CN101886064A - Acid amylase AMYA4 and gene and application thereof - Google Patents

Abstract

The invention relates to the field of gene engineering, in particular to acid amylase AMYA4 and gene and application thereof. The amino acid sequence of the acid amylase AMYA4 is expressed as SEQ ID NO.1. The optimal pH value of the AmyA4 is 4.2, the optimal temperature is 75 DEG C, and the AmyA4 has high activity at the temperature of between 55 and 75 DEG C and has strong raw starch degrading capability at the same time. The zymology properties meet the process requirement for preparing sugar by a double-enzyme method, so the acid amylase has great application potential. In the process for preparing sugar by simulating the double-enzyme method, no matter paste starch or raw starch is used as a substrate, the starch can reach good hydrolysis rate under the action of matching commercial saccharifying enzyme, so the AmyA4 has huge application potential.

Description

A kind of acid amylase AMYA 4 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 amylase AMYA 4 and gene thereof and application.

Background technology

(α-amylase) is one of present most important industrial enzyme preparation to α-Dian Fenmei, is present in widely in animals and plants and the microorganism, and it is a kind of inscribe glucuroide, is EC3.2.1.1 according to the standard of international enzyme committee's meeting (Enzyme Commission).When α-Dian Fenmei acts on starch,, starch molecule is degraded rapidly, generate dextrin and reducing sugar from inner α-1,4 key that cuts arbitrarily of starch molecule.α-Dian Fenmei acts on starch makes starch lose the dye-forming reaction of iodine usually.When the hydrolysis amylose starch, α-Dian Fenmei is cut α-1,4 key at random, makes starch be hydrolyzed into Oligomeric maltose and oligosaccharides, and then is hydrolyzed into maltose and glucose.When being substrate with the amylopectin, α-Dian Fenmei can be cut α-1,4 key at random, but can not cut α-1,6 key and near α-1,4 key, therefore in the hydrolysate except that maltose and glucose, also contain some limit dextrin (Penayanez et al., 1963; You Xin, 1997).The optical property of the product terminal glucose residue C1 carbon atom of α-Dian Fenmeishuixie starch is and is α-configuration, so claim α-Dian Fenmei (outstanding new, 1997).α-Dian Fenmei is one of present most important industrial enzyme preparation, is bringing into play huge effect in industry such as monosodium glutamate, maltose, maltose alcohol, dextrin, glucose, alcohol, beer, lactic acid, citric acid.Because some bacterials have characteristics such as high temperature resistant, acidproof, alkaline-resisting, more meet the various extreme conditions in the industrial production, therefore current widely used α-Dian Fenmei majority is the amylase that derives from bacterium (bacillus amyloliquefaciens and Bacillus licheniformis), and especially the most widely used in industry such as need pyritous fermentation is the bacterium high-temperature.

α-Dian Fenmei is a kind of crucial zymin, is one of enzyme of consumption maximum in the foodstuffs industry.α-Dian Fenmei with its good performance be widely used in grain processing, foodstuffs industry, brewage, fermentation, papermaking, industrial textile and pharmaceutical industries etc., it had once accounted for the half territory of the whole zymin market share.

In traditional starch refine dsugar technology, with purified starch or rice etc. is raw material, usually use acid hydrolysis legal system glucose, owing to need high temperature, high pressure and acid catalyst, therefore when producing glucose, must be with the compound decomposition reaction of glucose, produce some not fermentable sugar and a series of coloring matters thereof, this not only reduces the starch transformation efficiency, and because the liquid glucose of producing is of poor quality, to the follow-up refining disadvantageous effect of bringing, the use of a large amount of acid has simultaneously also brought very big pollution to environment.Utilizing double-enzyme method sugar making is to act on single-minded zymin as catalyzer, the reaction conditions gentleness, compound decomposition reaction is less, therefore adopt double-enzyme method to produce glucose, conversion of raw material and sugar concentrations such as starch or rice have been improved, having improved the liquid glucose quality, is present ideal method for manufacturing sugar.Enzyme liquefaction and enzyme glycolysis technology are called double-enzyme method (outstanding new, 1997).

