CN102127555A

CN102127555A - Cloning of beta-1,4-endo-glucanase gene and preparation of recombinase

Info

Publication number: CN102127555A
Application number: CN2010105812996A
Authority: CN
Inventors: 邬敏辰; 史红玲; 郭静; 韦敬土; 刘高磊
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2010-12-10
Filing date: 2010-12-10
Publication date: 2011-07-20

Abstract

The invention provides a cloning method for complete mRNA and DNA sequences of beta-1,4-endo-glucanase gene from Aspergillus usamii E001. The nucleotide sequences of the complete mRNA and DNA are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively. Bioinformatics analysis shows that the beta-1,4-endo-glucanase belongs to glycoside hydrolase family 12, is named Aus Cel12A, and has the amino acid sequence shown in SEQ ID NO:3; and the corresponding gene is named Aus cel12A. The invention also discloses a method for construction of Aus Cel12A engineering bacteria and high-efficiency expression and purification of recombinant Aus Cel12A. The most suitable operative temperature and pH of the prepared recombinant Aus Cel12A are respectively 60DEG C and 5.0; the recombinant Aus Cel12A is stable when the pH is 4.0-7.0 and the temperature is below 60DEG C, has higher industrial production potential and economic value, and lays a theoretical basis for researching other beta-endo-glucanase.

Description

A kind of β-1, the clone of 4-endo glucanase gene and the preparation of recombinase

Technical field

The present invention relates to be derived from the β-1 of a kind of glycoside hydrolase 12 families of Aspergillus usamii (Aspergillus usamii) E001 bacterial strain, the clone of complete mRNA of 4-endo glucanase gene and dna sequence dna, β-1, the structure of 4-endoglucanase engineering bacteria and recombinant beta-1, the method with purifying of efficiently expressing of 4-endoglucanase belongs to technical field of bioengineering.

Background technology

Mierocrystalline cellulose is the topmost constituent of plant cell wall, occupied the over half of renewable organic carbon source on the earth together with hemicellulose.Cellulase is meant energy hydrocellulose β-1,4 glucoside bonds generate the general name of one group of enzyme of cellobiose and glucose, mainly comprise inscribe dextran (endo-1,4-β-glucanase, EC 3.2.1.4, abbreviation EG), exoglucanase (exo-1,4-β-glucanase, EC 3.2.1.91, be called for short CBH) and beta-glucosidase (β-glucosidase, EC 3.2.1.21 is called for short BG) 3 big classes, under the synergy of these 3 kinds of enzymes, can become glucose to cellulose hydrolysis.Cellulase has been widely used in industrial circles such as food, feed, weaving, papermaking, biofuel and fermentation at present.

Cellulase of a great variety, (Glycoside Hydrolases GH) has distribution in the families such as the 3rd, 5,7,8,9,12, and wherein majority belongs to glycoside hydrolase the 5th family (GH5) and 12 families (GH12) at glycoside hydrolase; The source of cellulase is also very wide, most of cellulases are mainly from microorganism, since the sixties in 20th century, according to incompletely statistics, write down several thousand bacterial strains of approximately existing 53 genus of bacterial strain of cellulase-producing both at home and abroad altogether, mainly comprised bacterium, fungi and actinomycetes.Fungi is the important source of the plain enzyme of industrial fibre, mainly contain Trichodermareesei (Trichoderma reesei), viride (Trichoderma viride), healthy and free from worry wood mould (Trichoderma koningii), aspergillus niger (Aspergillus niger), Penicillium decumbens (Penicillium decumbens), chaetomium thermophilum (Chaetomium thermophile) and thermophilic ascomycete (Thermoascusaurantiacus) etc., study at most and be clear that most Trichodermareesei T.reesei at present.But about the research of the clone of β-endo glucanase gene (Aus cel12A) of deriving from Aspergillus usamii (Aspergillus usamii) GH12 and expression etc. does not see that report is arranged.

Summary of the invention

The purpose of this invention is to provide a kind of Aspergillus usamii E001 bacterial strain novel β-1, the complete mRNA of 4-endo glucanase gene and dna sequence dna clone, the method with purifying of efficiently expressing of the structure of β-endoglucanase engineering bacteria and recombinant beta-endoglucanase.Bioinformatic analysis shows that a kind of novel β-endoglucanase that derives from Aspergillus usamii E001 bacterial strain belongs to glycoside hydrolase the 12nd family, called after Aus Cel12A, and its corresponding unnamed gene is Aus cel12A.Aus Cel12A has advantages of high catalytic activity and thermostability, and bigger suitability for industrialized production and application potential and economic worth are arranged.

