CN103215300B - Method for producing trehalose synthase from integrated recombinant bacillus subtilis and manufacturing trehalose - Google Patents

Abstract

The invention discloses a method for producing trehalose synthase by taking integrated recombinant bacillus subtilis as a strain and producing trehalose from the trehalose synthase. The method specifically comprises the following steps of: integrating a trehalose synthase expression element in a bacillus subtilis chromosome to construct integrated recombinant bacillus subtilis, and fermenting in a nutrient culture medium to produce the trehalose synthase by taking the recombinant bacillus subtilis as a strain, wherein via simple separation, the trehalose synthase in the fermentation liquor can be directly used for manufacturing trehalose. The method disclosed by the invention has the advantages that the trehalose synthase is produced by virtue of a food safety expression system, the exogenous gene contained in the strain with integrated expression can be used for stable passage and expression, and the expressed trehalose synthase is secreted to the outside of cells, has no antimicrobial activity, and achieves the requirements of food applications for enzymic preparation, thus being beneficial to further manufacturing for trehalose.

Description

Produce TreP with integrated recombined bacillus subtilis and manufacture the method for trehalose

Technical field

The invention belongs to biological technical field, specifically produce TreP with integrated recombined bacillus subtilis and manufacture the method for trehalose, and TreP production obtained is for the manufacture of the method for trehalose.

Background technology

Trehalose is formed so that α, α-1,1-glycosidic link is connected by two molecule glucoses, the natural sex disaccharides be widespread in nature.Its self property is highly stable, and has provide protection to various bioactivators.In recent years, research finds that trehalose can be widely used in delicate flavour maintenance and the improved texture of varieties of food items.Along with the progress of society and the raising of people's living standard, people pay attention to increasingly to the quality of food and mouthfeel, which increase the demand of people to trehalose.Therefore, large-scale Cheap highly effective is produced trehalose and is had great economic worth, also more will can popularize the use of trehalose at field of food simultaneously.

Research has found that occurring in nature has multiple enzyme system can trehalose synthesis.Wherein one is all detectable TreP (Trehalose Synthase) in many microorganisms.Maltose directly can be transformed into trehalose by this enzyme, is relatively suitable for being applied to suitability for industrialized production trehalose.Research has found to derive from pimelobacter sp (Pimelobacter species), bending thermomonospora fusca (Thermomonospora curvata), Corynebacterium glutamicum (Corynebacterium glutamicum), maltose can be transformed into trehalose by the TreP of radioresistens abnormal cocci (Deinococcus radiodurans) and aquatic thermophile bacteria (Thermus aquaticus) efficiently.At present, existing reported success is all direct culture of isolated from original strain, or utilizes recombinant plasmid at expression in escherichia coli.From original strain, culture of isolated often affects scale operation because the enzyme that original strain is expressed is lived not high, and also there are some defects in escherichia expression system, as containing intracellular toxin, enterotoxin, be difficult to secreting, expressing, product needs brokenly born of the same parents to extract, and in order to keep the stability of plasmid, generally need in process of production to use microbiotic.Antibiotic agents residual in recombinant microorganism fermenting process is for the impact of food safety, strict restriction is had in the world to the anti-microbial activity of microbe-derived zymin, China's zymin that also clear stipulaties is microbe-derived in the national food safety standard (GB 25594-2010) of foodstuffs industry zymin must not detect anti-microbial activity, and this has higher requirement to food zymin.

