CN101851612B - Acid glucanase CELA and gene and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to acid glucanase CELA and gene and application thereof. The invention provides the glucanase CELA from Alicyclobacillus hesperidumA4 (CGMCC No. 3147), the amino acid sequence of the glucanase CELA is expressed as SEQ ID No.1, and the invention provides the gene CelA for coding the glucanase. The glucanase of the invention has the following properties of: optimal pH of 3.4, optimal temperature of 65 DEG C, and good thermal stability. The acid glucanase CELA serving as a novel enzyme preparation can be widely applied to industries of animal feed, food, energy and the like.

Description

A kind of acid glucanase CELA and gene thereof and application

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of acid glucanase CELA and gene thereof and application.

Background technology

Beta-glucan extensively is present in the aleurone layer and albuminous cell wall of cereal class (barley, oat, rye and wheat), structural non-starch polysaccharide in the platymiscium cell walls, space structure with line style, according to the species difference, the content of beta-glucan and proportion are also different.Wherein contained ratio is the highest in the middle of barley and oat.Dextranase is the general name that dextran can be degraded into the class of enzymes of oligose and glucose.At present dextranase is at food, feed, and beer, medicine and other fields have obtained increasingly extensive application.At present barley mainly be applied to beer brewage with feed in, therefore relevant dextranase has obtained using the most widely in brewage and fodder industry, especially in Europe, with barley, dregs of beans is in the feed formulation of main raw material, and the interpolation of dextranase is more extensive.In the barley germination process, dextran in the middle of the barley can not be decomposed fully by self endogenous dextranase, only can decompose 30%-70% usually, and residual dextran increases converted mash viscosity, filtering rate reduces, and finished beer forms blushing or incipient gel precipitation easily.In Fructus Hordei Germinatus is made, add high temperature resistant beta-glucanase and can significantly reduce beta-glucan content in the finished product Fructus Hordei Germinatus, reduce wort viscosity, improve wheat juice filtering velocity and yield, help improving beer flavor, be held in the non-biostability of sampling wine.In animal and fowl fodder, contain a large amount of beta-glucans based on barley, wheat, rye, oat, owing to do not digest the enzyme of beta-glucan in the monogastric animal body, therefore can not the central beta-glucan of hydrolysate feed.Beta-glucan after the absorption more water, has higher viscosity as a kind of non-starch viscous polysaccharide in enteron aisle, stop the enteron aisle Digestive system fully to contact with chyme, thereby influences the absorption of nutritive substance, becomes a kind of antinutritional factor.Add beta-glucanase, can effectively eliminate the anti-oxidant action of beta-glucan, improved the utilising efficiency of feed greatly.

In fodder industry, animal gastrointestinal tract is sour environment (as the gi tract of pig), and contains a large amount of endogenous proteases, in the course of processing of feed, a pyroprocess is in short-term arranged simultaneously.Therefore, obtain novel have good temperature stability and suitability, have the research that various proteolytic enzyme (particularly stomach en-) are had an acidic dextranase of resistance and be significant.The clone with separate the acidic dextranase with temperature stability and protease resistant can better application in feed, and can reduce production costs, it is necessary all to be that dextranase is applied to suitability for industrialized production.

Summary of the invention

The acidic dextranase that the purpose of this invention is to provide a kind of energy efficient application.

A further object of the present invention provides the gene of the above-mentioned acidic dextranase of coding.

Another object of the present invention provides the recombinant vectors that comprises said gene.

Another object of the present invention provides the recombinant bacterial strain that comprises said gene.

Another object of the present invention provides a kind of gene engineering method for preparing above-mentioned acidic dextranase.

Another object of the present invention provides the application of above-mentioned acidic dextranase.

The present invention is from alicyclic acid genus bacillus Alicyclobacillus hesperidum A4, (be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCC No.3147, preservation date: separate obtaining a kind of new acid glucanase CELA on June 29th, 2009).

The invention provides a kind of acid glucanase CELA, its aminoacid sequence is shown in SEQ ID NO.1.

