CN101838637B - Wide-temperature applicability acidic dextranase AGL9A, and gene and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to wide-temperature applicability acidic dextranase AGL9A, and a gene and application thereof. The invention provides the dextranase AGL9A from Alicyclobacillus hesperidum A4(CGMCC No.3147), and an amino acid sequence of the dextranase AGL9A is shown as SEQ ID NO.1. The invention also provides the gene Agl9A for coding the dextranase. The dextranase of the invention has the properties of most preferred pH of 5.8, most preferred temperature of 55 DEGC, extremely wide temperature applicability and relatively higher thermalstability, and can be widely used, as a novel enzyme preparation, in the industries of animal feeds, food, beer, energy sources and the like.

Description

A kind of acidic dextranase AGL 9 A of wide-temperature applicability 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 acidic dextranase AGL 9 A and gene and application of wide-temperature applicability.

Background technology

Beta-glucan is a nature synthetic saccharan; Extensively be present in occurring in nature; Structural non-starch polysaccharide in the platymiscium cell walls is to be fundamental unit with the D-glucose, belongs to the homopolysaccharide class in the polyose; Space structure with line style is present in the aleurone layer and albuminous cell wall of cereal class (barley, oat, rye and wheat).Different according to species, the content of beta-glucan and proportion are also different.Wherein contained ratio is the highest in the middle of barley and oat.LSD is the general name that can VISOSE be degraded into the class of enzymes of oligose and glucose.At present LSD is in food (like sugar-refining industry), feed (like fish, bird, domestic animal etc.), and beer (making like Fructus Hordei Germinatus), medicine fields such as (like protective foodss) have obtained increasingly extensive application.Because barley self, barley mainly be applied to beer brewage with feed in, therefore relevant LSD has obtained application the most widely in brewage and fodder industry.In the barley germination process, the decomposition of VISOSE is incomplete, and degree of decomposition depends on barley variety and germination technology etc.; Generally only can decompose 30%-70%; Residual VISOSE increases converted mash viscosity, and filtering rate reduces, and the raw material leaching yield is on the low side; The finished beer quality guaranteed period is short, forms blushing or incipient gel deposition.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 the animal and fowl fodder that is the basis with barley, wheat, rye, oat, contain a large amount of beta-glucans; Because do not digest the enzyme of beta-glucan in the monogastric animal body, beta-glucan 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 influence the absorption of nutritive substance, reduced the nutritive value of feed, become a kind of ANFs.VISOSE is the ANFs in the various wheat class feeds.Add beta-glucanase, can effectively eliminate the anti-oxidant action of beta-glucan, improved the utilising efficiency of feed greatly.

Alicyclic acid genus bacillus (Alicyclobacillus) is one type of thermophilic aciduric bacteria.Because alicyclic acid genus bacillus (A.acidocaldarius) etc. can cause the corruption of pasteurization fruit juice, produce the smell that is difficult to accept, add the viability that it is unique.Caused at present the very big concern of global foodstuffs industry and academia.

In fodder industry, animal gastrointestinal tract is sour environment (like the gi tract of pig), in the course of processing of feed, a pyroprocess is in short-term arranged simultaneously; In the Fructus Hordei Germinatus preparation and saccharifying of beer; Because the ph optimum environment of the endogenous enzyme in the middle of the Fructus Hordei Germinatus is pH5.0-6.0; In most cases in saccharifying with pH people for adjusting to pH5.6, in addition, the saccharifying of Fructus Hordei Germinatus is a process that temperature raises gradually.Therefore, obtaining novel research with acidic dextranase of good temperature stability and suitability is significant.The clone with separate have high-temperature stability and adaptive LSD can better application in feed and brewing industry, and can reduce production costs, it is necessary all to be that LSD 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 wide-temperature applicability of ability efficient application.

A purpose more of the present invention provides the gene of the acidic dextranase of the above-mentioned wide-temperature applicability 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 the acidic dextranase of above-mentioned wide-temperature applicability.

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

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: CGMCCNo.3147, preservation date: separate obtaining a kind of new wide-temperature applicability acidic dextranase AGL 9 A on June 29th, 2009).

The invention provides a kind of acidic dextranase AGL 9 A of wide-temperature applicability, its aminoacid sequence is shown in SEQID NO.1.