Subtilis has the history preparing leavened food for a long time, is nonpathogenic, and not producing intracellular toxin and pyrogenicity allergic protein matter, is a kind of bacterial classification of food safety.In addition, subtilis expression system also has the following advantages: 1, have very strong protein secreting function, does not need smudge cells to extract protein, only needs to process fermented supernatant fluid more simply and can obtain purer target protein.2, do not have obvious codon-bias, expression product is also not easy to form inclusion body simultaneously.3, fermentation condition is simple.Therefore, develop subtilis expression system production foodstuff additive and there are profound significance and wide market outlook.Different according to adopted bearer type, subtilis expression pattern can be divided into reproducible plasmid expression and chromosomal integration to express.Plasmid replication is usually not bery stable in subtilis, which has limited the high expression of foreign gene.Also also exist simultaneously and need in process of production to use antibiotic problem.Also do not find to utilize integrated subtilis expression system Expression product TreP at present, and then utilize the TreP obtained to manufacture the research report of trehalose.

Summary of the invention

The object of this invention is to provide a kind of method also utilizing the TreP manufacture trehalose of acquisition with integrated recombined bacillus subtilis for bacterial classification production TreP.Utilize integrated plasmid to be incorporated in subtilis chromogene group by TreP Expression element, build integrated recombined bacillus subtilis, utilize in this bacterium liquid medium within and ferment, obtain TreP.Through simple separation, the TreP in fermented liquid can be directly used in the manufacture of trehalose.

The technical scheme that the present invention solves the problems of the technologies described above is as follows:

1. can express a recombined bacillus subtilis bacterial strain for TreP in integrated mode, it is characterized in that, its preparation method is:

TreP Expression element is cloned on subtilis integrated plasmid, the recombinant plasmid transformed host subtilis obtained, selects foreign gene and be incorporated into recombined bacillus subtilis in subtilis karyomit(e) by double exchange;

Described TreP Expression element comprises following component: can efficient promotor gene is expressed in subtilis promotor, can in subtilis the signal peptide DNA segment of efficient secretory expression albumen and trehalose synthesize enzyme gene;

Described can efficient promotor gene is expressed in subtilis promotor for deriving from subtilis (Bacillus subtilis) overlapping Promoter P43, or derive from the promotor of Bacillus licheniformis (Baclicus lincheniformis) maltogenic amylase gene amyM;

Described can produce the beta-amylase gene signal peptide DNA segment of hydrogen sulfide clostridium (Clostridium thermosulfurogenes) by the signal peptide DNA segment of efficient secretory expression albumen in subtilis for deriving from high temperature, or derives from the ɑ-acetolactate decarboxylase gene signal peptide DNA segment of bacillus brevis (Bacillus brevis);

Described trehalose synthesize enzyme gene is for deriving from the trehalose synthesize enzyme gene of Corynebacterium glutamicum (Corynebacteriumglutamicum), derive from the trehalose synthesize enzyme gene of bending thermomonospora fusca (Thermomonosporacurvata), or derive from the trehalose synthesize enzyme gene of aquatic thermophile bacteria (Thermus aquaticus).

Described subtilis integrated plasmid is integrated plasmid pMLK83 and plasmid thereof;

Described host subtilis is subtilis 168 derivative strain, comprises 1A751, WB600, and WB800.

2. utilize the recombined bacillus subtilis bacterial strain of expressing TreP in integrated mode to produce TreP and to prepare the method for trehalose, it is characterized in that, processing step is as follows:

1) preparation of one-level kind: connect above-mentioned Bacillus subtilis genes engineering strain list bacterium colony in 4ml LB liquid nutrient medium 37 DEG C, 220rpm overnight incubation from the LB flat board containing Liu Suanyan NEOMYCIN SULPHATE 20ug/ml, the bacterial classification of gained is one-level kind;

2) preparation of secondary kind: one-level kind is inoculated in 800ml LB liquid nutrient medium, in 37 DEG C, it is about 0.6 (about 4 ~ 5 hours) that 220rpm is cultured to OD600;

3) preparation of three grades of kinds: secondary kind be inoculated in 80L LB liquid fermentation tank, controls pH about 7.0 with citric acid, NaOH by 37 DEG C, and ventilate and stir, dissolved oxygen controls 20 ~ 30%, and being cultured to OD600 is about 0.6 (about 5 ~ 6 hours);