SEQ ID NO.1：

MSPSGGVCVNRKQRTLKLGTLAATIVALSAVATPAVASADTTTAIASSTVHVTV

NAAAELGIVPNTALGVNTAVWDGHLLDAAIPSLLRGIGVTMLRYPGGSTSDE

YNWQTNTVTGGYADPNNTFDNFMGVVQKAGAQPIITVNAGTGTPSEAAAW

VQDANVTHHYGVKYWEIGNEMYGSWEAGNFANNPSGYAKEAVSFIQAMKA

VDPSIKIGVDLIAPGTGEDDWNATVLSTMHSLGVLPDFAIVHWYAQNPGGET

DAGLLSSTNQISTMMDTLKQQLSSYGTIPVFVTETNSVSYNPGRQSTSLVNAL

FLDDDMADWLESGAQNVDWWDLHNGIVTQQAGANVDPNLYGQYNYGDY

GLLSNGSSDNGISEPAANTPFPTYYGYQMLAAVMVPGATMIGAGSNNDLVAV

HATKLPNGAVDVMLINKDPKQAYTVDLQAEGFAAKGPAFTLFYGQGSNAVTP

GKLDNLQNVTLPPYSVTDIIIPAVPGHQPQGPQFTDKTTLSTPQVKPSANETLT

TTFTDTRGAVKDGTLDVEIYNPAGQLVGQQVQSGVTFTPGQSSQPITWNWTA

PDSPGTYTVKAFVFSQDGTSVYAADPSAATFTVTQPDPPTISATVQLSATTVK

VGTPVTITTTYTETAPTGYLNNGLLVQYAVYNNWTSSQQSNPTATLTPGQSVT

ETWTFTPEQAGTYTFPEGIFTSGWTQLQWINQNVTLTVTN

715 amino acid of this enzyme genes encoding, so the theoretical molecular of dextranase CELA is 75.4kDa.

Dextranase CELA of the present invention all has greater activity in acid range.The present invention screens the dextranase that a kind of alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 (CGMCCNo.3147) is produced, its recombinase optimum pH at pichia spp is 3.4, keeps the enzymic activity more than 80% in the scope of pH2.8～4.2; 37 ℃ are incubated 60 minutes in the scope of pH1.2～7.8, can keep the enzymic activity more than 80%.Optimum temperuture is 65 ℃, all has the enzyme activity more than 50% between 45 ℃-75 ℃; 70 ℃ of insulations 60 minutes, residual enzyme work reached more than 80%.The also unprecedented report of the dextranase of this character.

The invention provides the gene of the above-mentioned acid glucanase CELA of coding.Particularly, the gene order of this gene is shown in SEQ ID NO.2:

SEQ ID NO.2：

ATGTCACCTTCAGGGGGAGTCTGTGTGAACCGAAAACAGCGTACGTTAAAGTT

GGGAACGCTCGCAGCAACGATTGTGGCACTCTCAGCCGTGGCCACGCCTGCGG

TCGCCAGTGCGGATACGACGACGGCCATTGCGTCATCGACAGTTCATGTCACAG

TCAATGCCGCTGCTGAACTTGGAATCGTGCCCAATACTGCACTTGGTGTGAATA

CGGCCGTCTGGGACGGGCATTTACTCGATGCAGCCATTCCATCTCTGCTTCGTG

GCATTGGGGTAACCATGTTGCGATATCCCGGAGGATCGACTTCAGATGAGTACA

ATTGGCAAACGAATACCGTAACTGGGGGTTATGCAGATCCCAACAACACCTTTG

ACAACTTCATGGGAGTGGTCCAAAAGGCTGGTGCGCAACCCATTATTACGGTC

AACGCCGGCACGGGCACACCGAGTGAAGCTGCCGCATGGGTTCAAGATGCAA

ATGTCACGCACCACTACGGTGTCAAGTATTGGGAAATCGGAAATGAGATGTATG

GCAGCTGGGAAGCAGGGAATTTTGCAAATAACCCATCTGGTTATGCGAAAGAA

GCTGTATCCTTCATTCAGGCCATGAAAGCGGTTGATCCTTCTATTAAAATCGGCG

TGGACCTCATCGCACCTGGTACTGGAGAAGATGACTGGAATGCAACCGTGCTT

AGCACCATGCACAGCCTTGGGGTGCTTCCGGACTTCGCCATTGTGCACTGGTAT

GCGCAAAATCCGGGAGGCGAGACGGACGCTGGGCTGCTCAGTTCGACGAATC

AAATTTCGACGATGATGGATACCTTGAAGCAGCAATTGAGCTCCTATGGAACCA

TCCCGGTTTTCGTCACCGAAACGAATTCGGTTTCGTATAACCCTGGGCGCCAGA

GTACAAGTCTGGTTAATGCACTGTTCCTCGATGATGACATGGCAGACTGGCTTG

AGTCCGGAGCGCAAAACGTCGACTGGTGGGACTTGCATAACGGCATTGTTACA

CAACAGGCCGGTGCAAATGTCGATCCGAATCTGTATGGGCAGTATAACTACGGA

GACTATGGACTTTTGTCCAATGGCTCGAGTGACAATGGCATTTCAGAACCGGCT

GCCAATACACCATTCCCAACGTATTATGGATACCAAATGCTCGCAGCCGTCATGG

TGCCAGGAGCGACGATGATCGGTGCCGGATCGAACAATGATCTGGTGGCCGTG

CATGCGACCAAGTTGCCCAATGGTGCTGTCGACGTCATGTTGATCAACAAGGAT

CCGAAACAAGCGTATACCGTCGATTTGCAAGCTGAAGGATTTGCTGCTAAGGGT

CCTGCATTCACGTTATTCTACGGGCAAGGCAGTAACGCGGTGACGCCGGGCAA

ATTGGATAATCTGCAGAATGTGACACTACCGCCCTATTCTGTGACGGACATCATC

ATACCGGCGGTGCCCGGGCATCAGCCACAAGGGCCACAGTTTACGGACAAGAC

GACGTTATCCACTCCTCAGGTAAAGCCTAGTGCCAACGAGACTTTGACCACGA

CGTTTACCGACACGCGTGGTGCGGTCAAGGATGGTACACTCGACGTGGAAATC

TACAATCCAGCAGGGCAATTGGTTGGGCAACAAGTGCAGTCTGGCGTGACGTT

TACGCCTGGGCAATCATCTCAACCGATTACCTGGAACTGGACGGCGCCCGATTC

TCCTGGGACGTATACCGTGAAGGCGTTCGTCTTCAGCCAAGACGGAACAAGCG

TGTATGCGGCAGACCCGAGTGCAGCTACGTTCACGGTCACACAGCCGGATCCG

CCCACCATTTCGGCCACCGTTCAGCTGTCCGCAACTACGGTCAAAGTGGGTAC

ACCTGTGACCATCACGACGACTTACACCGAAACCGCGCCTACGGGGTACCTGA

ACAACGGGTTGCTTGTACAGTACGCCGTGTACAATAACTGGACATCATCGCAAC

AGTCCAATCCAACTGCGACATTGACTCCTGGGCAATCGGTGACTGAGACTTGG

ACATTTACGCCAGAGCAGGCCGGAACCTACACATTCCCTGAAGGCATCTTTACC

AGTGGATGGACACAATTGCAGTGGATTAATCAGAACGTGACCTTGACTGTGAC

AAACTAA

The method separating clone of the present invention by PCR glucanase gene CELA, the DNA complete sequence analysis is the result show, dextranase CELA structure gene CELA total length 2148bp contains a terminator TAA.The maturation protein theoretical molecular of dextranase CELA is 75.4kDa.Glucanase gene CELA sequence and the aminoacid sequence derived are carried out the BLAST comparison in GenBank.This gene is 44% with the highest consistence of cellulase (CAD86595) aminoacid sequence that derives from Alicyclobacillusacidocaldarius, illustrates that CELA is a kind of new dextranase.

The present invention also provides the recombinant vectors that comprises above-mentioned glucanase gene, is preferably pPIC9-CelA.Glucanase gene of the present invention is inserted between the suitable restriction enzyme site of expression vector, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, be preferably glucanase gene is inserted between the SnaBI and NotI restriction enzyme site on the plasmid pPIC9, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and control by it, obtain expression of recombinant yeast plasmid pPIC9-CelA.

The present invention also provides the recombinant bacterial strain that comprises above-mentioned glucanase gene, is preferably recombinant bacterial strain GS115/CelA.