Double-enzyme method sugar making starch transformation efficiency height, the sugared cost of ton is low, the concentration height of sugar, therefore liquid glucose quality height, is widely adopted in fermentation industries such as monosodium glutamate.Yet present industrial application is alpha-amylase and middle temperature saccharifying enzyme.The optimum temperature of alpha-amylase is 95 ℃, and the optimum temperature of middle temperature saccharifying enzyme is 60 ℃, and their optimum pH is also inequality simultaneously.The optimum pH of alpha-amylase is usually at 6.0-6.5, and the optimum pH of saccharifying enzyme is usually at 4.0-4.5.In double-enzyme method sugar making technology, pH about 4-5, therefore need adjust pH value and temperature usually repeatedly behind the starch dissolution, has caused the complicacy and the environmental problem (Liu and Xu, 2008) of producing.α-Dian Fenmei has become one of of paramount importance enzyme in the industrial application, but the large-scale commercial applications production of enzyme still is confined in several specific fungies and bacterium.Demand for α-Dian Fenmei efficiently is more and more.

The a plurality of alpha-amylase genes that derive from alicyclic acid genus bacillus (Alicyclobacillus) genus at present are purified, and clone and have expressed.Such as the α-Dian Fenmei that derives from Alicyclobacillus acidocaldarius (Matzke etal., 1997; Schwermann et al., 1994; Yuan et al., 2005).It is acid that their character all belongs to, heat-resistant alpha-amylase.

Also more and more for the farinose diastatic research of can degrading at present, separated with purifying the farinose amylase of a plurality of degradeds.Hayashida etc. (1986) are pure from Aspergillus ficum to obtain a living starch starch enzyme; Hayashida etc. (1988) are pure from Bacillus subtilis again subsequently to obtain a living starch starch enzyme, and produced amylolysis amylase successively was separated afterwards, purifying or clone come out (Gangadharan et al., 2009; Jeang et al., 2002; Jeang et al., 1995; Kim et al., 1990; Kim et al., 1989; Saha et al., 1988).The farinose ability yet the amylase that derives from the alicyclic acid bacillus of report is not degraded.

Summary of the invention

The acid starch enzyme 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 acid starch enzyme of coding.

Another object of the present invention provides the application of above-mentioned acid starch enzyme.

The present invention is from the disclosed alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 of CN200910235943.1 (open day 2010.05.05), (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 acid amylase AMYA 4 on June 29th, 2009).

According to acid amylase AMYA 4 of the present invention, its aminoacid sequence is shown in SEQ ID NO.1.