4) tank fermentation is produced: be inoculated in 3T fermentor tank by three grades of kinds, LB liquid nutrient medium, 36 ~ 38 DEG C, ventilate and stir, dissolved oxygen controls 20 ~ 30%, controls pH 6 ~ 8 with citric acid, NaOH, cultivate about 26 hours, 10000g centrifugal force is degerming, after molecular weight cut-off 5000 ~ 10000 ultra-filtration membrane concentrated supernatant, obtains TreP concentration stock solution;

5) with malt syrup or starch slurry for raw material, transform the method for producing trehalose by standard enzymatic and utilize above-mentioned restructuring TreP solution to manufacture trehalose.

TreP Expression element is incorporated in subtilis karyomit(e) by the present invention first, using this bacterium as bacterial classification, and conventional production technique, fermentative production TreP.The TreP produced can be directly used in manufacture trehalose through simple separation purifying.

Advantage of the present invention is: the present invention utilizes the expression system of food safety to produce TreP, foreign gene contained by the bacterial strain of integrated expression can be stablized and goes down to posterity and express, the TreP of expressing is secreted into outside born of the same parents, without anti-microbial activity, reach the requirement of food applications zymin, be conducive to the manufacture of next step trehalose.

Embodiment

The most effective way avoiding plasmid instability in expression system uses subtilis integrated plasmid, by exogenous origin gene integrator in subtilis karyomit(e), foreign gene copies with chromosomal and copy and express, and such foreign gene can keep good stability in host.The integrated plasmid pMLK83 purchased from American Ohio State University Bacillus heredity preservation center (BGSC that the present invention uses, http://www.bgsc.org), this plasmid have two sections with the DNA sequence dna (homology arm) of subtilis amylase gene homology, in these two sections of homology arms, have neomycin resistance gene; This plasmid has intestinal bacteria replicon and without subtilis replicon, therefore can copy in intestinal bacteria and can not copy in subtilis in addition.When Plastid transformation is to subtilis, selected by neomycin resistance, the recombinant bacterium be only incorporated in subtilis karyomit(e) could grow, and so just filters out recon.

Below in conjunction with embodiment, the invention will be further described.But it should be noted that, embodiment does not form the restriction to application claims protection domain.

Embodiment 1

This example will comprise and derive from subtilis (Bacillus subtilis) overlapping Promoter P43 promotor, and the ɑ-acetolactate decarboxylase gene signal peptide DNA segment deriving from bacillus brevis (Bacillus brevis) and the monocistronic TreP Expression element of trehalose synthesize enzyme gene deriving from Corynebacterium glutamicum (Corynebacterium glutamicum) are cloned into integrated plasmid pMLK83.

1. construction recombination plasmid pMLK83-P43

According to the Promoter P43 sequence annotated in Genbank, design upstream primer is 5 ' attgctggacgcttatggac 3 ' and downstream primer is 5 ' cgggatccattcctctcttacctataat 3 '.PCR reaction system 100ul:DNA template (subtilis 1A751 STb gene) 1ul (about 20ng), 5 × PrimeSTAR Buffer 20ul, 10pmol/ul dNTP 2ul, the forward and reverse primer of 10pmol/ul is respectively 2ul, 2.5U/ul PrimeSTAR HSDNA polysaccharase 1ul, adds ddH ₂o to 100ul.PCR response procedures: 94 DEG C of 5min; 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min; 4 DEG C of preservations.Be connected with T4 ligase enzyme after PCR fragment carries out double digestion respectively with plasmid pMLK83 restriction enzyme BamH I, Hind III, be transformed in bacillus coli DH 5 ɑ, obtain recombinant plasmid pMLK83-P43 through Screening and Identification.