The present invention also provides a kind of method for preparing acid glucanase CELA, may further comprise the steps:

1), gets recombinant bacterial strain with above-mentioned recombinant vectors transformed host cell;

2) cultivate recombinant bacterial strain, induce reorganization dextranase CELA to express; And

3) reclaim the also expressed dextranase CELA of purifying.

Wherein, preferred described host cell is pichia spp cell, cerevisiae or many types of inferior yeast cell, preferably the expression of recombinant yeast plasmid is transformed pichia spp cell (Pichic pastoris) GS115, obtains recombinant bacterial strain GS115/CelA.

The present invention also provides the application of above-mentioned acid glucanase CELA.

The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, provide a kind of character good, be suitable at food, particularly use new dextranase in the fodder industry.Dextranase optimal pH of the present invention is 3.4, and higher enzymic activity (more than 80%) is all arranged in the scope of pH2.8～4.2; PH good stability under the acidic conditions; Ability and anti-various proteolytic enzyme (particularly stomach en-) with better heat-resisting.Its superior heat resistance energy can effectively be reduced in the loss of enzyme activity in the feed course of processing, reduces the application cost of dextranase.Optimal pH be acid range and preferably protease resistant (particularly stomach en-) can improve the transformation efficiency of non-starchiness polysaccharide in the feed, reduce formulation cost, reduce environmental pollution; Can also be used for bioenergy, as the dextran in paper industry waste material and the agricultural wastes is converted into the D-glucose monomer, and then, change into valuable fuel by the most of microbe metabolism.Hydrolysate (glucose and oligomeric glucose) can be applicable to the protective foods industry; Dextran is used in combination with other material in pharmaceutical industry, can delay the release of pharmaceutical cpd.

Description of drawings

Fig. 1 analyzes at the SDS-PAGE of the reorganization dextranase of Pichia anomala expression, wherein, and 1: low molecular weight protein Marker; 2: the reorganization dextranase of purifying.

The recombinate optimal pH of dextranase of Fig. 2.

The recombinate pH stability of dextranase of Fig. 3.

The recombinate optimum temperuture of dextranase of Fig. 4.

The recombinate thermostability of dextranase of Fig. 5.

The recombinate various protease resistants of dextranase of Fig. 6

Alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 CGMCC3147, be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCCNo.3147, preservation date: 2009 06 month No. 29.

Embodiment

Test materials and reagent

1, bacterial strain and carrier: alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 separates acquisition by the inventor, be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCCNo.3147.Yeast expression vector pPIC9 and bacterial strain GS115 are available from Invitrogen company.

2, enzyme and other biochemical reagents: restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.Barley is available from Sigma company, and other all is domestic reagent (all can buy from common biochemical reagents company and obtain).

3, substratum:

(1) alicyclic acid genus bacillus AlicyclobacillushesperidumA4CGMCC3147 substratum consists of: 0.2% peptone, 0.1% yeast extract, 0.2% glucose, pH3.0.

(2) BMGY substratum: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V).

(3) BMMY substratum: replace glycerine divided by 0.5% methyl alcohol, all the other compositions are all identical with BMGY.

Illustrate: make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.

The separation and purification of embodiment 1 alicyclic acid genus bacillus AlicyclobacillushesperidumA4 (CGMCCNo.3147) and product enzyme characteristic thereof

Alicyclic acid genus bacillus AlicyclobacillushesperidumA4 is seeded to product enzyme culture medium flat plate (yeast extract 1.0g, Tryptones 2.0g, oat beta-glucan 5.0g, agar 30, Congo red 0.5g, transfer pH3.0 with hydrochloric acid) 60 ℃ cultivate after 72 hours, congo red staining with 0.5% has or not according to transparent circle that it has dextranase activity with big or small preliminary identification.60 ℃ of cultivations of alicyclic acid genus bacillus AlicyclobacillushesperidumA4 multiparity enzyme substratum (yeast extract 1.0g, Tryptones 2.0g oat beta-glucan 5.0g transfer pH3.0 with sulfuric acid) after 48 hours, are measured the dextranase activity of supernatant liquor.Prove that it has dextranase activity.