Technical scheme of the present invention: a kind of nucleotide sequence that is derived from Aspergillus usamii (Aspergillus usamii) E001 bacterial strain new A us Cel12A gene complete mRNA and DNA is respectively SEQ ID NO:1 and SEQ ID NO:2.The flow process of obtaining complete mRNA and dna sequence dna as shown in Figure 1.

A.usamii E001 bacterial strain is by Southern Yangtze University screening and preservation, in the Vol.31 at " food and fermentation industries " magazine in 2005, and No.4, Pg.50 is open, and the inventor promises to undertake that this bacterial strain provided to the public in 20 years.

Described Aus Cel12A aminoacid sequence of being derived by complete mRNA sequence is SEQ ID NO:3.

The activity determination method of described reorganization Aus Cel12A:

In 25mL tool plug test tube A and B, the mass concentration that each adding is prepared with pH 5.0, citric acid-phosphate sodium dihydrogen buffer solution is beta-glucan (Sigma, USA) solution 2.4mL, 50 ℃ of preheating 10min of 0.5%, in the A pipe, add the suitably enzyme liquid of dilution of 0.1mL, 50 ℃ of accurate response 20min; Respectively add 2.5mL 3 ' immediately, 5 '-dinitrosalicylic acid (DNS) reagent, in the B pipe, add 0.1mL enzyme liquid again, A and B pipe all boil 7min; Respectively add deionized water 5mL after the cooling, shake up; 540nm sentences the B pipe and measures A pipe absorbance for blank, and finds corresponding reducing sugar (with glucose meter) content and be converted to unit of enzyme activity from the glucose typical curve.Unit of enzyme activity's definition: under this condition determination, produce the required enzyme amount of 1 μ mol reducing sugar with per minute and be defined as 1 β-1,4-dextranase activity unit (U).

The clone and the expression method of complete mRNA of described Aus cel12A and dna sequence dna:

(1) clone of Aus cel12A 3 ' end mRNA sequence: at first β-endoglucanase sequence of Aspergillus kawachii (GenBank D12901), Aspergillus niger (GenBank AJ224451) and Aspergillus terreus (GenBank XM_001214697) GH12 is carried out the homology comparison, find out two sections about 10 amino acid whose conserved sequences; MRNA to these two sections aminoacid sequences carries out the homology comparison again, designs two forward degenerated primer Cel12A-3F1 and Cel12A-3F2.

Cel12A-3F1：5′-TA(T/C)GACAG(T/C)GC(G/C)TCGAGCCC-3′

Cel12A-3F2：5′-GTGAA(A/G)AGCTACTC(T/G)AACTC-3′

Extract total RNA of A.usamii E001, carry out the RT-PCR amplification according to TaKaRa RNA PCR Kit (Ver.3.0) specification sheets.With the two-wheeled pcr amplification product with 1% agarose gel electrophoresis analysis, rubber tapping is reclaimed the purpose band and is connected (pUCm-T-cel12A3 ') with the pUCm-T carrier, transform JM109, after enzyme is cut evaluation correctly, serve the Hai Shenggong order-checking, obtain Aus cel12A3 ' end mRNA sequence.

(2) clone of Aus cel12A 5 ' end mRNA sequence:, design two reverse primer Cel12A-5R1 and Cel12A-5R2 based on the above-mentioned Aus cel12A 3 ' end mRNA sequence that obtains.

Cel12A-5R1：5′TTGTCCTGCTTCCATTCCAC-3′

Cel12A-5R2：5′ACATCACTCACGAGCTTCTT-3′

According to TaKaRa 5 '-Full RACE Kit specification sheets carry out 5 '-RACE.Two-wheeled PCR product with 1% agarose gel electrophoresis analysis, is tapped rubber and reclaimed the purpose band and be connected (pUCm-T-cel12A5 ') with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve Hai Shenggong and check order, obtain Aus cel12A 5 ' end mRNA sequence.