2. construction recombination plasmid pMLK83-P43-cgTreS

The following DNA segment of synthetic:

1 GGATCCATGA AAAAAAATAT CATCACTTCT ATCACATCTC TGGCTCTGGT

51 TGCCGGGCTG TCTTTGACTG CTTTTGCAGC TACAACGGTG CTGCAAACCT

101 CCTGGCATTT CTCTATCCTG GCAGGCATGA CTGATACCTC TCCGTTTGAA

151 TTCTCAGCCG AGTGCAGATC ACCACCCTGA TCACGCGGCT CGCCCAGTTC

201 TTGATGCCCA CGGCTTGATC GTTGAGCACG AATCGGAAGA GTTTCCAGTC

251 CCCGCACCCG CTCCCGGTGA ACAGCCCTGG GAGAAGAAAA ACCGCGAGTG

301 GTACAAAGAC GCCGTTTTCT ACGAAGTGCT GGTTCGTGCC TTCTACGATC

351 CAGAAGGCAA CGGAGTCGGA TCGTTGAAAG GCCTGACCGA AAAACTGGAT

401 TACATCCAGT GGCTCGGCGT GGATTGCATT TAGATCCCAC CGTTTTATGA

451 TTCCCCACTG CGCGACGGCG GTTACGATAT CCGCAACTTC CGTGAAATCC

501 TGCCCGAATT CGGCACCGTC GATGACTTCG TGGAACTCGT TGACCACGCC

551 CACCGCCGTG GCCTGCGTGT TATCACCGAC TTGGTCATGA ATCACACCTC

601 CGACCAGCAC GCATGGTTCC AAGAATCCCG GCGCGACCCA ACCGGCCCCT

651 ACGGAGATTT CTATGTGTGG AGCGATGATC CCACCCTGTA CAACGAAGCC

701 CGCATCATCT TTGTAGATAC AGAAGAATCC AACTGGACCT ATGATCCGGT

751 GCGTGGCCAG TACTTCTGGC ACCGCTTCTT CTCCCACCAA CCAGACCTCA

801 ACTACGACAA CCCCGCAGTC CAAGAGGCCA TGCTAGATGT CTTGCGTTTC

851 TGGCTGGACC TGGGACTTGA TGGTTTCCGA CTAGATGCCG TTCCTTATCT

901 TTTTGAACGC GAAGGCACCA ACGGCGAAAA CCTCAAAGAA ACCCACGATT

951 TCCTCAAACT GTGTCGCTCT GTCATTGAGA AGGAATACCC CGGCCGAATC

1001 CTGCTCGCAG AAGCCAACCA ATGGCCCCAA GATGTGGTCG AATACTTCGG

1051 TGAAAAAGAC AAAGGCGATG AATGCCACAT GGCCTTCCAC TTCCCTTTGA

1101 TGCCGCGCAT CTTCATGGGA GTTCGCCAAG GTTCACGCAC CCCGATCAGT

1151 GAGATCCTGG CCAACACCCC GGAGATTCCC AAGACTGCCC AATGGGGTAT

1201 TTTCCTGCGT AATCATGATG AGCTCACCCT TGAAATGGTC TCCGATGAGG

1251 AACGCAGCTA CATGTACTCC CAATTCGCCT CCGAACCTCG CATGCGCGCC

