
(1) actication of culture
The slant strains of intact bacillus acidocldarius 701 is inoculated in slant medium, cultivates 36h for 45 DEG C and carry out actication of culture, so activation 3 times;
Described slant medium consists of: extractum carnis 10g, sodium-chlor 12g, peptone 20g, glucose 5g, agar 20g, herbal mediciment powder 10g, distilled water 1000mL, pH value 10,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take the Radix Astragali 30 parts, Radix Codonopsis 18 parts, radix bupleuri 15 parts, the root of large-flowered skullcap 15 parts, respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 6 times of weight in container, control temperature 90 DEG C keeps 4h, is then cooled to 60 DEG C, add mixing enzyme preparation and carry out enzymolysis, be 6 by lactic acid adjust ph, enzymolysis 4h, finally adds the mixture of mixture 3 times of w ethanol and propyl alcohol, control temperature to 78 DEG C keeps 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
Described mixed enzyme addition is 10% of mixture gross weight.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 20 parts, outer beta-glucanase 20 parts, beta-glucosidase 15 parts, zytase 20 parts, pentosanase 20 parts, Pullulanase 30 parts, beta-amylase 15 parts, neutral protease 15 parts, aspartic protease 15 parts, superoxide-dismutase 10 parts, glucose oxidase 10 parts, acid phosphatase 10 parts.
The mass ratio of described ethanol and propyl alcohol is 1:1.5.
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: slant strains 2 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 43 DEG C, incubation time 15h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 43 DEG C, incubation time 15h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 45 DEG C, stirring velocity 400rpm, ventilation (V/V) 1:2, tank pressure 0.05Mpa, incubation time 20h;
Described one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.5%, glucose 1.5%, peptone 0.5%, extractum carnis 0.8%, dipotassium hydrogen phosphate 1.5%, herbal mediciment powder 2%, trehalose 3%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.3%, insufficient section pure water is supplied, pH value 10,123 DEG C of sterilizing 40min.
Described seed tank culture basic weight amount consists of:
Maltodextrin 15%, yeast powder 0.8%, herbal mediciment powder 2%, trehalose 3%, peptone 0.5%, corn steep liquor 0.5%, dipotassium hydrogen phosphate 1.5%, magnesium sulfate 0.1%, Trisodium Citrate 0.5%, birch xylan 0.8%, insufficient section pure water is supplied, pH value 10,123 DEG C of sterilizing 40min.
Described seeding tank fermented liquid cell concentration is 8.0x10
8individual/mL;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 45 DEG C, stirring velocity 700rpm, ventilation (V/V) 1:3, incubation time 15h; Then with 2 DEG C/h rate of temperature fall slow cooling to 15 DEG C, constant temperature culture 20h; Continue with 2 DEG C/h rate of temperature fall slow cooling to 5 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 30h; Finally slowly be warming up to 15 DEG C, constant temperature culture 20h with 2 DEG C/h temperature rise rate; Continue slowly to be warming up to 45 DEG C, constant temperature culture 20h with 2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 30%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 10;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/mL-8mg/mL, start to add supplemented medium, feed supplement amount is to maintain fermented liquid reducing sugar content for 2mg/mL-5mg/mL;
Put tank standard: the old and feeble self-dissolving of 80% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 150g, Semen Maydis powder 60g, soybean cake powder 25g, herbal mediciment powder 50g, trehalose 40g, yeast powder 8g, corn steep liquor 5g, ammonium sulfate 3g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 5g, birch xylan 15, defoamer 1g, pure water 1000mL, pH value 10,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 30%, Semen Maydis powder 20%, bean powder 25%, herbal mediciment powder 10%, and insufficient section pure water is supplied, pH value 10,123 DEG C of sterilizing 40min.
The concocting method of described fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 10, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 30min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 30min and liquefy, finally add other raw material, stir, adjust initial pH10,123 DEG C of sterilizing 40min are for subsequent use.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry high temperature resistant alkalescent xylanase.
Described allocation process does not add any sanitas, can add concentrated enzyme liquid gross weight 5% herbal mediciment powder.
The high temperature resistant alkalescent xylanase liquid enzymes Rate activity obtained through above-mentioned preparation method is 420IU/mL.
Embodiment 5 alkalescent xylanase of the present invention is used for the bleaching of Straw Pulp
1. ferment treatment condition
Starch dense 5%; Temperature 75 DEG C; Treatment time 60min; PH value 11; Enzyme dosage 20IU/g.
2. hypochlorite bleach condition
Starch dense 6%; Temperature 40 DEG C; Bleaching time 120min; Bleaching liquor is clorox, and concentration is 26.25g/L; Chlorine dosage is 5%, 2.5%.
3. paper pulp through ferment treatment and hypo(chlorite)bleaching result as shown in table 1, wherein h-0: without ferment treatment, only with the paper pulp of hypochlorite bleach; H-1: through ferment treatment, the paper pulp not using hypochlorite bleach; H-2, h-3: through ferment treatment, the paper pulp using sodium hypochlorite bleaching in poaching engine; H-2, h-0 conditions of bleaching is identical.