SEQ?ID?NO.1：

MNDAMFICLSQLCKRLHRQTTLFTPLHTRIDACGKTTQRGIFVGQIKKTTIAALIAL

SAALPTAGSVKPVLADANDSTPSIQITSGNDAKAGVASDTLTVAVSGMDIVGTTPD

VTLAGADGSTRNLTTDTTVADNSTLHVELPMGDSGLGAGTYTLTVSAGGASASAT

LTIEPYTSASTIQWDGIYTDDSATYVSIPNPSPGQNVTIRLRAYSGNLTKVVLKAYD

TAQSKSFDVEMAPTSTFGPYQLWSATVPASNGGTIYYRFDLYDDNDFACLSGDGL

HTSDDTNQNFPLPVGAVTLSTTDANPGDTVTASDPVGDFSGSGQSQTTVNFLDQS

GNVVATTTGSNASWSSVDFTVPKDVPNGLYTVDLDTTAKDADGVTNVSLDRQVP

LLVGPEPAWMQSFYHDSYSSFYRSPFGAVATGTPVILRLRGPVDLKSALLRLWGAN

GNGSELDLPMQPLAMSATEIEQATGTPDATQYSWWTVTIPASDVTTAGTMWYQFA

GQLASGQTVYYDDNGNQLEGPGQPSFSAGGPSYQLSVYNQGFTTPDWLKHAVIY

EIMPDRFYNGDIANDENPKTQKGIYTDAVGQETLGPIQFHQDWNSQPYDPNIPASS

DPAIQALRGNGQWNIDFFGGDLKGIQDKLDYLKSLGVNTLYLMPVFEAESNHKY

DTADYMKIDPGFGTAQDWLNLAKAAHADGFHILLDGVFEDTGSDSVYFNKFSNM

GSLGAWQAYMQNQPNLSPYYSWYEWTNNPANPYNGWWNNDTLPQTDTNNPSF

QQFIYGGKDAVAKHWLALGADGWRLDSADNSNYNVTWWSNFRNAVKSIDPNAA

IVGEIWNTATNDNGTDWLTGSTFDSVMNYSFRNAVIDFFRGTYNDGSVQHHAVDA

AGFNQELMRLYSEYPLQSFYAMMNLVDSQDTMRILTVLENAPEPGSMSALQQATY

QPTATDQQLGIKRLELVSDLQFGFPGDPTIWYGDEAGVSGYSDPLSRDTYPWGHE

NEALLNHYRLLGAIRAANPVLQTGTFTPVYAQGEVYAFARTIQGGQDVFGKPAAD

ASAIVALNNQNQTQTVNLPVAGVIANGTKLLDELNDQWYTVENGAVQLTLAPYE

GAILVTPTANPVAYLQTINGQTSIAWTPVAGANGYLLLRHQGDVWVPVGRPLSAN

TLSSPVTQGATAVDYAIAALPPVAPGVGLSSGQPLQAVTVPAASLGQPQVQVDVQH

SGVSLHITPVPNATQYVVYLQQPDGSYQAVATVAAHGDVHLRLPVAPGTQSISVRV

AAQNEDGQAVTDPMVITVNPSAKAQR

This enzyme comprises 1290 amino acid, so the theoretical molecular of amylase AMYA4 is 138kDa.

Amylase AMYA4 of the present invention all has greater activity in acid range, its optimum pH is 4.2 behind the purifying, keeps the enzymic activity more than 80% in the scope of pH 2.8～4.2; 37 ℃ are incubated 60 minutes in the scope of pH1.2～7.8, can keep the enzymic activity more than 80%.Optimum temperuture is 75 ℃, all has the enzyme activity more than 50% between 55 ℃-80 ℃; 75 ℃ of insulations 60 minutes, residual enzyme work reached more than 80%.In addition, this enzyme also has the farinose ability of hydrolysis.The also unprecedented report of the amylase of this character.

The invention provides the gene of the above-mentioned acid amylase AMYA 4 of coding.Particularly, the gene order of this gene is shown in SEQ ID NO.2:

SEQ?ID?NO.2：

ATGAATGATGCAATGTTCATATGCCTCAGTCAGTTGTGCAAACGATTGCACAGA

CAAACCACTCTCTTCACACCATTGCACACCCGCATCGACGCTTGTGGCAAAAC

AACACAAAGGGGGATTTTTGTGGGCCAAATCAAGAAAACGACCATCGCAGCA

CTCATCGCCCTGTCCGCGGCTCTGCCAACAGCCGGTTCCGTAAAGCCAGTGCTC

GCGGATGCAAACGATTCGACACCATCCATCCAAATTACGTCGGGAAACGACGC

TAAGGCTGGCGTGGCGAGTGACACCCTCACCGTCGCCGTGAGTGGTATGGACA

TTGTCGGAACCACCCCAGACGTCACCTTGGCAGGCGCAGACGGCTCGACGCG

CAATCTCACGACCGACACGACCGTCGCGGACAATTCGACACTGCATGTCGAAT

TGCCCATGGGGGACAGTGGTTTAGGGGCGGGCACGTATACCCTGACGGTCTCT

GCTGGTGGCGCCAGCGCATCCGCGACGCTCACCATTGAGCCGTACACCAGCGC

AAGCACCATTCAGTGGGACGGTATCTACACGGACGATTCTGCAACCTACGTTTC

GATCCCCAATCCAAGCCCGGGGCAAAACGTCACCATTCGCCTTCGCGCCTACA

GCGGCAATTTGACCAAAGTCGTGCTGAAGGCGTACGACACTGCACAGAGCAAA

TCGTTTGACGTCGAAATGGCGCCGACGTCGACGTTTGGCCCTTATCAGTTGTGG

TCGGCGACTGTGCCGGCGTCGAATGGAGGTACCATCTATTATCGGTTTGACCTG

TATGATGACAATGACTTCGCTTGTCTTTCCGGCGATGGTCTGCACACAAGCGAC

GACACGAATCAAAACTTCCCCCTCCCTGTAGGCGCTGTGACGCTGTCCACAAC

CGACGCAAATCCAGGAGACACGGTGACGGCCTCCGATCCCGTAGGGGATTTCT

CGGGCAGCGGCCAGAGCCAAACGACCGTCAACTTCCTTGACCAAAGCGGGAA

CGTCGTCGCAACGACGACCGGATCAAACGCAAGCTGGTCGAGTGTCGATTTCA

CAGTGCCAAAAGATGTACCCAACGGACTCTACACGGTCGATCTCGATACCACC

GCGAAAGACGCGGATGGTGTGACAAACGTCTCGCTGGATCGGCAGGTGCCACT

TTTGGTCGGTCCGGAACCAGCCTGGATGCAGAGCTTTTACCACGACTCGTACA

GTTCGTTTTATCGCAGTCCGTTTGGTGCTGTGGCCACCGGCACACCCGTGATCC

TGCGCCTGCGTGGACCTGTAGATTTAAAATCGGCGCTCCTTCGGCTATGGGGTG

CAAACGGAAACGGGTCTGAACTCGACTTGCCGATGCAGCCACTCGCGATGTCC

GCTACGGAGATCGAACAGGCAACAGGCACGCCCGATGCTACACAATACAGTTG

GTGGACCGTGACCATCCCCGCCTCCGATGTGACCACTGCGGGAACCATGTGGTA

TCAGTTCGCAGGACAACTGGCAAGCGGCCAAACCGTGTACTACGACGACAAC

GGAAACCAGCTGGAGGGCCCTGGCCAACCGAGCTTTTCTGCCGGCGGGCCCA

GCTATCAGTTATCTGTCTATAACCAAGGATTTACGACACCCGACTGGTTGAAAC

ATGCGGTCATCTACGAGATTATGCCGGATCGATTCTACAACGGCGACATAGCGA

ACGACGAAAATCCAAAGACGCAAAAGGGTATCTACACCGATGCCGTTGGTCAG

GAAACGCTGGGTCCTATTCAATTTCATCAGGATTGGAATAGTCAACCATATGATC

CAAACATCCCAGCTTCCAGCGATCCAGCCATCCAGGCCCTGCGTGGCAACGGT

CAATGGAACATCGACTTTTTCGGTGGCGACCTGAAGGGAATTCAGGATAAGCT

CGATTACCTAAAAAGCCTTGGCGTCAATACCCTTTATCTCATGCCTGTGTTTGAG

GCCGAGTCCAATCATAAATACGACACGGCCGATTACATGAAGATCGATCCGGGG

TTTGGCACCGCCCAGGACTGGCTGAACCTCGCAAAGGCTGCCCATGCAGACGG

ATTTCACATCCTCCTGGACGGCGTGTTTGAAGACACCGGATCGGACAGCGTGTA

TTTCAACAAATTCAGCAACATGGGTAGCCTTGGCGCCTGGCAGGCCTATATGCA

GAATCAACCGAATCTCTCGCCCTACTATAGCTGGTACGAGTGGACCAACAACCC

AGCGAATCCGTACAACGGCTGGTGGAACAACGACACGTTGCCGCAGACAGATA

CGAATAACCCGTCATTTCAGCAATTTATTTACGGCGGCAAAGACGCCGTCGCGA

AACACTGGTTGGCGCTTGGCGCCGATGGCTGGCGCCTCGATTCGGCGGACAAC

AGCAACTATAACGTCACTTGGTGGAGCAATTTCCGCAACGCCGTAAAATCTATT

GAT℃CTAACGCAGCAATCGTCGGGGAAATATGGAACACTGCGACAAACGACAA

CGGCACGGACTGGCTCACCGGATCGACCTTCGACAGCGTCATGAATTACTCATT

TCGCAACGCCGTGATCGACTTTTTCCGTGGAACGTACAACGACGGAAGCGTCC

AACATCACGCGGTCGACGCGGCGGGATTCAACCAGGAACTGATGCGCCTGTAC

AGCGAGTATCCTTTGCAGTCGTTCTACGCGATGATGAACCTCGTCGACTCCCAA

GATACGATGCGGATTTTGACGGTTCTCGAAAACGCGCCCGAGCCAGGCTCCAT

GAGCGCCTTGCAACAGGCCACCTACCAGCCCACGGCAACCGATCAACAACTGG

GTATCAAGCGCTTAGAGCTCGTGTCTGACCTGCAGTTTGGCTTCCCGGGCGACC

CGACCATCTGGTATGGTGACGAAGCGGGCGTTTCGGGTTACAGTGATCCGTTGT

