CN103667151B - Bacillus thermophilus capable of producing high-temperature and alkali resistant xylanase and application of bacillus thermophilus - Google Patents
Bacillus thermophilus capable of producing high-temperature and alkali resistant xylanase and application of bacillus thermophilus Download PDFInfo
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Abstract
The invention discloses a bacillus thermophilus 701 capable of producing high-temperature and alkali resistant xylanase and belongs to the technical field of microbial fermentation. The bacillus thermophilus is obtained through the steps that a soil sample is collected from a paper mill, a bacillus thermophilus is separated from the soil sample, and ultraviolet mutagenesis and nitrosoguanidine mutagenesis, and screening are performed repeatedly. The xylanase produced by the bacillus thermophilus has the characteristics of strong high-temperature resistance and alkali resistance. The bacillus thermophilus is preserved in the China Center for Type Culture Collection on October 12, 2013, the preservation No. is CCTCC M2013537. The high-temperature and alkali resistant xylanase prepared from the bacillus thermophilus 701 is 350-420 IU/ml in specific activity, 40-75 DEG C in applicable temperature range, 65 DEG C in optimum reaction temperature, completely stable in enzyme activity at the temperature of 75 DEG C, 5.0-11.0 in applicable reaction pH value, completely stable in enzyme activity at the pH value of 11.0, and is 10.0 in optimum reaction pH value.
Description
Technical field
The invention belongs to technical field of microbial fermentation, specifically a kind of bacillus acidocldarius and application thereof of producing 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 a kind of strain excellent producing high temperature resistant alkalescent xylanase of isolation and selection is a kind of necessary.
Summary of the invention
The object of this invention is to provide a kind of bacillus acidocldarius (Bacillusthermophilus) 701 producing high temperature resistant alkalescent xylanase.
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 (Bacillusthermophilus) 701.This bacterial strain is preserved in China typical culture collection center on November 3rd, 2013 and (is 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 NO:M2013537, and Classification And Nomenclature is bacillus acidocldarius (Bacillus thermophilus) 701.
Bacillus acidocldarius 701 provided by the invention is Gram-positives, and bacillus is aerobic, produces gemma, and LB solid medium cultivates 24h for 37 DEG C, and bacterium colony is rounded, and colony diameter is about 1.0-2.0 ㎜, oyster white, and smooth surface is opaque, the irregular expansion in edge.Peritrichous, can move, without pod membrane, have gemma, thalline size is (0.7 ~ 0.9) μm × (3 ~ 3.5) μm, gemma (0.6 ~ 0.9) μm × (1.0 ~ 1.5) μm, ellipticity, be positioned at thalline central authorities or slightly inclined, after sporulation, thalline does not expand.When growing in liquid nutrient medium, form wrinkle mould.Casein hydrolysis experiment, Starch Hydrolysis experiment, gelatine liquefication experiment, nitrate reduction experiment are the positive; Thalline can utilize glucose, fructose, sucrose, can not utilize lactose and N.F,USP MANNITOL; H
2the experiment of S, catalase, indole test experiment are feminine gender.
Bacillus acidocldarius 701 provided by the invention carries out 16sRNA gene sequencing, 16SrDNA sequence existing in obtained sequence Blast software and Genbank is carried out tetraploid rice, software MEGA4.0 is utilized to build evolutionary tree, it is one that result shows that bacterial strain of the present invention and bacillus acidocldarius (Bacillus thermophilus) gather, sequence similarity is 98%, the morphological feature of comprehensive 701 bacterial strains, physiological and biochemical property and 16S rDNA nucleotide sequence homology analytical results, bacterial strain 701 is accredited as bacillus acidocldarius (Bacillus thermophilus).
High temperature resistant alkalescent xylanase Rate activity prepared by bacillus acidocldarius 701 provided by the 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.
By mutagenesis screening scheme, mutant strain step-sizing is eliminated, finally to strain excellent through leavening property test screen, obtain the bacterial strain bacillus acidocldarius 701 that high temperature resistant alkalescent xylanase is produced in a strain, fermented liquid alkalescent xylanase Rate activity is 350-420IU/mL, the thermostability of enzyme is analyzed, at crude enzyme liquid is placed in 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C respectively, lives every 10 minutes sampling and measuring enzymes.At 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 minutes enzymes are lived and are not declined.At 60 DEG C and 65 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 85% in 95%, 60 minutes.At 70 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minutes.At 75 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 70% in 80%, 60 minutes.
Another object of the present invention is the application of bacillus acidocldarius 701 in the high temperature resistant alkalescent xylanase of preparation.
Beneficial effect:
High temperature resistant alkalescent xylanase Rate activity prepared by bacillus acidocldarius 701 of 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.