Interpretation of result from table 1: brown stock is through ferment treatment, significantly can improve the hypo(chlorite)bleaching performance of Straw Pulp, when bleaching consumption is 5%, bleaching magma improves 18.4% through ferment treatment than without pulp brightness after ferment treatment ticket, when being floated to about 65.0%, magma bleaching chlorine dosage is 5%, and the paper pulp through ferment treatment can be saved with chlorine 50% than control group.
Table 1: through the result of different treatment post-bleach paper pulp
| Numbering | h-1 | h-2 | h-3 | h-0 |
| Enzyme dosage (IU/g) | 20 | 20 | 20 | — |
| Yield (%) | 98.98 | 95.98 | 96.9 | — |
| KMnO 4Value | 9.89 | 9.98 | 9.74 | — |
| KMnO 4Value declines (%) | 7.26 | 6.68 | 8.94 | — |
| Chlorine dosage (%) | — | 5.0 | 2.5 | 5.0 |
| Residual chlorine (g/L) | — | 0.30 | 0.071 | 0.036 |
| Chlorine consumption (%) | — | 90.69 | 97.77 | 98.87 |
| Endpoint pH | 11 | 8.62 | 8.38 | 8.19 |
| Whiteness (%) | — | 84 | 65.6 | 65.6 |
Claims (6)
1. a preparation method for high temperature resistant alkalescent xylanase, is characterized in that, described zytase with produce high temperature resistant alkalescent xylanase bacillus acidocldarius (
bacillus thermophilus) CCTCC M 2013537 is starting strain, and prepared through liquid submerged fermentation by Optimal Medium composition and zymotechnique, comprise the steps: by activated for the slant strains of intact bacillus acidocldarius CCTCC M 2013537 and one, two, three seed culture and first class seed pot step by step enlarged culturing obtain seed liquor, with 6% inoculum size access fermentor tank, culture temperature 37-45 DEG C, stirring velocity 200-700rpm, ventilation 1:1-3 V/V, incubation time 10-15h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 10-15 DEG C, constant temperature culture 15-20h; Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, seeding tank seed liquor is added access fermentor tank with 4% inoculum size, constant temperature culture 20-30h; Finally slowly be warming up to 10-15 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Continue slowly to be warming up to 37-45 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Fermentation liquor is filtered, concentrated, allotment, essence filter, dry high temperature resistant alkalescent xylanase, described allocation process adds concentrated enzyme liquid gross weight 0.5-5% herbal mediciment powder;
The preparation method of described herbal mediciment powder is as follows: with weight parts, takes Radix Astragali 20-30 part, Radix Codonopsis 10-18 part, radix bupleuri 10-15 part, root of large-flowered skullcap 10-15 part; Respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then 45-60 DEG C is cooled to, the mixing enzyme preparation adding mixture gross weight 5-10% carries out enzymolysis, be 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 1:1-1.5, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains herbal mediciment powder;
The parts by weight of described mixing enzyme preparation consist of: endo-beta-glucanase 10-20 part, outer beta-glucanase 10-20 part, beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta-amylase 10-15 part, neutral protease 10-15 part, aspartic protease 10-15 part, superoxide-dismutase 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part.
2. the preparation method of a kind of high temperature resistant alkalescent xylanase as claimed in claim 1, it is characterized in that, when preparing zytase, seed tank culture basic weight amount consists of: maltodextrin 5-15%, yeast powder 0.4-0.8%, herbal mediciment powder 1.5-2%, trehalose 1-3%, peptone 0.1-0.5%, corn steep liquor 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, Trisodium Citrate 0.1-0.5%, birch xylan 0.1-0.8%, insufficient section pure water is supplied, pH value 5-11,121-123 DEG C of sterilizing 30-40min.
3. the preparation method of a kind of high temperature resistant alkalescent xylanase as claimed in claim 1, is characterized in that, when preparing zytase, seeding tank fermented liquid cell concentration is 7.0 × 10
8-8.0 × 10
8individual/ml.
4. the preparation method of a kind of high temperature resistant alkalescent xylanase as claimed in claim 1, it is characterized in that, when preparing zytase, fermention medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, soybean cake powder 15-25 g, herbal mediciment powder 30-50g, trehalose 30-40g, birch xylan 5-15g, yeast powder 4-8g, corn steep liquor 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium primary phosphate 1-2 g, Trisodium Citrate 1-5g, defoamer 0.1-1g, pure water l000 mL, pH value 5-11,121 DEG C of sterilizing 20 min.
5. the preparation method of a kind of high temperature resistant alkalescent xylanase as claimed in claim 4, it is characterized in that, the concocting method of described fermention medium is: accurately take raw material in proportion, by the pure water in raw material, Semen Maydis powder, soybean cake powder drops in material-compound tank, adjust ph 5-11, add middle temperature amylase and the 30u alpha-amylase of every gram of Semen Maydis powder 3u, DEG C insulation 15-30min of warming while stirring to 70 simultaneously, then be slowly warming up to 90 DEG C of insulation 15-30min to liquefy, finally add other raw material, stir, adjust initial pH5-11, 121 DEG C of sterilizing 20min are for subsequent use.