CCCGTGACACGTACCCATGGGGCCATGAAAATGAGGCCCTGCTCAACCACTATC

GCTTGCTTGGCGCGATTCGCGCGGCCAACCCCGTGCTGCAAACCGGCACGTTT

ACGCCCGTTTACGCCCAAGGCGAGGTCTATGCGTTCGCACGCACCATCCAAGG

TGGCCAGGACGTATTTGGCAAACCCGCAGCCGATGCGTCGGCGATTGTGGCATT

GAACAACCAAAACCAAACGCAAACCGTGAACCTTCCGGTTGCGGGCGTGATC

GCCAATGGAACGAAGCTTCTGGACGAACTCAACGACCAATGGTACACCGTGGA

GAACGGGGCTGTGCAGTTGACGCTGGCGCCATACGAAGGTGCTATTCTGGTCA

CGCCCACAGCGAATCCTGTCGCATACCTGCAGACAATCAATGGGCAAACCTCG

ATTGCCTGGACGCCGGTTGCTGGGGCGAACGGATACTTGTTGCTGCGACACCA

AGGCGATGTATGGGTACCCGTAGGCCGCCCGCTTTCGGCGAACACGCTGTCTTC

GCCGGTAACCCAAGGTGCAACCGCCGTCGACTATGCGATTGCGGCCCTGCCAC

CCGTGGCACCAGGCGTCGGCCTTAGCTCGGGCCAACCGCTACAGGCGGTTACG

GTGCCGGCCGCATCCCTCGGTCAGCCCCAAGTGCAAGTGGATGTACAGCATAG

CGGGGTCAGTCTGCACATCACACCGGTGCCAAACGCGACACAATATGTCGTCTA

TCTACAACAGCCCGATGGATCTTATCAGGCCGTCGCAACCGTGGCGGCACATGG

CGATGTCCACCTCAGGTTGCCTGTGGCGCCTGGAACACAGTCCATATCCGTCCG

CGTGGCGGCGCAAAATGAAGACGGCCAGGCTGTGACCGATCCGATGGTGATTA

CGGTAAATCCATCCGCCAAAGCACAGCGCTAA

The method separating clone of the present invention by PCR amylase gene AMYA4, the DNA complete sequence analysis is the result show, amylase AMYA4 structure gene AMYA4 total length 3873bp contains a terminator TAA.The maturation protein theoretical molecular of amylase AMYA4 is 138kDa.Amylase gene AMYA4 sequence and the aminoacid sequence derived are carried out the BLAST comparison in GenBank.This gene is 63% with the highest consistence of amylase (CAA44638|) aminoacid sequence that derives from Alicyclobacillusacidocaldarius, illustrates that AMYA4 is a kind of new amylase.

The present invention also provides the application of above-mentioned acid amylase AMYA 4.

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 the new amylase of using in the double-enzyme method sugar making technology particularly.Amylase optimal pH of the present invention is 4.2, 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 has very strong living starch degradation ability.Optimal pH is 4.2 close with the middle temperature saccharifying enzyme of present industrial application, therefore can adjust the pH of starch wine with dregs repeatedly, reduce the usage quantity of soda acid in the saccharifying, its optimum temperature is 75 ℃, close with the optimum temperature of middle temperature saccharifying enzyme, can adjust repeatedly the starch wine with dregs saccharification temperature, therefore can cut down the consumption of energy, it all has very high activity in 55-75 ℃ of scope, has very strong living starch degradation ability simultaneously, can make the starch wine with dregs make technical process simpler through the gelatinization and the liquefaction process in early stage, energy consumption consumption is lower.In simulated dual enzyme process sugar refining technology, no matter be to be substrate with the pasted starch or to be substrate to give birth to starch, under the effect that cooperates the commercialization saccharifying enzyme, can reach good percent hydrolysis, this is indicating the application potential that AmyA4 is huge.