(3) clone of Aus cel12A 5 ' end promoter sequence: the carrier mediated PCR method of a kind of T that adopts previous our invention, with treated A.usamii E001 genomic dna is template, first round PCR is a primer with T-PrimerF and Cel12A-5R1, and second to take turns PCR be primer with T-PrimerF and Cel12A-5R2.Two-wheeled PCR product with 1% agarose gel electrophoresis analysis, is reclaimed the purpose band and is connected (pUCm-T-cel12AP) with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve the Hai Shenggong order-checking, obtain Aus cel12A 5 ' end promoter sequence.

(4) clone of Aus cel12A 3 ' end transcription termination region:, design two forward primer Cel12A-3F3 and Cel12A-3F4 based on the above-mentioned Aus cel12A 3 ' end mRNA sequence that obtains.

Cel12A-3F3：5′-GTGTCAGAAAGCCCTATCAA-3′

Gel12A-3F4：5′-CAGCTATCTCACTCAGAACC-3′

Adopting the carrier mediated PCR method of a kind of T of previous our invention, is template with treated A.usamii E001 genomic dna, and first round PCR is a primer with Cel12A-3F3 and T-PrimerR, and second to take turns PCR be primer with Cel12A-3F4 and T-PrimerR.With two-wheeled PCR product with 1% agarose gel electrophoresis analysis, rubber tapping is reclaimed the purpose band and is connected (pUCm-T-cel12A3T) with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve the Hai Shenggong order-checking, obtain Aus cel12A 3 ' end Transcription Termination region sequence.

(5) bioinformatic analysis of Aus cel12A: 5 of Aus cel12A ' and 3 ' end mRNA sequence is spliced, obtain from transcribing initiation site to the complete mRNA sequence of poly (A); On NCBI, open reading frame (Open ReadingFrame) is analyzed, and then derived the aminoacid sequence of Aus Cel12A; Carry out signal peptide prediction.Coding region dna sequence dna and its both sides sequence of Aus cel12A are spliced, obtain global DNA sequence, and the function of its 5 ' end promoter region and 3 ' end transcription termination region is predicted.

(6) mensuration of Aus cel12A intron sequences:, design a pair of primer Cel12A-F1 and Cel12A-R based on the complete mRNA sequence of the above-mentioned Aus cel12A that obtains.

Cel12A-F1:5 ' GAATTCATGAAGCTCCCTGTGACAC-3 ' contains EcoR I restriction enzyme site

Cel12A-R:5 ' GCGGCCGCCTAGTTGACACTGGCGGTC-3 ' contains Not I restriction enzyme site

With A.usamii E001 genomic dna is template, Cel12A-F1 and Cel12A-R are that primer carries out PCR, with its product with 1% agarose gel electrophoresis analysis, reclaim the purpose band and be connected (pUCm-T-cel12A-DNA) with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve the Hai Shenggong order-checking, obtain the coding region dna sequence dna of Aus cel12A.Coding region dna sequence dna and mRNA sequence are carried out sequence alignment with DNAMAN 6.0, draw 2 intron sequences of Aus cel12A.

(7) contain the structure of coding Aus Cel12A mature peptide expression of gene plasmid:, design a forward primer Cel12A-F based on the signal peptide sequence of complete mRNA sequence of the above-mentioned Aus cel12A that obtains and prediction.

Cel12A-F:5 '- GAATTCCAGACGATGTGCTCTCAAT-3 ' contains EcoR I restriction enzyme site

Carry out RT-PCR according to TaKaRa RNA PCR Kit (Ver.3.0) working instructions.With Oligo dT-Adaptor Primer is article one chain that primer carries out the synthetic cDNA of reverse transcription; With M13 Primer M4 and Cel12A-F is that primer carries out first round PCR; With Cel12A-F and Cel12A-R is that primer carries out second and takes turns PCR.Two-wheeled PCR product with 1% agarose gel electrophoresis analysis, is tapped rubber and reclaimed the purpose band and be connected (pUCm-T-cel12A) with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve Hai Shenggong and check order.

PUCm-T-cel12A that sequencing result is correct and pPIC9K plasmid all carry out double digestion with EcoR I and Not I, the enzyme that rubber tapping is reclaimed is cut product and is connected under the effect of T4 dna ligase, obtain recombinant plasmid pPIC9K-cel12A (Fig. 2), and recombinant plasmid is carried out sequencing.