6. the preparation method of a kind of high temperature resistant alkalescent xylanase as claimed in claim 1, is characterized in that, comprise the steps:
(1) actication of culture
The slant strains of intact bacillus acidocldarius CCTCC M 2013537 is inoculated in slant medium, cultivates 36h for 45 DEG C and carry out actication of culture, so activation 3 times;
Described slant medium consists of: extractum carnis 10g, sodium-chlor 12g, peptone 20g, glucose 5g, agar 20g, herbal mediciment powder 10g, distilled water l000 mL, pH value 10,121 DEG C of sterilizing 20 min;
The preparation method of described herbal mediciment powder is as follows:
Take the Radix Astragali 30 parts; Radix Codonopsis 18 parts; Radix bupleuri 15 parts; The root of large-flowered skullcap 15 parts; Respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 6 times of weight in container, control temperature 90 DEG C keeps 4h, then being cooled to 60 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 6 by lactic acid adjust ph, enzymolysis 4h, finally add the mixture of mixture 3 times of w ethanol and propyl alcohol, control temperature to 78 DEG C keeps 4h, filters; Filter vacuum concentrates postlyophilization and obtains herbal mediciment powder;
Described mixing enzyme preparation addition is 10% of mixture gross weight;
The parts by weight of described mixing enzyme preparation consist of: endo-beta-glucanase 20 parts, outer beta-glucanase 20 parts, beta-glucosidase 15 parts, zytase 20 parts, pentosanase 20 parts, Pullulanase 30 parts, beta-amylase 15 parts, neutral protease 15 parts, aspartic protease 15 parts, superoxide-dismutase 10 parts, glucose oxidase 10 parts, acid phosphatase 10 parts;
The mass ratio of described ethanol and propyl alcohol is 1:1.5;
(2) liquid seeds enlarged culturing
first order seed is cultivated: slant strains 2 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 43 DEG C, incubation time 15h;
secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 43 DEG C, incubation time 15h;
first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 45 DEG C, stirring velocity 400rpm, ventilation (V/V) 1:2, tank pressure 0.05Mpa, incubation time 20h;
One-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.5%, glucose 1.5%, peptone 0.5%, extractum carnis 0.8%, dipotassium hydrogen phosphate 1.5%, herbal mediciment powder 2%, trehalose 3%, calcium sulfate 1g, magnesium chloride 2g, Trisodium Citrate 3g, insufficient section pure water is supplied, pH value 10,123 DEG C of sterilizing 40min;
Seed tank culture basic weight amount consists of:
Maltodextrin 15%, yeast powder 0.8%, herbal mediciment powder 2%, trehalose 3%, peptone 0.5%, corn steep liquor 0.5%, dipotassium hydrogen phosphate 1.5%, magnesium sulfate 0.1%, Trisodium Citrate 0.5%, birch xylan 0.8%, insufficient section pure water is supplied, pH value 10,123 DEG C of sterilizing 40min;
Seeding tank fermented liquid cell concentration is 8.0 × 10
8individual/ml;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 45 DEG C, stirring velocity 700r/m, ventilation 1:3 V/V, incubation time 15h; Then with 2 DEG C/h rate of temperature fall slow cooling to 15 DEG C, constant temperature culture 20h; Continue with 2 DEG C/h rate of temperature fall slow cooling to 5 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 30h; Finally slowly be warming up to 15 DEG C, constant temperature culture 20h with 2 DEG C/h temperature rise rate; Continue slowly to be warming up to 45 DEG C, constant temperature culture 20h with 2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 30%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 10;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/mL-8mg/mL, start to add supplemented medium, to maintain fermented liquid reducing sugar content for 2mg/mL-5mg/mL;
Put tank standard: the old and feeble self-dissolving of 80% thalline, enzyme activity increasess slowly;
Fermention medium consists of: maltodextrin 150g, Semen Maydis powder 60g, soybean cake powder 25g, herbal mediciment powder 50g, trehalose 40g, yeast powder 8g, corn steep liquor 5g, ammonium sulfate 3g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2 g, Trisodium Citrate 5g, birch xylan 15, defoamer 1g, pure water l000 mL, pH value 10,121 DEG C of sterilizing 20 min;
Supplemented medium weight consists of: maltodextrin 30%, Semen Maydis powder 20%, bean powder 25%, herbal mediciment powder 10%, and insufficient section pure water is supplied, pH value 10,123 DEG C of sterilizing 40min;
The concocting method of fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 10, add middle temperature amylase 3u/g Semen Maydis powder and alpha-amylase 30u/g Semen Maydis powder, DEG C insulation 30min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 30min and liquefy, finally add other raw material, stir, adjust initial pH10,121 DEG C of sterilizing 20min are for subsequent use;
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry high temperature resistant alkalescent xylanase, described allocation process does not add any sanitas, adds concentrated enzyme liquid gross weight 5% herbal mediciment powder.
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| CN104560833B (en) * | 2015-01-23 | 2017-06-23 | 深圳大学 | A kind of basophilic micrococcus luteus and its alkalescent xylanase and the application of generation |
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