Description of drawings

The diastatic SDS-PAGE of the former enzyme purification of Fig. 1 analyzes, wherein, and 1: low molecular weight protein Marker;

2: the reorganization amylase of purifying.

The optimal pH of Fig. 2 purifying starch enzyme.

The pH stability of Fig. 3 purifying starch enzyme.

The optimum temperuture of Fig. 4 purifying starch enzyme.

The thermostability of Fig. 5 purifying starch enzyme.

Embodiment

Test materials and reagent

1, bacterial strain and carrier: 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.

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

3, substratum:

Alicyclic acid genus bacillus AlicyclobacillushesperidumA4CGMCC3147 produces the enzyme substratum and consists of:

0.2% peptone, 0.1% yeast extract, 0.5% Zulkovsky starch, pH3.0.

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 clone of embodiment 1 alicyclic acid genus bacillus AlicyclobacillushesperidumA4 (CGMCCNo.3147) amylase encoding gene AMYA4

The acquisition of gene order

The amylase gene sequence of having reported according to the alicyclic acid bacillus, mass spectrum sequence (GNGQWNIDFFGGDLK with reference to above-mentioned purifying starch enzyme, HWLALGADGW), directly design Auele Specific Primer AF and AR are that template is carried out pcr amplification with the total DNA of alicyclic acid genus bacillus Alicyclobacillus hesperidum A4 (CGMCC No.3147).The PCR reaction parameter is: 94 ℃ of sex change 5min, and 60 ℃ of annealing 30sec, 72 ℃ are extended 1.5min.Last 72 ℃ of insulation 10min.Obtain an about 480bp 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. amylase AMYA4TAIL-PCR Auele Specific Primer

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

By finding this full length gene 3873bp, encode 1290 amino acid and a terminator codon behind the genome sequence of comparative starches enzyme.Infer that the aminoacid sequence of the gene A MYA4 that and the amylase gene sequence on the GeneBank carry out homology relatively, the highest consistence is 63%, illustrate that AMYA4 is a kind of new amylase, show that the diastatic gene of coding 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.

The activation analysis of embodiment 2 purifying starch enzymes

The method of separation and purification acid amylase AMYA 4 from Alicyclobacillus hesperidum A4 may further comprise the steps:

1) after 55 ℃ of shakes of seed culture medium are cultivated 48 hours, 1% inoculum size is to producing the enzyme substratum, and 55 ℃, 200rpm shake and cultivate after 48 hours, and bacterium liquid is centrifugal, get supernatant and are used for purifying;

2) culture centrifuging and taking supernatant is about 101, and the hollow fiber ultrafiltration membrane by 6K is to about 600ml down for ice bath then, and the ultra-filtration membrane bag with 5K further is concentrated into 200ml again;

3) go above-mentioned concentrated solution 10ml at buffer A (20mmol/L citric acid-Na ₂HPO ₄PH 3.5) middle dialyzed overnight, cation-exchange chromatography: get and use buffer A equilibrated HiTrap SP XL cation seperation column on the 2.0mL concentrated solution in advance, carry out gradient elution with containing 1.0mol/L NaCl buffer B and buffer A then, elution requirement is: 8 column length volumes (0-100%), flow velocity 2.0mL/min.Every pipe 1.0mL carries out fraction collection.

The DNS method: concrete grammar is as follows: at pH 4.2 (0.1M SODIUM PHOSPHATE, MONOBASIC-citric acid), under 75 ℃ 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.Amylase behind the purifying has carried out tryptic enzyme digestion reaction, analyzes by MALDI-TOF-MS, obtained 3 sections in the aminoacid sequence of peptide, sequence is respectively: NH ₂-G-N-G-Q-W-N-I-D-F-F-G-G-D-L-K, NH ₂-H-W-L-A-L-G-A-D-G-W and NH ₂-N-A-V-I-D-F-F-R.Utilize software inquiry AlicyclobacillushesperidumA4 genome sequence, protein library to obtain gene order, prove that the amylase behind the purifying is acid amylase AMYA 4 of the present invention.