The clone of embodiment 2 alicyclic acid genus bacillus AlicyclobacillushesperidumA4 (CGMCCNo.3147) glucanase coding gene as well as CELA

The acquisition of gene order

Conservative (YWEIGNE and AMKAVD) sequences Design according to the 51st family's glucanase gene has been synthesized degenerated primer Cel51F, Cel51R such as table 1

With the total DNA of alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 (CGMCC No.3147) is that template is carried out pcr amplification.The PCR reaction parameter is: 94 ℃ of sex change 5min; Preceding 10 circulations, 94 ℃ of sex change 30sec, 50 ℃ of-55 ℃ of touchdown (0.5 ℃/circulation) annealing 30sec, 72 ℃ are extended 0.5min, and 25 cycling conditions are back 94 ℃ of sex change 30sec then, 55 ℃ of annealing 30sec, 72 ℃ are extended 1.5min.Last 72 ℃ of insulation 10min.Obtain an about 180bp fragment, this fragment recovery back is linked to each other with the pEASY-T3 carrier send the order-checking of three rich Bioisystech Co., Ltd.

The nucleotide sequence that obtains according to order-checking, each three TAIL-PCR specificity nested primers of design upstream and downstream: design direction is for needing the zone of ignorance direction of amplification, and with they difference called after usp1, usp2, usp3 (upstream Auele Specific Primer), dsp1, dsp2, dsp3 (downstream Auele Specific Primer) sees Table 1.

Table 1. dextranase CelA TAIL-PCR Auele Specific Primer

N represents A, G, C or T; H represents T, A or C; R represents A or G; Y represents C or T

Obtain the flanking sequence of known sequence by TAIL-PCR, amplification obtains product and reclaims the back order-checking.

By this full length gene of discovery 2148bp behind the genome sequence that compares dextranase, encode 715 amino acid and a terminator codon, N end 1-39 amino-acid residue is a signal peptide sequence.Infer that the aminoacid sequence of the gene C ELA that and the glucanase gene sequence on the GeneBank carry out homology relatively, the highest consistence is 44%, illustrate that CELA is a kind of new dextranase, the gene that shows the coding dextranase that separating clone obtains from AlicyclobacillushesperidumA4 (CGMCCNo.3147) is new gene.

Extract alicyclic acid genus bacillus AlicyclobacillushesperidumA4 (CGMCCNo.3147) genomic dna:

With 2 days bacterium liquid centrifuging and taking thalline of liquid culture, add the 1mL N,O-Diacetylmuramidase, handle 60min for 37 ℃, add lysate again, 65 ℃ of water-bath cracking 30min, every the 10min mixing once, at 4 ℃ of centrifugal 5min of following 10000rpm.Get supernatant extrct foreigh protein removing in phenol/chloroform, get supernatant again and add the equal-volume Virahol, after room temperature leaves standstill 5min, 4 ℃ of centrifugal 10min of following 10000rpm.Abandon supernatant, precipitation is with 70% washing with alcohol twice, and vacuum-drying adds an amount of TE and dissolves, place-20 ℃ standby.

Primer CelAF and CelAR such as table 1 have been synthesized in design according to the CelA sequence information.

With the total DNA of alicyclic acid genus bacillus AlicyclobacillushesperidumA4 (CGMCCNo.3147) is that template is carried out pcr amplification.The PCR reaction parameter is: 95 ℃ of sex change 5min, and 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 120s, 30 circulations.Last 72 ℃ of insulation 10min.

The preparation of embodiment 3 reorganization dextranases.

Expression vector pPIC9 is carried out double digestion (SnaBI and NotI), to encode the simultaneously gene C elA double digestion (SnaBI and NotI) of dextranase, the gene fragment that cuts out the coding dextranase is connected with expression vector pPIC9, acquisition contains the recombinant plasmid pPIC9-CelA of glucanase gene CelA and transforms pichia spp GS115, obtains recombinant pichia yeast strain GS115/CelA.

Get the GS115 bacterial strain that contains recombinant plasmid, be inoculated in the 400mL BMGY nutrient solution, behind 30 ℃ of 250rpm shaking culture 48h, centrifugal collection thalline.Resuspended in the 200mLBMMY substratum then, 30 ℃ of 250rpm shaking culture.After inducing 48h, centrifugal collection supernatant.Measure the vigor of dextranase.The expression amount of reorganization dextranase is 20.41U/mL.SDS-PAGE result (Fig. 1) shows that the reorganization dextranase has obtained expression in pichia spp.