SEQ?ID?NO.1：

MEATMQAAIHINQLGYRPQDRKRFYVSGCGGSFAVFRADGSEVFAGELQAVGDD

KATGMQVYIGDISDVIEVGTYQLTVPGIGQSVSLAIHPDVYANLHKGLQKFFYFQR

CGVELSAEHAGEFSHAACHVEKAHVYAEPSRRIDCHGGWHDAGDYGKYVVPAA

KAVADLLLAYEFYPKAFMSPVGIPESGAGLPDVLSEVRFELEWMLRMQDEQTGGV

YHKVTTYRFPPLSTMPEDDRGELVLSPISYAATATFAAALAHAARIYRAFDADFATR

CLNAAARAYEWCTSHDAEPFHNPDGVATGEYGDKKLSDEHYWAAASLLQATGE

DRYHQACLQAFATGGFSLTELGWADVGGYGTIAYLRADRAVTQAQVWEHLRKA

WLTEADRLVSLGACTAFGIPLAEHDYIWGSNMVLLNRGMHLLIAYALEPSDLYVD

GALQTIHYMLGENALQQCYVSGYGRKPLLHPHHRPSVADAVEAPVPGMLAGGPN

RNLQDTVARERLQNQPAARCFLDHEESYSTNEVAVYWNSPAVFVVSHFVGV

541 amino acid of this enzyme genes encoding, so the theoretical molecular of dextranase AGL 9 A is 59.4kDa.

Dextranase AGL 9 A of the present invention all has greater activity in neutrality and acid range.The present invention screens the LSD that a kind of alicyclic acid genus bacillus Alicyclobacillus hesperidumA4 (CGMCCNo.3147) is produced; Its recombinase optimum pH at pichia spp is 5.8, in the scope of pH5.0～7.0, keeps the enzymic activity more than 75%; 37 ℃ are incubated 60 minutes in the scope of pH4.2～10.4, can keep the enzymic activity more than 75%.Optimum temperuture is 55 ℃, between 35 ℃-65 ℃, all has the enzyme activity more than 55%; 60 ℃ of insulations 60 minutes, residual enzyme work reached more than 70%.The also unprecedented report of the LSD of this character.

The invention provides the gene of the above-mentioned wide-temperature applicability acidic dextranase AGL 9 A of coding.Particularly, the gene order of this gene is shown in SEQ ID NO.2:

SEQ?ID?NO.2：

ATGGAGGCGACTATGCAAGCAGCAATACATATAAACCAGTTGGGCTATCGTCCA

CAGGATCGGAAACGTTTCTATGTAAGCGGTTGCGGAGGTTCGTTTGCCGTCTTC

CGGGCGGATGGTAGCGAGGTTTTCGCTGGCGAATTGCAGGCAGTCGGAGATGA

CAAGGCAACCGGAATGCAGGTGTACATCGGCGACATCAGTGACGTCATCGAAG

TCGGCACATATCAACTCACAGTGCCGGGCATAGGACAATCGGTCTCTTTGGCCA

TTCATCCTGACGTGTATGCCAATCTGCATAAAGGCTTACAGAAGTTTTTCTACTT

TCAGCGCTGTGGCGTCGAGTTGTCTGCTGAGCACGCCGGCGAATTCTCGCATG

CCGCTTGTCACGTGGAAAAGGCACATGTGTATGCCGAACCATCGCGGCGTATCG

ACTGTCACGGCGGATGGCATGACGCTGGGGACTACGGGAAATACGTTGTGCCG

GCGGCGAAAGCGGTTGCGGATCTGTTGCTCGCCTATGAATTCTATCCGAAGGCA

TTTATGAGCCCGGTCGGTATCCCAGAGTCGGGAGCCGGCTTGCCTGACGTGCTT

AGTGAGGTTCGCTTTGAACTCGAATGGATGTTGCGTATGCAGGATGAGCAGAC

GGGAGGCGTCTATCACAAGGTCACAACTTATCGGTTCCCTCCTTTGTCAACGAT

GCCTGAGGACGATCGTGGGGAACTGGTGCTCTCGCCTATTTCATACGCAGCTAC

TGCGACATTCGCGGCTGCACTTGCCCATGCGGCTCGCATCTATCGGGCGTTTGA

TGCGGATTTTGCAACCCGATGTTTGAACGCTGCTGCGCGGGCCTACGAATGGTG

CACGAGTCATGACGCCGAACCGTTTCACAATCCGGATGGCGTAGCCACAGGCG

AGTATGGCGATAAGAAGCTGAGCGATGAACACTACTGGGCAGCGGCTTCGCTT

TTGCAAGCGACGGGAGAAGATCGCTATCATCAGGCATGCTTGCAGGCCTTCGC

AACTGGAGGGTTTTCCCTGACTGAGCTAGGCTGGGCTGACGTTGGCGGTTACG

GCACAATCGCGTATTTGCGGGCGGATCGTGCCGTTACGCAGGCGCAGGTTTGG

GAACATCTGCGCAAAGCTTGGCTGACAGAGGCGGATAGGTTGGTCAGCCTTGG

TGCATGTACGGCATTTGGCATTCCGCTCGCGGAGCACGATTACATTTGGGGAAG

CAACATGGTGTTGTTAAATCGTGGCATGCACTTGCTGATTGCATACGCACTGGA

ACCTTCCGATCTGTACGTCGACGGAGCTTTACAGACTATTCATTACATGCTGGGC

GAAAATGCCTTACAACAGTGCTACGTTTCAGGGTATGGGAGAAAGCCGCTTCTT

CATCCTCACCATCGTCCATCGGTGGCTGATGCAGTCGAGGCTCCTGTGCCAGGC

ATGTTGGCCGGCGGCCCCAATCGCAATTTGCAAGATACAGTAGCTCGCGAGCGT

CTGCAGAACCAGCCTGCAGCACGATGTTTTCTCGATCACGAAGAAAGTTATTCG

ACCAATGAAGTGGCTGTCTACTGGAACTCGCCAGCCGTCTTTGTGGTTTCTCAT

TTTGTGGGTGTCTGA

The method separating clone of the present invention through PCR glucanase gene AGL9A, the DNA complete sequence analysis is the result show, dextranase AGL 9 A structure gene AGL9A total length 1626bp contains a terminator TGA.The maturation protein theoretical molecular of dextranase AGL 9 A is 59.4kDa.Glucanase gene AGL9A sequence and the aminoacid sequence derived are carried out the BLAST comparison in GenBank.This gene is 48% with the highest consistence of cellulase (CAC34051) aminoacid sequence that derives from the 9th family of Alicyclobacillusacidocaldarius, explains that AGL9A is a kind of new LSD.

The present invention also provides the recombinant vectors that comprises above-mentioned glucanase gene, is preferably pPIC9-Agl9A.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 a 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-Agl9A.

The present invention also provides the recombinant bacterial strain that comprises above-mentioned glucanase gene, is preferably recombinant bacterial strain GS115/Agl9A.

The present invention also provides a kind of method for preparing acidic dextranase AGL 9 A, may further comprise the steps:

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

2) cultivate recombinant bacterial strain, induce the reorganization dextranase AGL 9 A to express; And

3) reclaim the also expressed dextranase AGL 9 A of purifying.

Wherein, preferred said 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/Agl9A.

The present invention also provides the application of above-mentioned acidic dextranase AGL 9 A.

The present invention's technical problem at first to be solved is the deficiency that overcomes prior art, provide a kind of character good, be suitable at feed, food is particularly used new LSD in the brewing industry.LSD ph optimum of the present invention is 5.8, at pH 5.4～6.0 higher enzymic activity (more than 90%) is arranged all; The pH good stability; Ability with better heat-resisting.Its superior heat resistance ability can effectively be reduced in the loss of enzyme activity in the brewer's malt making processes, reduces the application cost of LSD.Higher pH accommodation improves the transformation efficiency of non-starchiness polysaccharide in the feed, reduces formulation cost, reduces environmental pollution; Can be applicable to brewing industry, reduce the viscosity of wort, improve filtration efficiency, increase the wort productive rate; Can also be used for bioenergy, as the VISOSE 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; VISOSE 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 LSD of Pichia anomala expression, wherein, and 1: low molecular weight protein Marker; 2: the purified recombinant LSD.

The recombinate ph optimum of LSD of Fig. 2.

The recombinate pH stability of LSD of Fig. 3.

The recombinate optimum temperuture of LSD of Fig. 4.

The recombinate thermostability of LSD of Fig. 5.

The recombinate various protease resistants of LSD of Fig. 6

Alicyclic acid genus bacillus Alicyclobacillus hesperidumCGMCC3147; 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, pH4.0.

Explain: 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, the 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.Prove that it has dextranase activity.

The clone of embodiment 2 alicyclic acid genus bacillus AlicyclobacillushesperidumA4 (CGMCCNo.3147) glucanase coding gene as well as AGL9A

The acquisition of gene order

Conservative (IPESG and TGEYGD) sequences Design according to the 9th family's glucanase gene has been synthesized degenerated primer Agl9F, Agl9R 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, 53 ℃ of annealing 30sec, 72 ℃ are extended 1.5min.Last 72 ℃ of insulation 10min.Obtain an about 354bp 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; Design each three TAIL-PCR specificity nested primers of upstream and downstream: the zone of ignorance direction of design direction for needing to increase, and with they difference called after usp1, usp2; Usp3 (upper reaches Auele Specific Primer); Dsp1, dsp2, dsp3 (downstream Auele Specific Primer) sees table 1.