1301 AACGTAGGAA TCCGCAGGCG CCTTTCCCCA CTGCTTGAAG GCGACCGCAA

1351 CCAGCTGGAA CTCCTTCACG GTTTGTTGCT GTCTCTACCT GGCTCACCCG

1401 TGTTGTATTA CGGTGATGAA ATTGGCATGG GCGACAATAT CTGGCTCCAC

1451 GACCGCGACG GAGTGCGCAC CCCCATGCAG TGGTCCAACG ACCGCAACGG

1501 TGGTTTCTCC AAAGCTGATC CTGAACGCCT GTACCTTCCA GCGATCCAAA

1551 ATGATCAATA CGGCTACGCC CAAGTAAACG TGGAAAGCCA ACTCAACCGC

1601 GAAAACTCCC TGCTGCGCTG GCTCCGAAAC CAAATCCTTA TCCGCAAGCA

1651 GTACCGCGCA TTTGGTGCCG GAACCTACCG TGAAGTGTCC TCCACCAATG

1701 AGTCAGTGTT GACATTTTTA CGAGAACACA AGGGCCAAAC CATTTTGTGT

1751 GTCAACAACA TGAGCAAATA TCCTCAGGCA GTCTCGCTTG ATTTGCGTGA

1801 ATTTGCAGGA CACACCCCTC GAGAGATGTC GGGCGGGCAG CTGTTCCCTA

1851 CCATTGCTGA ACGGGAGTGG ATTGTCACTT TAGCCCCTCA CGGATTCTTC

1901 TGGTTTGATC TCACCGCCGA TGAAAAGGAC GATATGGAAT GACCGCGG

The DNA fragmentation of synthesis is carried out being connected with T4 ligase enzyme after double digestion with restriction enzyme BamH I and Sac II with plasmid pMLK83-P43, be transformed in bacillus coli DH 5 ɑ competent cell, obtain recombinant plasmid pMLK83-p43-cgTreS. through Screening and Identification

Embodiment 2

This example will comprise and derive from subtilis (Bacillus subtilis) overlapping Promoter P43 promotor, derive from the beta-amylase gene signal peptide DNA segment that high temperature produces hydrogen sulfide clostridium (Clostridiumthermosulfurogenes) and the monocistronic TreP Expression element of trehalose synthesize enzyme gene deriving from bending thermomonospora fusca (Thermomonospora curvata) is cloned into integrated plasmid pMLK83.

The following DNA segment of synthetic:

1 GGATCCATGA TTGGAGCTTT TAAAAGGTTG GGTCAAAAAT TGTTTTTGAC

51 ATTGTTAACG GCATCATTAA TTTTTGCATC TTCTATAGTA ACTGCTAATG

101 CAGTGCAGAT GACCGGGGAC CCCATCCCCG ACACCTTCAC CCACGAAAAG

151 CCGCGCGACC CCTACTGGTA CAAGCACGCG GTCTTCTACG AGGTGCTGGT

201 GCGCGGGTTC AGCGACTCCA ACGACGACGG CACCGGAGAC CTGCGCGGCC

251 TCATCAACCG GCTGGACTAT CTGCAGTGGC TGGGCATCGA CTGCATCTGG

301 CTGCTGCCGA TCTACCAGTC GCCGCTGCGG GACGGCGGCT ACGACATCAG

351 CGACTACACC AAGATCCTGC CGGAGTTCGG CGATCTGGGC GACTTCGTGG

401 AGCTGGTCGA TGAGGCGCAC CGGCGGGGCA TCCGCGTCAT CGCCGACCTG

451 GTGATGAACC ACACCAGCGA CCAGCACCCC TGGTTCCAGG CGTCCCGCAC

501 GGACCCCGAC GGGCCGTACG GCGACTTCTA CATGTGGTCC GACACCGACG

551 ACAAGTACCC GGACGCGCGG ATCATCTTCG TCGACACCGA GGTGTCCAAC

601 TGGACCTACG ACCCGGTGCG CGGCCAGTAC TACTGGCACC GCTTCTTCTC

651 CCACCAGCCG GACCTCAACT ACGACAACCC GGCGGTGCAG GAGGCGATGC

701 TGGAGGTGCT GCGGTTCTGG CTGGACCTGG GCATCGACGG GTTCCGGTTG

751 GACGCGGTGC CCTACCTGTA CGCCCGCGAG GGCACCAACT GCGAGAACCT

801 GCCGGAGACC CACGCCTATC TGAAGCGGGT GCGCGCCGAA GTGGACCGGC

851 TGTACCCGGA CCGGGTGCTG CTGGCCGAGG CCAACCAGTG GCCGGCCGAC

901 GTGGTGGAGT ACTTCGGCGA CCCGGCCACC GGCGGGGACG AGTGCCACAT

951 GGCCTTCCAC TTCCCGGTGA TGCCACGGAT CTTCATGGCG GTGCGGCGCG

1001 AGCAGCGCTA CCCCATCTCC GAGATCATGG CGCAGACCCC CAAGATCCCC

1051 GAGAACTGCC AGTGGGGCAT CTTCCTGCGC AACCACGATG AGCTGACGCT

1101 GGAGATGGTC ACCGACGAAG AGCGGGACTA CATGTACGCC GAGTACGCCA

1151 AGGACCCACG GATGAAGGCC AACATCGGCA TCCGGCGCCG GCTGGCCCCG

1201 CTGCTGGACA ACGACCGCAA CCAGCTGGAG CTGTTCACCG CGCTGCTGCT

1251 GTCGCTGCCG GGATCGCCGG TGCTGTACTA CGGCGACGAG ATCGGCATGG

1301 GCGACAACAT CTGGCTGGGC GACCGCGACA GCGTGCGCAC CCCGATGCAG

1351 TGGACGCCCG ACCGCAACGC CGGCTTCTCC CGCTGCGACC CGGCCCGGCT

1401 CTACCTGCCG GTGATCATGG ACCCGATCTA CGGGTACCAG GCGGTCAACG

1451 TGGAGGCCCA GCAGCGCAAC CCCGGCTCGC TGCTGAACTG GACCCGCAAG

1501 ATGATCGAGA TCCGCAAGCG GCACCCGGTG TTCGGGCTGG GCTCGTATGT

1551 GGAGCTGCCG GCCTCCAACC CCAGCGTGCT GGCCTTCGTG CGCGAGTACG

1601 GCGACGACCG GGTGCTGTGC GTCAACAACC TCTCGCGCTT CCCGCAGCCG

1651 GTGGAGCTGG ACCTGCGCCG CTTCGAAGGC TGCACCCCGG TGGAGTGCAT

1701 GGGCGGGGTG CAGTTCCCGG CGATCGGCGA GCTGCCCTAC CTGCTCACCC

1751 TGCCCGGTCA CGGCTTCTAC TGGTTCGTGC TGCCGCCGCC GCCCGGCGGC

1801 TGGCCCGACG GCTCCGGCGC CGCCGAGCAG TCACTGCGGG AACAGTCCGA

1851 CCGGGGTGCG GACTTCCCCG CAGATCCCTC GCAGGAGACC CGGCAGACGG

1901 CGAAGTAACC GCGG

The DNA fragmentation of synthesis is carried out being connected with T4 ligase enzyme after double digestion with restriction enzyme BamH I and Sac II with plasmid pMLK83-P43, be transformed in bacillus coli DH 5 ɑ competent cell, obtain recombinant plasmid pMLK83-p43-tcTreS. through Screening and Identification

Embodiment 3

This example will comprise the promotor deriving from Bacillus licheniformis (Baclicus lincheniformis) maltogenic amylase gene amyM, and the ɑ-acetolactate decarboxylase gene signal peptide DNA segment deriving from bacillus brevis (Bacillus brevis) and the monocistronic TreP Expression element of trehalose synthesize enzyme gene deriving from aquatic thermophile bacteria (Thermusaquaticus) are cloned into integrated plasmid pMLK83.