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 screening of starting strain
(1) from the waste paper pulp of Xue Li paper mill, Hunan Province, 2g soil sample is gathered, be added to and cool in the little triangular flask of sterilized water containing 50mL, shaking table 200r/min, shake 20min, then be placed in 80 DEG C of water-baths, water-bath 10min, constantly shake triangular flask mixing soil sample, leave standstill 5min Aspirate supernatant 100 μ L, gradient dilution is to 10 successively
-1~ 10
-9concentration, chooses 10
-3, 10
-4, 10
-5, 10
-6concentration coating beef extract-peptone is dull and stereotyped, cultivates 24h for 37 DEG C, continues 30 DEG C and cultivates 24h.
(2) according to the shape of microbe colony, size, surface tissue, marginal texture, quality, gloss, transparency, color and generation can the feature of the aspect such as lysochrome, with transfering loop, isolated single bacterium colony is chosen, move on on screening culture medium flat board, and number, cultivate 48h for 34 DEG C.
Described screening culture medium consists of: extractum carnis 3g, sodium-chlor 5g, peptone 10g, agar powder 15g, distilled water l000mL, pH7.2,121 DEG C of sterilizing 20min.
(3) after bacterial strain cultivates 48h on flat board, be inoculated on screening culture medium inclined-plane by the dominant colony on difference numbering flat board, preservation uses for multiple sieve.
(4), after the bacterial strain that the difference of preservation is numbered being inoculated into respectively slant activation 2 times, by the bacterial classification access seed culture medium activated, 180r/min, cultivates 18h by 37 DEG C.Fermention medium is inoculated into again, 200r/min, 28 DEG C, fermentation 48h by the inoculum size of 5%.Remove thalline by centrifugal for gained fermented liquid 10000r/min, fermented liquid supernatant liquid carries out the experiment of alkalescent xylanase vitality test.
Described seed fermentation substratum consists of: glucose 2g, peptone 1g, NaH
2pO
42H
2o0.2g, Na
2hPO
42H
2o0.4g, MgSO
47H
2o0.05g, distilled water 100mL, pH7.4,121 DEG C of sterilizing 20min.
Described fermention medium consists of: glucose 2g, peptone 2g, NaH
2pO
42H
2o0.02g, Na
2hPO
42H
2o0.04g, MgSO
47H
2o0.01g, CaCl
20.01g, MnSO
40.002g, distilled water l00mL pH7.4,121 DEG C of sterilizing 20min.
(5) select the bacterial strain producing alkalescent xylanase enzyme activity strong and carry out glycerine preservation, obtain the starting strain HYX0021 of bacterial strain of the present invention.
The mutagenesis screening of embodiment 2 bacterial strain
(1) ultraviolet mutagenesis
Draw starting strain HYX0021 and cultivate 10-12h bacterium liquid, in injection plane ware, the bacterium liquid measure of each plane ware is 10mL.Be placed in by the plate filling bacterium liquid on the magnetic stirring apparatus in mutagenesis case under ultraviolet lamp tube, plate is 30cm apart from the vertical range of ultraviolet lamp tube.Open ultraviolet lamp, preheating 30min, light wave is stablized.Regulate the rotating speed of stirrer, after stabilization of speed, open plate lid and irradiate, and start timing, the irradiation time of plate is respectively 1,2,3,4,5,6,7,8,9,10min.From each plate, take out the bacteria suspension of 0.1mL, do suitable dilution, obtain different dilution bacteria suspension.It is dull and stereotyped that bacteria suspension 0.3mL after absorption dilution is coated with isolation medium, is placed in thermostat container and cultivates (for avoiding photoreactivation plate need wrap up with black paper or kraft paper, whole mutagenesis operating process is all carried out under ruddiness).
(2) nitrosoguanidine mutagenesis
A little for nitrosoguanidine powder is placed in plate culture medium central authorities, in culturing process, nitrosoguanidine progressively spreads, the concentration gradient formed on flat board outside mediad, can occur that with flat board central authorities be the concentric(al) circles bacterium colony group in the center of circle through cultivating, dull and stereotyped central close region colony number is zero, and plate edge region bacterium colony increases successively.The bacterial classification that sets out produces the bacterial classification that makes a variation in various degree under different lethality rate.
(3) dull and stereotyped primary dcreening operation
Transparent circle size is utilized to carry out primary dcreening operation.By the bacterium colony dibbling that grows after mutagenesis on separating plate, 34-35 DEG C cultivate 10-15h after, observe transparent circle size around it, choose birch xylan transparent circle diameter and the larger bacterium colony of colony diameter ratio and carry out shaking flask and sieve again.
Described separating plate substratum consists of: yeast powder 0.2%, birch xylan 0.2%, peptone 0.5%, extractum carnis 0.7%, dipotassium hydrogen phosphate 1.0%, sodium-chlor 0.1%, agar 1.8%, insufficient section pure water is supplied, pH7.0-8.0,121-123 DEG C of sterilizing 30-40min.
(4) shaking flask is sieved again
The colony inoculation obtained by primary dcreening operation, in multiple sieve substratum, is cultivated 48 hours for 34 DEG C, detects alkalescent xylanase enzyme and lives.