Table 1. Xylanase XYNA 4 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 through TAIL-PCR, amplification obtains product and reclaims the back order-checking.

Through this full length gene of discovery 1626bp behind the genome sequence that compares LSD, encode 541 amino acid and a terminator codon, the N end does not have the signal peptide sequence of prediction.The aminoacid sequence of measured Gene A L9A and the glucanase gene sequence on the GeneBank are carried out homology relatively; The highest consistence is 48%; Explain that AGL9A is a kind of new LSD, the gene that shows the coding LSD 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, whenever once at a distance from the 10min mixing, 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, deposition is with 70% washing with alcohol twice, and vacuum-drying adds an amount of TE and dissolves, place-20 ℃ subsequent use.

Primer (Agl9AF:10GCATACGTAATGGAGGCGACTATGCAAGCAGC and Agl9AR:GAAGCGGCCGCTCAGACACCCACAAAATGAGAAACCAC) has been synthesized in design according to the Agl9A 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, 58 ℃ of annealing 30sec, 72 ℃ are extended 90s, 30 circulations.Last 72 ℃ of insulation 10min.

The preparation of embodiment 3 reorganization LSDs.

Expression vector pPIC9 is carried out double digestion (SnaBI and NotI); To encode the simultaneously Gene A gl9A double digestion (SnaBI and NotI) of LSD; The gene fragment that cuts out the coding LSD is connected with expression vector pPIC9; Acquisition contains the recombinant plasmid pPIC9-Agl9A of glucanase gene Agl9A and transforms pichia spp GS115, obtains recombinant pichia yeast strain GS115/Agl9A.

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 200mL BMMY substratum then, 30 ℃ of 250rpm shaking culture.After inducing 48h, centrifugal collection supernatant.Measure the vigor of LSD.The expression amount of reorganization LSD is 2.14U/mL.SDS-PAGE result (Fig. 1) shows that the reorganization LSD has obtained expression in pichia spp.

The activation analysis of embodiment 4 reorganization LSDs

The DNS method: concrete grammar is following: at pH5.8 (0.1M SODIUM PHOSPHATE, MONOBASIC-Hydrocerol A), under 55 ℃ 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 PM discharges 1 μ mol reducing sugar.

The property testing of embodiment 5 reorganization dextranase AGL 9 As

1, the measuring method of the ph optimum of reorganization dextranase AGL 9 A and pH stability is following:

Embodiment 3 purified recombinant LSDs are carried out enzymatic reaction to measure its ph optimum under different pH.The substrate VISOSE is with damping fluid (the 0.1M glycocoll-hydrochloric acid pH1.2-2.6 of different pH; 0.1mol/L Hydrocerol A-Sodium phosphate, dibasic pH3.0-7.4; 0.1MTris-HClpH7.8-8.6; 0.1M glycocoll-sodium hydroxide pH9.0-12.0), under 60 ℃, carry out the LSD vitality test.Result (Fig. 2) shows that the ph optimum of AGL9A is 5.8, in the scope of pH5.0～7.0, keeps the enzymic activity more than 75%.LSD is 37 ℃ of processing 60min in the damping fluid of above-mentioned various different pH, in the pH7.0 buffer solution system, measure enzymic activity again under 55 ℃, with the pH patience of research enzyme.Result (Fig. 3) shows that LSD is all very stable between pH4.2-10.4, and the residual enzyme activity is more than 75% behind the processing 60min in this pH scope, and this this enzyme of explanation has pH stability preferably.

2, the optimum temperuture of LSD and thermal stability determination method are following:

Enzymatic reaction is carried out in being determined as under Hydrocerol A-Sodium phosphate, dibasic damping fluid (pH5.8) buffer solution system and differing temps of the optimum temperuture of LSD.Temperature tolerance is determined as LSD and under differing temps, handles different time, under 60 ℃, carries out enzyme assay again.The enzyme reaction optimum temperuture is measured result (Fig. 4) and is shown that its optimum temperuture is 55 ℃.The thermostability property test of enzyme shows (Fig. 5), and recombinase stability in the time of 60 ℃ is very good.60 ℃ are incubated 60min down, and the residual enzyme activity is 72%, and said enzyme all has the enzyme activity more than 55% between 35 ℃-65 ℃.