1. construction recombination plasmid pMLK83-amyM

According to the promotor amyM sequence annotated in Genbank, design upstream primer is 5 ' cccaagcttctgtacacttgcgtcctcca 3 ' and downstream primer is 5 ' cgggatcctctcctcccctttcaatgtg 3 '.PCR reaction system 100ul:DNA template (Bacillus licheniformis ATCC 14580 STb gene) 1ul (about 20ng), 5 × PrimeSTARBuffer 20ul, 10pmol/ul dNTP 2ul, the forward and reverse primer of 10pmol/ul is respectively 2ul, 2.5U/ul PrimeSTAR HS archaeal dna polymerase 1ul, adds ddH ₂o to 100ul.PCR response procedures: 94 DEG C of 5min; 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C of 10min; 4 DEG C of preservations.Be connected with T4 ligase enzyme after PCR fragment carries out double digestion respectively with plasmid pMLK83 restriction enzyme BamH I, Hind III, be transformed in bacillus coli DH 5 ɑ, obtain recombinant plasmid pMLK83-amyM through Screening and Identification.

2. construction recombination plasmid pMLK83-amyM-taTreS

The following DNA segment of synthetic:

1 GGATCCATGA AAAAAAATAT CATCACTTCT ATCACATCTC TGGCTCTGGT

51 TGCCGGGCTG TCTTTGACTG CTTTTGCAGC TACAACGGTG GACCCCCTCT

101 GGTACAAGGA CGCGGTGATC TACCAGCTCC ACGTCCGCTC CTTCTTTGAC

151 GCCAACAACG ACGGCTACGG GGACTTTGAG GGCCTGAGGC GGAAGCTTCC

201 CTACCTGGAG GAGCTCGGGG TCAACACCCT CTGGCTCATG CCCTTCTTCC

251 AGTCCCCCTT GAGGGACGAC GGGTACGATA TCTCCGACTA CTACCAGATC

301 CTCCCCGTCC ACGGGACCCT GGAGGACTTC ACCGTGGACG AGGCCCACGG

351 CCGGGGGATG AAGGTGATCA TTGAGCTCGT CCTGAACCAC ACCTCCATTG

401 ACCACCCTTG GTTCCAGGAG GCGAGGAAGC CGAATAGCCC CATGCGGGAC

451 TGGTACGTGT GGAGCGACAC CCCGGAGAAG TACAAGGGGG TCCGGGTCAT

501 CTTCAAGGAC TTTGAAACCT CCAACTGGAC CTTTGACCCC GTGGCCAAGG

551 CCTACTACTG GCACCGCTTC TACTGGCACC AGCCCGACCT CAACTGGGAC

601 AGCCCCGAGG TGGAGAAGGC CATCCACCAG GTCATGTTCT TCTGGGCCGA

651 CCTGGGGGTG GACGGCTTCC GCCTGGACGC CATCCCCTAC CTCTACGAGC

701 GGGAGGGGAC CTCCTGCGAG AACCTCCCCG AGACCATTGA GGCGGTGAAG

751 CGCCTGAGGA AGGCCCTGGA GGAGCGCTAC GGCCCCGGGA AGATCCTCCT

801 CGCCGAGGTC AACATGTGGC CGGAGGAGAC CCTCCCCTAC TTCGGGGACG

851 GGGACGGGGT CCACATGGCC TACAACTTCC CCCTGATGCC CCGGATCTTC

901 ATGGCCCTAA GGCGGGAGGA CCGGGGTCCC ATTGAAACCA TGCTCAAGGA