The property testing of embodiment 3 purifying starch enzyme AMYA4

1, the measuring method of the optimal pH of purifying starch enzyme AMYA4 and pH stability is as follows:

The amylase of embodiment 2 purifying is carried out enzymatic reaction to measure its optimal pH under different pH.The substrate Zulkovsky starch is with damping fluid (the 0.1mol/L citric acid-Sodium phosphate dibasic pH 3.0-7.4 of different pH; 0.1MTris-HCl pH 7.8-8.6; 0.1M glycine-sodium hydroxide pH 9.0-11.0; ), under 75 ℃, carry out amylase activity and measure.Result (Fig. 2) shows that its suitableeest action pH is 4.2, at the relative activity of pH 3.8～5.8 maintenances more than 70%.The stable p H scope of this enzyme is 3.0～10.0,37 ℃ of effect 2h, and enzyme activity remains on more than 85%.The experimental result of diastatic optimum temperature and thermostability is seen Fig. 3.

2, diastatic optimum temperuture 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 (pH4.2) buffer solution system and differing temps of diastatic optimum temperuture.Temperature tolerance is determined as amylase and handles different time under differing temps, carries out enzyme assay again under 75 ℃.The diastatic optimum temperature of enzyme reaction optimum temperuture measurement result (Fig. 4) is 75 ℃.This enzyme can keep good stability at 75 ℃, and the remaining activity more than 90% is arranged behind effect 1h under 75 ℃, and this enzyme acts on 10min down at 80 ℃, and enzymic activity can also keep more than 50%.

3, different metal ion chemistry reagent is as follows to AmyA4 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 75 ℃, pH4.2 condition, measure enzymic activity.Result's (table 1) shows that most metal ion is less to diastatic activity influence, Hg ²⁺, Fe ²⁺, Cu ²⁺With SDS this enzymic activity is suppressed more serious.The diastatic activity of it should be noted that and having reported all has Ca ²⁺The dependency difference, Ca ²⁺AmyA4 do not had obvious facilitation.Even when 10mM concentration, also has certain restraining effect.

The various chemical reagent of table 1 are to the influence of amylase AMYA4 vigor

The hydrolysising experiment of embodiment 4 purifying starch enzymes in simulated dual enzyme process sugar refining technology is right

With AmyA4 and commercialization amylase, saccharifying enzyme is a research object, has tested aging starch and farinose transformation efficiency, experimental result such as table 2.

Table 2 simulated dual enzyme process sugar refining technology result

Sample 1: add earlier commercial a-amylase at 90 ℃, pH 6.0 effects 2 hours are added into the commercialization saccharifying enzyme then 60 ℃ of times that effect is different after regulating pH value to 4.2 then; Sample 2: at first add AmyA4 60 ℃ of effects 2 hours, be added into the commercialization saccharifying enzyme then 60 ℃ of times that effect is different; Sample 3: add AmyA4 simultaneously and be in the same place, 4.2,60 ℃ of different times of effect of pH with the commercialization saccharifying enzyme; Sample 4: add AmyA4 simultaneously and be in the same place, 4.2,60 ℃ of different times of effect of pH with the commercialization saccharifying enzyme.

The result shows, cooperates the commercialization saccharifying enzyme with AmyA4, is substrate to give birth to starch, can reach 96% transformation efficiency.

Claims (7)

1. an acid amylase AMYA 4 is characterized in that, its aminoacid sequence is shown in SEQ ID NO.1.

2. an acid starch enzyme gene A MYA4 is characterized in that, the described acid amylase AMYA 4 of coding claim 1.

3. acid starch enzyme gene A MYA4 as claimed in claim 2 is characterized in that its base sequence is shown in SEQ IDNO.2.

4. the recombinant vectors that comprises the described acid starch enzyme of claim 2 gene A MYA4.

5. the recombinant bacterial strain that comprises the described acid starch enzyme of claim 2 gene A MYA4.

6. recombinant bacterial strain as claimed in claim 5 is characterized in that, described bacterial strain is yeast, intestinal bacteria, aspergillus, genus bacillus or lactobacillus.

7. the application of the described acid amylase AMYA 4 of claim 1.

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