3, the K of LSD _mValues determination method is following:

Use the barley of different concns to be substrate, in Hydrocerol A-Sodium phosphate, dibasic damping fluid (pH5.8) buffer solution system, measure enzymic activity down, calculate its k under 55 ℃ for 55 ℃ _mValue.Through measuring, this LSD is the k of substrate with the VISOSE under 55 ℃ _mValue is 4.82mg ml ^-11.90mg/mL, maximum reaction velocity V _MaxBe 74.57 μ mol/minmg

4, different metal ion chemistry reagent is measured as follows the influence of XYLA4 enzyme work:

In enzymatic reaction system, add the different metallic ion and the chemical reagent of different concns, study its influence to enzymic activity, various material final concentrations are 2 and 20mmol/L.Under 55 ℃, pH5.8 condition, measure enzymic activity.Result's (table 1) shows, most of ions and chemical reagent do not have considerable change at the vigor of concentration reorganization LSD during for 2mmol.Pb ²⁺, Cr ³⁺, can partly suppress its vigor, but Ag ⁺, Hg ²⁺With SDS its vigor of strongly inhibited almost.Fe when concentration is 20mmo ³⁺, Pb ²⁺, EDTA, 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 1.19 and 1.56 times when 1mmol and 10mmol respectively.

The various chemical reagent of table 1 are to the influence of dextranase AGL 9 A vigor

The simulation Fructus Hordei Germinatus hydrolysising experiment of embodiment 6 reorganization LSDs

Concrete grammar is following: the 10g malt meal, be dissolved in pH5.6 (0.1M SODIUM PHOSPHATE, MONOBASIC-Hydrocerol A) damping fluid of 90ml, and add the enzyme liquid (20u/ml) of 1ml, not enzyme-added liquid is as contrast.Under the water-bath under the 100rpm rotating speed according to following routine processes:

Handle 30min for 45 ℃, handle 30min for 50 ℃ then, handle 60min for 55 ℃; Handle 60min for 60 ℃ and handle under the 5min condition for last 100 ℃, be cooled to 25 ℃, behind the centrifugal 5min of 3000rpm; Get supernatant (10ml) and survey filtering rate, measure viscosity with capillary viscosimeter with middling speed filter paper.

The result shows: the viscosity ratio contrast of handling through enzyme liquid has descended 6.12%, and filtration time has shortened 26.71%.Explain that Agl9A can finely be applied in the brewing industry

Sequence table

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

< 120>a kind of acidic dextranase AGL 9 A of wide-temperature applicability and gene thereof and application