(8) structure of GS115/cel12A, expression, product purification and determination of activity: with Sal I pPIC9K-cel12A is carried out linearizing, carry out electricity according to the Pichia anomala expression handbook and change, screen, obtain the pichia spp recon GS115/cel12A of high copy; Carry out abduction delivering according to the normal process on the handbook, measure β-1,4-endoglucanase activity with the DNS method; Fermented liquid obtains electrophoretically pure recombinant beta-1 through frozen centrifugation, ultrafiltration, ion exchange chromatography and gel permeation chromatography, the 4-endoglucanase.

Beneficial effect of the present invention: the invention provides a kind of Aspergillus usamii E001 bacterial strain novel β-1, complete mRNA of 4-endo glucanase gene and dna sequence dna clone, the method with purifying of efficiently expressing of the structure of β-endoglucanase engineering bacteria and recombinant beta-endoglucanase, and sequence carried out bioinformatic analysis.Bioinformatic analysis shows that a kind of novel β-endoglucanase that derives from Aspergillus usamii E001 belongs to the 12nd family of glycoside hydrolase, called after Aus Cel12A, and its corresponding unnamed gene is Aus cel12A.Aus Cel12A has advantages of high catalytic activity and thermostability, and bigger suitability for industrialized production, application potential and economic worth are arranged.

Description of drawings

Fig. 1: the schema that obtains A.usamii E001 Cel12A gene complete mRNA and dna sequence dna

Fig. 2: the structure synoptic diagram of recombinant plasmid pPIC9K-cel12A

Embodiment

Below in conjunction with specific embodiment, further set forth working method of the present invention.But these embodiment only are used to describe in detail the present invention, limit the scope of the invention and be not used in.

The clone of embodiment 1Aus cel12A 3 ' end mRNA sequence

With Oligo dT-Adaptor Primer is article one chain of the synthetic cDNA of primer reverse transcription; With M13 Primer M4 and Cel12A-3F1 is that primer carries out first round PCR, and its reaction conditions is: 94 ℃ of 2min, 30 circulations (94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 90s), 72 ℃ of 10min; With M13 Primer M4 and Cel12A-3F2 is that primer carries out second and takes turns PCR, and its reaction conditions is: 94 ℃ of 2min, 30 circulations (94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 90s), 72 ℃ of 10min.Two-wheeled PCR product with 1% agarose gel electrophoresis analysis, is reclaimed the purpose band and is connected (pUCm-T-cel12A3 ') with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve the Hai Shenggong order-checking.

The clone of embodiment 2Aus cel12A 5 ' end mRNA sequence

With 5 '-Outer Primer and the Cel12A-5R1 of Full RACE Kit is that primer carries out first round PCR, its reaction conditions is: 94 ℃ of 3min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min; With Inner Primer and Cel12A-5R2 is that primer carries out second and takes turns PCR, and its reaction conditions is: 94 ℃ of 3min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min.Two-wheeled PCR product with 1% agarose gel electrophoresis analysis, is reclaimed the purpose band and is connected (pUCm-T-cel12A5 ') with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve the Hai Shenggong order-checking.

The clone of embodiment 3Aus cel12A 5 ' end promoter sequence

With treated A.usamii E001 genomic dna is template, and first round PCR is a primer with T-PrimerF and Cel12A-5R1, and reaction conditions is: 94 ℃ of 4min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min; Second to take turns PCR be primer with T-PrimerF and Cel12A-5R2, and reaction conditions is: 94 ℃, and 4min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min.Two-wheeled PCR product with 1% agarose gel electrophoresis analysis, is tapped rubber and reclaimed the purpose band and be connected (pUCm-T-cel12AP) with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve Hai Shenggong and check order.

The clone of embodiment 4Aus cel12A 3 ' end transcription termination region

With treated A.usamii E001 genomic dna is template, and first round PCR is a primer with Cel12A-3F3 and T-PrimerR, and reaction conditions is: 94 ℃ of 4min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min; Second to take turns PCR be primer with Cel12A-3F4 and T-PrimerR, and reaction conditions is: 94 ℃ of 4min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min.Two-wheeled PCR product with 1% agarose gel electrophoresis analysis, is tapped rubber and reclaimed the purpose band and be connected (pUCm-T-cel12A3T) with pUCm-T, transform JM109, after enzyme is cut evaluation correctly, serve Hai Shenggong and check order.