The activation analysis of embodiment 4 reorganization dextranases

The DNS method: concrete grammar is as follows: at pH3.4 (0.1M SODIUM PHOSPHATE, MONOBASIC-citric acid), under 65 ℃ of conditions, the reaction system of 1mL comprises 100 μ L suitable dilution enzyme liquid and 900 μ L (1%, w/v) substrate, reaction 10min adds the 1.5mLDNS termination reaction, and boiling water boils 5min.Cooling back 540nm measures the OD value.1 enzyme unit (U) that lives is defined as the enzyme amount that under given condition per minute discharges 1 μ mol reducing sugar.

The property testing of embodiment 5 reorganization dextranase CELA

1, the measuring method of the optimal pH of reorganization dextranase CELA and pH stability is as follows:

The reorganization dextranase of embodiment 3 purifying is carried out enzymatic reaction to measure its optimal pH under different pH.The substrate dextran is with damping fluid (the 0.1M glycine-hydrochloric acid pH 1.2-2.6 of different pH; 0.1mol/L citric acid-Sodium phosphate dibasic pH 3.0-7.4; 0.1MTris-HCl pH 7.8-8.6), under 65 ℃, carry out the dextranase vitality test.Result (Fig. 2) shows that the optimal pH of CELA is 3.4, keeps the enzymic activity more than 80% in the scope of pH2.8～4.2.Dextranase is 37 ℃ of processing 60min in the damping fluid of above-mentioned various different pH, measure enzymic activity again under 65 ℃ in the pH3.4 buffer solution system, with the pH patience of research enzyme.Result (Fig. 3) shows that dextranase is all very stable between pH1.2-7.8, and the residual enzyme activity is more than 80% behind the processing 60min in this pH scope, and this illustrates that this enzyme has pH stability preferably.

2, the optimum temperuture of dextranase and thermal stability determination method are as follows:

Enzymatic reaction is carried out in being determined as under citric acid-Sodium phosphate dibasic damping fluid (pH3.4) buffer solution system and differing temps of the optimum temperuture of dextranase.Temperature tolerance is determined as dextranase and handles different time under differing temps, carries out enzyme assay again under 65 ℃.Enzyme reaction optimum temperuture measurement result (Fig. 4) shows that its optimum temperuture is 65 ℃.The thermostability test of enzyme shows (Fig. 5), and recombinase stability in the time of 70 ℃ is very good.70 ℃ are incubated 60min down, and the residual enzyme activity is 84%, and described enzyme all has the enzyme activity more than 50% between 45 ℃-75 ℃.

3, different metal ion chemistry reagent is as follows to XYLA4 enzyme influence mensuration alive:

Add the different metal ions and the chemical reagent of different concns in enzymatic reaction system, study its influence to enzymic activity, various material final concentrations are 1 and 10mmol/L.Under 65 ℃, pH3.4 condition, measure enzymic activity.Result's (table 1) shows, except SDS, most of ions and chemical reagent do not have considerable change at the vigor of concentration reorganization dextranase during for 1mmol.Co when concentration is 10mmo ²⁺, Cr ³⁺, Fe ²⁺, Pb ²⁺, Cu ²⁺, Ag ⁺, Hg ²⁺' and its vigor of the equal strongly inhibited of SDS.Beta-mercaptoethanol can make the recombinase vigor be increased to original 1.16 and 1.39 times when 1mmol and 10mmol respectively.