<160>2

<210>1

<211>541

<212>PRT

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

<400>1

MEATMQAAIH?INQLGYRPQD?RKRFYVSGCG?GSFAVFRADG?SEVFAGELQA

VGDDKATGMQ 60

VYIGDISDVI?EVGTYQLTVP?GIGQSVSLAI?HPDVYANLHK?GLQKFFYFQR

CGVELSAEHA 120

GEFSHAACHV?EKAHVYAEPS?RRIDCHGGWH?DAGDYGKYVV?PAAKAVADLL

LAYEFYPKAF 180

MSPVGIPESG?AGLPDVLSEV?RFELEWMLRM?QDEQTGGVYH?KVTTYRFPPL

STMPEDDRGE 240

LVLSPISYAA?TATFAAALAH?AARIYRAFDA?DFATRCLNAA?ARAYEWCTSH

DAEPFHNPDG 300

VATGEYGDKK?LSDEHYWAAA?SLLQATGEDR?YHQACLQAFA?TGGFSLTELG

WADVGGYGTI 360

AYLRADRAVT?QAQVWEHLRK?AWLTEADRLV?SLGACTAFGI?PLAEHDYIWG

SNMVLLNRGM 420

HLLIAYALEP?SDLYVDGALQ?TIHYMLGENA?LQQCYVSGYG?RKPLLHPHHR

PSVADAVEAP 480

VPGMLAGGPN?RNLQDTVARE?RLQNQPAARC?FLDHEESYST?NEVAVYWNSP

AVFVVSHFVG 540

V 541

<210>2

<211>1626

<212>DNA

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

<400>2

atggaggcga?ctatgcaagc?agcaatacat?ataaaccagt?tgggctatcg?tccacaggat 60

cggaaacgtt?tctatgtaag?cggttgcgga?ggttcgtttg?ccgtcttccg?ggcggatggt 120

agcgaggttt?tcgctggcga?attgcaggca?gtcggagatg?acaaggcaac?cggaatgcag 180

gtgtacatcg?gcgacatcag?tgacgtcatc?gaagtcggca?catatcaact?cacagtgccg 240

ggcataggac?aatcggtctc?tttggccatt?catcctgacg?tgtatgccaa?tctgcataaa 300

ggcttacaga?agtttttcta?ctttcagcgc?tgtggcgtcg?agttgtctgc?tgagcacgcc 360

ggcgaattct?cgcatgccgc?ttgtcacgtg?gaaaaggcac?atgtgtatgc?cgaaccatcg 420

cggcgtatcg?actgtcacgg?cggatggcat?gacgctgggg?actacgggaa?atacgttgtg 480

ccggcggcga?aagcggttgc?ggatctgttg?ctcgcctatg?aattctatcc?gaaggcattt 540

atgagcccgg?tcggtatccc?agagtcggga?gccggcttgc?ctgacgtgct?tagtgaggtt 600

cgctttgaac?tcgaatggat?gttgcgtatg?caggatgagc?agacgggagg?cgtctatcac 660

aaggtcacaa?cttatcggtt?ccctcctttg?tcaacgatgc?ctgaggacga?tcgtggggaa 720

ctggtgctct?cgcctatttc?atacgcagct?actgcgacat?tcgcggctgc?acttgcccat 780

gcggctcgca?tctatcgggc?gtttgatgcg?gattttgcaa?cccgatgttt?gaacgctgct 840

gcgcgggcct?acgaatggtg?cacgagtcat?gacgccgaac?cgtttcacaa?tccggatggc 900

gtagccacag?gcgagtatgg?cgataagaag?ctgagcgatg?aacactactg?ggcagcggct 960

tcgcttttgc?aagcgacggg?agaagatcgc?tatcatcagg?catgcttgca?ggccttcgca 1020

actggagggt?tttccctgac?tgagctaggc?tgggctgacg?ttggcggtta?cggcacaatc 1080

gcgtatttgc?gggcggatcg?tgccgttacg?caggcgcagg?tttgggaaca?tctgcgcaaa 1140

gcttggctga?cagaggcgga?taggttggtc?agccttggtg?catgtacggc?atttggcatt 1200

ccgctcgcgg?agcacgatta?catttgggga?agcaacatgg?tgttgttaaa?tcgtggcatg 1260

cacttgctga?ttgcatacgc?actggaacct?tccgatctgt?acgtcgacgg?agctttacag 1320

actattcatt?acatgctggg?cgaaaatgcc?ttacaacagt?gctacgtttc?agggtatggg 1380

agaaagccgc?ttcttcatcc?tcaccatcgt?ccatcggtgg?ctgatgcagt?cgaggctcct 1440

gtgccaggca?tgttggccgg?cggccccaat?cgcaatttgc?aagatacagt?agctcgcgag 1500

cgtctgcaga?accagcctgc?agcacgatgt?tttctcgatc?acgaagaaag?ttattcgacc 1560

aatgaagtgg?ctgtctactg?gaactcgcca?gccgtctttg?tggtttctca?ttttgtgggt 1620

gtctga 1626

Claims (9)

1. an acidic dextranase AGL 9 A that between 35-65 ℃, is suitable for is characterized in that its aminoacid sequence is shown in SEQ ID NO.1.

2. an acidic dextranase Gene A gl9A is characterized in that, the described acidic dextranase AGL 9 A that between 35-65 ℃, is suitable for of coding claim 1.

3. the acidic dextranase Gene A gl9A that between 35-65 ℃, is suitable for 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 A gl9A that between 35-65 ℃, is suitable for of claim 3.

5. the recombinant vectors of the acidic dextranase Gene A gl9A that between 35-65 ℃, is suitable for according to claim 4 is characterized in that said recombinant vectors is pPIC9-Agl9A.

6. the recombinant bacterial strain that comprises the described acidic dextranase Gene A gl9A that between 35-65 ℃, is suitable for of claim 3.

7. recombinant bacterial strain as claimed in claim 6 is characterized in that, said bacterial strain is a Pichia yeast.

8. a method that is prepared in the acidic dextranase AGL 9 A that is suitable between 35-65 ℃ 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 LSD to express; And

3) reclaim the also expressed dextranase AGL 9 A of purifying.

9. the application of the said acidic dextranase AGL 9 A that between 35-65 ℃, is suitable for of claim 1.

Images