Embodiment 5 contains the structure of coding Aus Cel12A mature peptide expression of gene plasmid

With Oligo dT-Adaptor Primer is article one chain that primer carries out the synthetic cDNA of reverse transcription; With Cel12A-F and M13 Primer M4 is that primer carries out first round PCR, and reaction conditions is: 94 ℃ of 2min, 30 circulations (94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min; With Cel12A-F and Cel12A-R is that primer carries out second and takes turns PCR, and reaction conditions is: 94 ℃ of 2min, 30 circulations (94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 45s), 72 ℃ of 10min.Take turns the PCR product with 1% agarose gel electrophoresis analysis with second, reclaim the purpose band and is connected (pUCm-T-cel12A), transform JM109, cut through enzyme and identify that serving Hai Shenggong after correct checks order with pUCm-T.PUCm-T-cel12A that sequencing result is correct and pPIC9K plasmid all carry out double digestion with EcoR I and Not I, the enzyme that reclaims is cut product and is connected under the effect of T4 dna ligase, obtain recombinant plasmid pPIC9K-cel12A, and recombinant plasmid is carried out sequencing.

The structure of embodiment 6GS115/cel12A, expression, product purification and determination of activity

With Sal I pPIC9K-cel12A is carried out linearizing, carry out electricity according to the Pichia anomala expression handbook and transform, screen, obtain the pichia spp recon GS115/cel12A of high copy.This genetic engineering bacterium is expressed 96h with 1.0% methanol induction, adopts the DNS method to record that recombinant beta-endoglucanase activity reaches 240U/mL in the fermented liquid.The supernatant liquor of fermented liquid after centrifugal is reorganization AusCel12A crude enzyme liquid, this crude enzyme liquid is that the ultra-filtration membrane of 10kDa concentrates through molecular weight cut-off, again through DEAE-Sepharose FastFlow ion exchange chromatography and Sephadex G-75 gel permeation chromatography purifying, purifying is single band after SDS-PAGE detects, and shows that the molecular weight of reorganization Aus Cel12A is 27kDa.60 ℃ of the optimum temperatures of this reorganization Aus Cel12A, the suitableeest action pH 5.0, this enzyme is at pH 4.0-7.0, stable below 60 ℃.

Sequence table

＜110〉Southern Yangtze University

＜120〉a kind of β-1, the clone of 4-endo glucanase gene and the preparation of recombinase

<140>201010581299.6

<141>2010-12-10

<210>SEQ?ID?NO：1

<211>1020

<212>mRNA

＜213〉Aspergillus usamii (Aspergillus usamii) E001

<220>

<221>CDS

<222>(91)...(810)

<220>

<221>sig_peptide

<222>(91)...(138)

<220>

<221>mat_peptide

<222>(139)...(807)