The various chemical reagent of table 1 are to the influence of dextranase CELA vigor

Embodiment 6 reorganization dextranases in simulated gastric fluid to the influence of barley dregs of beans type daily ration viscosity

The following 1g barley of concrete grammar dregs of beans type is dissolved in the simulated gastric fluid that contains 1U CelA of 9mL, and mixing solutions is placed on 37 ℃ of shaking table 180rpm vibration 1h.In order to determine in digestive process, accumulative effect (the Haraldsson et al. that causes hydrolysate along with the variation of pH condition, 2005), the pH gradient former of describing according to Minekus et al. (1995), to CelA in simulated gastric fluid from barley dregs of beans type daily ration the accumulative effect of hydrolysis carried out experimental simulation research.Whole pH gradient is carried out pH 2.0,20min according to following strategy; PH 2.3,20min; PH 2.8,20min; PH 3.8,10min; PH 4.6,10min; PH 5.5,10min.The pH of solution is placed on ice and regulates by HCl or NaOH in this process.The final accumulative total effect of effect is represented by measuring viscosity.The mensuration of viscosity is measured with capillary viscosimeter.Particularly, will be good by filter paper of Xinhua through the feed liquid of above-mentioned processing, get the filtered solution of 5ml, measure by capillary viscosimeter.

The result shows: the barley bean-dregs feed viscosity ratio contrast of handling through enzyme liquid has descended 36.72%.Show that CelA can finely be applied in the fodder industry.

Sequence table

＜110〉Institute of Feeds,China Academy of Agriculture Sciences

＜120〉a kind of acid glucanase CELA and gene thereof and application

<160>2

<210>1

<211>715

<212>PRT

＜213〉alicyclic acid genus bacillus (Alicyclobacillus hesperidum A4)

<400>1

MSPSGGVCVN RKQRTLKLGT LAATIVALSA VATPAVASAD TTTAIASSTV

HVTVNAAAEL 60

GIVPNTALGV NTAVWDGHLL DAAIPSLLRG IGVTMLRYPG GSTSDEYNWQ

TNTVTGGYAD 120

PNNTFDNFMG VVQKAGAQPI ITVNAGTGTP SEAAAWVQDA NVTHHYGVKY

WEIGNEMYGS 180

WEAGNFANNP SGYAKEAVSF IQAMKAVDPS IKIGVDLIAP GTGEDDWNAT

VLSTMHSLGV 240

LPDFAIVHWY AQNPGGETDA GLLSSTNQIS TMMDTLKQQL SSYGTIPVFV

TETNSVSYNP 300

GRQSTSLVNA LFLDDDMADW LESGAQNVDW WDLHNGIVTQ QAGANVDPNL

YGQYNYGDYG 360

LLSNGSSDNG ISEPAANTPF PTYYGYQMLA AVMVPGATMI GAGSNNDLVA

VHATKLPNGA 420

VDVMLINKDP KQAYTVDLQA EGFAAKGPAF TLFYGQGSNA VTPGKLDNLQ

NVTLPPYSVT 480

DIIIPAVPGH QPQGPQFTDK TTLSTPQVKP SANETLTTTF TDTRGAVKDG

TLDVEIYNPA 540

GQLVGQQVQS GVTFTPGQSS QPITWNWTAP DSPGTYTVKA FVFSQDGTSV

YAADPSAATF 600

TVTQPDPPTI SATVQLSATT VKVGTPVTIT TTYTETAPTG YLNNGLLVQY

AVYNNWTSSQ 660

QSNPTATLTP GQSVTETWTF TPEQAGTYTF PEGIFTSGWT QLQWINQNVT

LTVTN 715

<210>2

<211>2148

<212>DNA

＜213〉alicyclic acid genus bacillus (Alicyclobacillus hesperidum A4)