The described substratum that sieves again consists of: yeast powder 0.2%, glucose 1%, peptone 0.4%, Semen Maydis powder 0.6%, dipotassium hydrogen phosphate 0.8%, insufficient section pure water is supplied, pH7.0-8.0,121-123 DEG C of sterilizing 30-40min.
(5) strain stability detects:
Multiple sieve gained live high-enzyme strain is carried out Secondary Culture, and follow the tracks of shaking flask detect its enzyme live.Sieve again through shaking flask, finally determine a strain and produce bacterium, and by its called after 701, the characteristic of this bacterial strain is that product alkalescent xylanase efficiency is high, good stability.
The shake flask results that superior strain 701 continuous passage is 6 times is as shown in table 1:
Table 1. bacterial strain 701 Detection of Stability result
| Shaking flask enzyme activity (IU/mL) | Enzyme activity (%) | |
| F1 | 380 | 100 |
| F2 | 382 | 100.6 |
| F3 | 388 | 102.0 |
| F4 | 378 | 99.5 |
| F5 | 388 | 102.3 |
| F6 | 387 | 101.9 |
Embodiment 3: identification of strains
Bacterial strain Physiology and biochemistry is identified: carry out the tests such as utilization of carbon source test, catalase test, hydrogen sulfide production test, indole test, NO3-N and NO2-N reduction test according to " uncle Jie Shi Bacteria Identification handbook " (the 9th edition) method; Morphological Identification: dibbling is on beef-protein medium solid plate respectively by bacterial strain to be identified, and 37 DEG C of cultivation 2d, describe and record bacterium colony cultural characteristic and morphological features.Physiology and biochemistry identification mark is as shown in table 2.Result contrast in table " uncle Jie Shi Bacteria Identification handbook " belongs to feature with thermophilic bacillus species and conforms to.The method of extraction reference " Molecular Cloning: A Laboratory guide " (the 3rd edition) of molecular biology identification: bacterial genomes DNA.According to primers the most conservative in bacterial 16 S rDNA.Primers F: 5'-AGAGTT TGA TCM TGGCTC AG-3'; Primer R:5'-AAG GAG GTG WTCCAR CC-3' is synthesized by Shanghai Sheng Gong biotechnology company limited.PCR reaction conditions: 94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C of 40s, 72 DEG C of 2min, 30 circulations, 72 DEG C of ends extend 10min.Pcr amplification product is delivered to Beijing three and is won the order-checking of polygala root Bioisystech Co., Ltd.After sequencing result carries out two-way splicing, the 16S rDNA sequence obtained is submitted in NCBI nucleic acid database and carries out BLAST on-line analysis.Part genus bacillus 16S rDNA sequence afterwards from the GenBank nucleic acid database of NCBI in random selecting report, application Clustal Ⅹ 1.81 and Phylodraw082 software building systematic evolution tree analyzing.In conjunction with the physiology and morphology biochemical character of bacterial strain, 701 bacterial strains are fixed tentatively as bacillus acidocldarius by preliminary evaluation.
Table 2 bacterial strain 701 morphology and physio-biochemical characteristics
Note: result "+" represents positive; “ – " represent negative.
Embodiment 4: the thermal stability analysis of alkalescent xylanase
The thermostability of enzyme is analyzed, at embodiment 2 crude enzyme liquid is placed in 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C respectively, lives every 10 minutes sampling and measuring enzymes.At crude enzyme liquid 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 minutes enzymes are lived and are not declined.At 60 DEG C and 65 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 85% in 95%, 60 minutes.At 70 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minutes.At 75 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 70% in 80%, 60 minutes, and compared with prior art reach same enzyme and live, tolerable temperature is higher.
Embodiment 5: alkalescent xylanase pH stability analysis
The pH stability of enzyme is analyzed, embodiment 2 crude enzyme liquid is placed in respectively pH value 4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 times, live every 10 minutes sampling and measuring enzymes.Crude enzyme liquid pH value 6.0,7.0,8.0,9.0 times, 60 minutes enzymes are lived and are not declined.PH value 5.0 and 10.0 times, what within 30 minutes, drop to that constitutive enzyme lives drops to 85% in 95%, 60 minutes.PH value 4.0 and 11.0 times, what within 30 minutes, drop to that constitutive enzyme lives drops to 70% in 80%, 60 minutes.It is obviously more wide in range than prior art bacterial strain that similarity condition is issued to same enzyme resistance to pH value alive.
Claims (2)
1. one kind is produced the bacillus acidocldarius (Bacillus thermophilus) 701 of high temperature resistant alkalescent xylanase, and this bacterial strain is preserved in China typical culture collection center on November 3rd, 2013, and preserving number is CCTCC NO:M2013537.
2. the application of bacillus acidocldarius (Bacillus thermophilus) 701 as claimed in claim 1 in the high temperature resistant alkalescent xylanase of preparation.
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