<400>1

ccctccgtgt?tggtgtctta?tcctgaacac?ttatcctctt?attcctttcg?aatttccctt 60

gattgccgct?cctccgctct?aacgcccaac?atgaagctcc?ccgtgtcact?tgctatgctt 120

gcggccaccg?ccatgggcca?gacgatgtgc?tctcaatatg?acagtgcctc?gagccccccg 180

tattcagtga?accagaacct?ctggggcgag?taccaaggca?ccggcagcca?gtgtgtatat 240

gtcgacaaac?tctccagcag?tggtgcatcc?tggcacaccg?aatggacctg?gagcggtggt 300

gagggaacag?tgaaaagcta?ctctaactct?ggcgttacat?ttaacaagaa?gctcgtgagt 360

gatgtatcaa?gcatccccac?cttggtggaa?tggaagcagg?acaacaccaa?cgtcaacgcc 420

gatgtcgcgt?atgatctttt?caccgcggcg?aatgtggacc?atgccacttc?tagcggcgac 480

tatgaactga?tgatttggct?tgcccgctac?ggcaacatcc?agcccattgg?caagcaaatt 540

gccacggcca?cagtgggagg?caagtcctgg?gaggtgtggt?atggcagcac?cacccaggcc 600

ggtgcggagc?agaggacata?cagcttcgtg?tcagaaagcc?ctatcaactc?atacagtggg 660

gacatcaatg?catttttcag?ctatctcact?cagaaccaag?gctttcccgc?cagctctcag 720

tacttgatca?atctgcagtt?tggaactgag?gcgttcaccg?ggggcccggc?aaccttcacg 780

gttgacaact?ggaccgccag?tgtcaactag?ggttctagaa?gtagcctttg?aggcagaatc 840

tgggtaaatt?gactccagct?cgggagaatg?atagcttgtt?tcttcgttct?ggaacgttgg 900

gcgtgtgaga?gctaaaaagt?cgtacccact?ctgattggaa?agacttattc?aacattggtc 960

cttcccttct?gttgggcaag?gcatagttag?tgattagaca?agtcaaggtc?atggtggatc 1020

＜110〉Southern Yangtze University

<140>201010581299.6

<141>2010-12-10

<210>SEQ?ID?NO：2

<211>1568

<212>DNA

＜213〉Aspergillus usamii (Aspergillus usamii) E001

<220>

<221>CDS

<222>(523)...(929)，(980)...(1212)，(1279)...(1358)

<220>

<221>mat_peptide

<222>(571)...(929)，(980)...(1212)，(1279)...(1358)

<220>

<221>Promoter

<222>(395)...(442)

<220>

<221>TATA_signal

<222>(408)...(413)