<400>2

atgtcacctt cagggggagt ctgtgtgaac cgaaaacagc gtacgttaaa gttgggaacg 60

ctcgcagcaa cgattgtggc actctcagcc gtggccacgc ctgcggtcgc cagtgcggat 120

acgacgacgg ccattgcgtc atcgacagtt catgtcacag tcaatgccgc tgctgaactt 180

ggaatcgtgc ccaatactgc acttggtgtg aatacggccg tctgggacgg gcatttactc 240

gatgcagcca ttccatctct gcttcgtggc attggggtaa ccatgttgcg atatcccgga 300

ggatcgactt cagatgagta caattggcaa acgaataccg taactggggg ttatgcagat 360

cccaacaaca cctttgacaa cttcatggga gtggtccaaa aggctggtgc gcaacccatt 420

attacggtca acgccggcac gggcacaccg agtgaagctg ccgcatgggt tcaagatgca 480

aatgtcacgc accactacgg tgtcaagtat tgggaaatcg gaaatgagat gtatggcagc 540

tgggaagcag ggaattttgc aaataaccca tctggttatg cgaaagaagc tgtatccttc 600

attcaggcca tgaaagcggt tgatccttct attaaaatcg gcgtggacct catcgcacct 660

ggtactggag aagatgactg gaatgcaacc gtgcttagca ccatgcacag ccttggggtg 720

cttccggact tcgccattgt gcactggtat gcgcaaaatc cgggaggcga gacggacgct 780

gggctgctca gttcgacgaa tcaaatttcg acgatgatgg ataccttgaa gcagcaattg 840

agctcctatg gaaccatccc ggttttcgtc accgaaacga attcggtttc gtataaccct 900

gggcgccaga gtacaagtct ggttaatgca ctgttcctcg atgatgacat ggcagactgg 960

cttgagtccg gagcgcaaaa cgtcgactgg tgggacttgc ataacggcat tgttacacaa 1020

caggccggtg caaatgtcga tccgaatctg tatgggcagt ataactacgg agactatgga 1080

cttttgtcca atggctcgag tgacaatggc atttcagaac cggctgccaa tacaccattc 1140

ccaacgtatt atggatacca aatgctcgca gccgtcatgg tgccaggagc gacgatgatc 1200

ggtgccggat cgaacaatga tctggtggcc gtgcatgcga ccaagttgcc caatggtgct 1260

gtcgacgtca tgttgatcaa caaggatccg aaacaagcgt ataccgtcga tttgcaagct 1320

gaaggatttg ctgctaaggg tcctgcattc acgttattct acgggcaagg cagtaacgcg 1380

gtgacgccgg gcaaattgga taatctgcag aatgtgacac taccgcccta ttctgtgacg 1440

gacatcatca taccggcggt gcccgggcat cagccacaag ggccacagtt tacggacaag 1500

acgacgttat ccactcctca ggtaaagcct agtgccaacg agactttgac cacgacgttt 1560

accgacacgc gtggtgcggt caaggatggt acactcgacg tggaaatcta caatccagca 1620

gggcaattgg ttgggcaaca agtgcagtct ggcgtgacgt ttacgcctgg gcaatcatct 1680

caaccgatta cctggaactg gacggcgccc gattctcctg ggacgtatac cgtgaaggcg 1740

ttcgtcttca gccaagacgg aacaagcgtg tatgcggcag acccgagtgc agctacgttc 1800

acggtcacac agccggatcc gcccaccatt tcggccaccg ttcagctgtc cgcaactacg 1860

gtcaaagtgg gtacacctgt gaccatcacg acgacttaca ccgaaaccgc gcctacgggg 1920

tacctgaaca acgggttgct tgtacagtac gccgtgtaca ataactggac atcatcgcaa 1980

cagtccaatc caactgcgac attgactcct gggcaatcgg tgactgagac ttggacattt 2040

acgccagagc aggccggaac ctacacattc cctgaaggca tctttaccag tggatggaca 2100

caattgcagt ggattaatca gaacgtgacc ttgactgtga caaactaa 2148

Claims (9)

1. an acid glucanase CELA is characterized in that, its aminoacid sequence is shown in SEQ ID NO.1.

2. an acidic dextranase gene C elA is characterized in that, the described acid glucanase CELA of coding claim 1.

3. acidic dextranase gene C elA as claimed in claim 2 is characterized in that its base sequence is shown in SEQ ID NO.2.

4. the recombinant vectors that comprises the described acidic dextranase gene C of claim 3 elA.

5. recombinant vectors according to claim 4 is characterized in that, the described recombinant vectors that comprises acidic dextranase gene C elA is pPIC9-CelA.

6. the recombinant bacterial strain that comprises the described acidic dextranase gene C of claim 3 elA.

7. recombinant bacterial strain as claimed in claim 6 is characterized in that, described bacterial strain is a pichia spp.

8. a method for preparing acid glucanase CELA is characterized in that, may further comprise the steps:

1) with the recombinant vectors transformed host cell of claim 4, gets recombinant bacterial strain;

2) cultivate recombinant bacterial strain, induce the reorganization dextranase to express; And

3) reclaim the also expressed dextranase CELA of purifying.

9. the described acid glucanase CELA of claim 1 is in the application of fodder industry.

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