<400>2

agcttcttac?ccgtgagctt?cgtatcagtc?aaaacaccta?agccctgttg?atatttcacc 60

gacaaaaggc?cggtccctaa?gccatgctca?tctaaattga?gttggtctat?ccttctaaat 120

taatatcgtc?agcacaagca?ggaccctttt?cttgtttccc?atcagatccc?gttctccacg 180

ttgttcggtt?accgactgcc?acagaccatc?actaacttta?aacggaaagt?gctccgacat 240

agggtctcat?tttgaaacgg?aggatgtgca?tgtctgttgc?agattggcgt?gagatggcgg 300

cgcaacccgc?aatagctagt?tggcttactt?cttcttgaat?gccatattgg?acacggaaat 360

tcggacaatc?ataatggcaa?tagtaagcat?tgttgattag?attcaggtat?ttaagctgcc 420

tcatctgccg?ctccctccgt?gttggtgtct?tatcctgaac?acttatcctc?ttattccttt 480

cgaatttccc?ttgattgccg?ctcctccgct?ctaacgccca?acatgaagct?ccccgtgtca 540

cttgctatgc?ttgcggccac?cgccatgggc?cagacgatgt?gctctcaata?tgacagtgcc 600

tcgagccccc?cgtattcagt?gaaccagaac?ctctggggcg?agtaccaagg?caccggcagc 660

cagtgtgtat?atgtcgacaa?actctccagc?agtggtgcat?cctggcacac?cgaatggacc 720

tggagcggtg?gtgagggaac?agtgaaaagc?tactctaact?ctggcgttac?atttaacaag 780

aagctcgtga?gtgatgtatc?aagcatcccc?accttggtgg?aatggaagca?ggacaacacc 840

aacgtcaacg?ccgatgtcgc?gtatgatctt?ttcaccgcgg?cgaatgtgga?ccatgccact 900

tctagcggcg?actatgaact?gatgatttgg?tatgtgagct?cgcaaagaat?caaagaatac 960

agaaatctaa?caaccccagg?cttgcccgct?acggcaacat?ccagcccatt?ggcaagcaaa 1020

ttgccacggc?cacagtggga?ggcaagtcct?gggaggtgtg?gtatggcagc?accacccagg 1080

ccggtgcgga?gcagaggaca?tacagcttcg?tgtcagaaag?ccctatcaac?tcatacagtg 1140

gggacatcaa?tgcatttttc?agctatctca?ctcagaacca?aggctttccc?gccagctctc 1200

agtacttgat?cagtaagatt?ccctgattct?accagcggac?gccgcagcga?aacagtcact 1260

aacagaagtg?atgtgtagat?ctgcagtttg?gaactgaggc?gttcaccggg?ggcccggcaa 1320

ccttcacggt?tgacaactgg?accgccagtg?tcaactaggg?ttctagaagt?agcctttgag 1380

gcagaatctg?ggtaaattga?ctccagctcg?ggagaatgat?agcttgtttc?ttcgttctgg 1420

aacgttgggc?gtgtgagagc?taaaaagtcg?tacccactct?gattggaaag?acttattcaa 1480

cattggtcct?tcccttctgt?tgggcaaggc?atagttagtg?attagacaag?tcaaggtcat 1560

ggtggatc 1568

＜110〉Southern Yangtze University

<140>201010581299.6

<141>2010-12-10

<210>SEQ?ID?NO：3

<211>239

<212>PRT

＜213〉Aspergillus usamii (Aspergillus usamii) E001

<400>3

Met?Lys?Leu?Pro?Val Ser?Leu?Ala?Met?Leu Ala?Ala?Thr?Ala?Met

5 10 15

Gly?Gln?Thr?Met?Cys Ser?Gln?Tyr?Asp?Ser Ala?Ser?Ser?Pro?Pro

20 25 30

Tyr?Ser?Val?Asn?Gln Asn?Leu?Trp?Gly?Glu Tyr?Gln?Gly?Thr?Gly

35 40 45

Ser?Gln?Cys?Val?Tyr Val?Asp?Lys?Leu?Ser Ser?Ser?Gly?Ala?Ser

50 55 60

Trp?His?Thr?Glu?Trp Thr?Trp?Ser?Gly?Gly Glu?Gly?Thr?Val?Lys

65 70 75

Ser?Tyr?Ser?Asn?Ser Gly?Val?Thr?Phe?Asn Lys?Lys?Leu?Val?Ser

80 85 90

Asp?Val?Ser?Ser?Ile Pro?Thr?Leu?Val?Glu Trp?Lys?Gln?Asp?Asn

95 100 105

Thr?Asn?Val?Asn?Ala Asp?Val?Ala?Tyr?Asp Leu?Phe?Thr?Ala?Ala

110 115 120

Asn?Val?Asp?His?Ala Thr?Ser?Ser?Gly?Asp Tyr?Glu?Leu?Met?Ile

125 130 135

Trp?Leu?Ala?Arg?Tyr Gly?Asn?Ile?Gln?Pro Ile?Gly?Lys?Gln?Ile

140 145 150

Ala?Thr?Ala?Thr?Val Gly?Gly?Lys?Ser?Trp Glu?Val?Trp?Tyr?Gly

155 160 165

Ser?Thr?Thr?Gln?Ala Gly?Ala?Glu?Gln?Arg Thr?Tyr?Ser?Phe?Val

170 175 180

Ser?Glu?Ser?Pro?Ile Asn?Ser?Tyr?Ser?Gly Asp?Ile?Asn?Ala?Phe

185 190 195

Phe?Ser?Tyr?Leu?Thr Gln?Asn?Gln?Gly?Phe Pro?Ala?Ser?Ser?Gln

200 205 210

Tyr?Leu?Ile?Asn?Leu Gln?Phe?Gly?Thr?Glu Ala?Phe?Thr?Gly?Gly

215 220 225

Pro?Ala?Thr?Phe?Thr Val?Asp?Asn?Trp?Thr Ala?Ser?Val?Asn

230 235 239

Claims

1. novel β-1 who derives from Aspergillus usamii (Aspergillus usamii) E001 bacterial strain glycoside hydrolase the 12nd family (GH12), 4-endo glucanase gene (Aus cel12A), the nucleotide sequence of its complete mRNA and DNA are respectively SEQ ID NO:1 and SEQ ID NO:2.

2. by the novel β-1 of the described glucanase gene of claim 1 (Aus cel12A) coding, 4-endoglucanase (AusCel12A), its complete aminoacid sequence is corresponding in the sequence table being SEQ ID NO:3.

3.Aus the acquisition methods of complete mRNA of cel12A and dna sequence dna:

(1) clone of Aus cel12A 3 ' end mRNA sequence: at first the aminoacid sequence to β-endoglucanase of Aspergillus kawachii, Aspergillus niger and Aspergillus terreus GH12 carries out the homology comparison, find out two sections about 10 amino acid whose conserved sequences, mRNA to these two sections aminoacid sequences carries out the homology comparison again, designs two forward degenerated primer Cel12A-3F1 and Cel12A-3F2;

Cel12A-3F1：5′-TA(T/C)GACAG(T/C)GC(G/C)TCGAGCCC-3′

Cel12A-3F2：5′-GTGAA(A/G)AGCTACTC(T/G)AACTC-3′

With the total RNA of A.usamii E001 is template, and Oligo dT-Adaptor Primer is article one chain of the synthetic cDNA of primer reverse transcription; With M13 Primer M4 and Cel12A-3F1 is that primer carries out first round PCR, and its reaction conditions is: 94 ℃ of 2min, 30 circulations (94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 90s), 72 ℃ of 10min; With M13 Primer M4 and Cel12A-3F2 is that primer carries out second and takes turns PCR, and its reaction conditions is: 94 ℃ of 2min, 30 circulations (94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 90s), 72 ℃ of 10min; The purpose band obtains Aus cel12A 3 ' end mRNA sequence through clone, order-checking;

(2) clone of Aus cel12A 5 ' end mRNA sequence:, design two reverse primer Cel12A-5R1 and Cel12A-5R2 based on the above-mentioned Aus cel12A 3 ' end mRNA sequence that obtains;

Cel12A-5R1：5′-TTGTCCTGCTTCCATTCCAC-3′

Cel12A-5R2：5′-ACATCACTCACGAGCTTCTT-3′

With reverse transcription synthetic cDNA article one chain is that template, Outer Primer and Cel12A-5R1 are that primer carries out first round PCR, and its reaction conditions is: 94 ℃ of 3min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min; With Inner Primer and Cel12A-5R2 is that primer carries out second and takes turns PCR, and its reaction conditions is: 94 ℃ of 3min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min; The purpose band obtains Aus cel12A 5 ' end mRNA sequence through clone, order-checking;

(3) clone of Aus cel12A 5 ' end promoter sequence: with treated A.usamii E001 genomic dna is template, first round PCR is a primer with T-PrimerF and Cel12A-5R1, reaction conditions is: 94 ℃ of 4min, 30 circulation (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min; Second to take turns PCR be primer with T-PrimerF and Cel12A-5R2, and reaction conditions is: 94 ℃, and 4min, 30 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s), 72 ℃ of 10min; The purpose band obtains Aus cel12A 5 ' end promoter sequence through clone, order-checking;

(4) clone of Aus cel12A 3 ' end Transcription Termination region sequence:, design two forward primer Cel12A-3F3 and Cel12A-3F4 based on the above-mentioned Aus cel12A 3 ' end mRNA sequence that obtains;

Cel12A-3F3：5′-GTGTCAGAAAGCCCTATCAA-3′

Cel12A-3F4：5′-CAGCTATCTCACTCAGAACC-3′

With treated A.usamii E001 genomic dna is template, and first round PCR is a primer with Cel12A-3F3 and T-PrimerR, and second to take turns PCR be primer with Cel12A-3F4 and T-PrimerR; The purpose band obtains Auscel12A 3 ' end Transcription Termination region sequence through clone, order-checking;

4.Aus the structure of Cel12A engineering bacteria and expression method:

(1) contain the structure of coding Aus Cel12A mature peptide expression of gene plasmid: with M13 Primer M4 and Cel12A-F is that primer carries out first round PCR (94 ℃ of 2min; 30 circulations, 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 60s; 72 ℃ of 10min); With Cel12A-F and Cel12A-R is that primer second is taken turns PCR (94 ℃ of 2min; 30 circulations, 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 45s; 72 ℃ of 10min); The second purpose band of taking turns PCR is cloned, checked order; Check order correct pUCm-T-cel12A and pPIC9K plasmid all carries out double digestion with EcoR I and Not I, the enzyme that reclaims is cut product and is connected under the effect of T4 dna ligase, obtain recombinant plasmid pPIC9K-cel12A, and to the recombinant plasmid evaluation of checking order;

(2) structure of GS115/cel12A, expression, product purification and determination of activity: with Sal I pPIC9K-cel12A is carried out linearizing, carry out electricity according to the Pichia anomala expression handbook and transform, screen, obtain the pichia spp recon GS115/cel12A of high copy; This project bacterium is with 1.0% methanol induction 96h, and the DNS method records that the recombined xylanase activity reaches 240IU/mL in the fermented liquid.The supernatant liquor of fermented liquid after centrifugal is recombinant beta-endoglucanase crude enzyme liquid, through molecular weight cut-off is that the ultra-filtration membrane of 10kDa concentrates, again through DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel permeation chromatography purifying, purifying is single band after SDS-PAGE detects, and shows that reorganization dextranase molecular weight is 27kDa; 60 ℃ of this reorganization dextranase optimum temperatures, the suitableeest action pH 5.0, this enzyme is stable below 3.0～7.0,60 ℃ at pH.