951 GGCGGAGGGA ATCCCCGAAA CCGCCCAGTG GGCCCTCTTC CTCCGCAACC

1001 ACGACGAGCT CACCCTGGAG AAGGTCACGG AGGAGGAGCG GGAGTTCATG

1051 TACGAGGCCT ACGCCCCCGA CCCCAAGTTC CGCATCAACC TGGGAATCCG

1101 CCGCCGCCTC ATGCCCCTCC TCGGGGGCGA CCGCAGGCGG TACGAGCTCC

1151 TCACCGCCCT CCTCCTCACC CTAAAGGGCA CGCCCATCGT CTACTACGGG

1201 GACGAGATCG GCATGGGGGA CAACCCCTTC CTCGGGGACC GGAACGGTGT

1251 CAGGACCCCC ATGCAGTGGT CCCAAGACCG CATCGTCGCC TTCTCCCGCG

1301 CCCCCTACCA CGCCCTCTTC CTTCCCCCCG TGAGCGAGGG GCCCTACAGC

1351 TACCACTTCG TCAACGTGGA GGCCCAGCGG GAAAACCCCC ACTCCCTCCT

1401 GAGCTTCAAC CGCCGCTTCC TCGCCCTGAG GAACCAGCAC GCCAAGATCT

1451 TCGGCCGGGG GAGCCTCACC CTTCTCCCCG TGGAGAACCG GCGCGTCCTC

1501 GCCTACCTGA GGGAGCACGA GGGGGAGCGG GTCCTGGTGG TGGCCAACCT

1551 CTCCCGCTAC ACCCAGGCCT TTGACCTCCC CTTGGAGGCC TACCAAGGCC

1601 TCGTCCCCGT GGAGCTCTTC TCGCAGCAAC CCTTCCCCCC GGTGGAGGGG

1651 CGCTACCGCT TGACCCTGGG CCCCCACGGC TTCGCCCTCT TCGCCCTGAA

1701 GCCCGTGGAG GCGGTGCTCC ACCTCCCCTC CCCCGACTGG GCCGAGGAGC

1751 CCGCCCCCGA GGAGGCCGAC CTGCCCCGGG TCCACATGCC CGGGGGGCCG

1801 GAGGTCCTCC TGGTGGACAC CCTGGTCCAC GAAAGGGGGC GGGAGGAGCT

1851 CCTAAACGCC CTCGCCCAGA CCCTGAAGGA GAAGAGCTGG CTCGCCCTCA

1901 AGCCGCAGAA GGTGGCCCTC CTGGACGCCC TCCGCTTCCA GAAGGACCCG

1951 CCCCTTTACC TCACCCTGCT CCAGCTGGAG AACCACAGGA CCCTCCAGGT

2001 CTCCCTCCCC CTCCTCTGGT CCCCCCAGAG GCGGGAAGGC CCCGGCCTCT

2051 TCGCCCGCAC CCACGGCCAG CCCGGCTACT TCTACGAGCT CTCCTTGGAC

2101 CCAGGCTTCT ACCGCCTCCT CCTCGCCCGC CTTAAGGAGG GGTTTGAGGG

2151 GCGGAGCCTC CGGGCCTACT ATCGCGGCCG CCACCCGGGT CCCGTGCCCG

2201 AGGCCGTGGA CCTCCTCCGG CCGGGACTCG CGGCGGGGGA GGGGGTCTGG

2251 GTCCAGCTCG GCCTCGTCCA AGACGGGGGC CTGGACCGCA CGGAGCGGGT

2301 CCTCCCCCGC CTGGACCTCC CCTGGGTTCT CCGGCCCGAA GGGGGCCTCT

2351 TCTGGGAGCG GGGCGCCTCC AGAAGGGTCC TCGCCCTCAC GGGAAGCCTC

2401 CCCCCGGGCC GCCCCCAGGA CCTCTTCGCC GCCCTGGAGG TCCGGCTCCT

2451 GGAAAGCCTT CCCCGCCTCC GGGGGCACGC CCCCGGGACC CCAGGCCTCC

2501 TTCCCGGGGC CCTGCACGAG ACCGAAGCCC TGGTCCGCCT CCTCGGGGTG

2551 CGCCTCGCCC TCCTCCACCG GGCCCTTGGG GAGGTGGAGG GGGTGGTGGG

2601 GGGCCACCCC CTCCTAGGAC GCGGCCTCGG GGCCTTCCTG GAGCTGGAGG

2651 GGGAGGTGTA CCTCGTGGCC CTGGGCGCGG AAAAGCGGGG CACGGTGGAG

2701 GAGGACCTGG CCCGCCTGGC CTACGACGTG GAGCGGGCCG TGCACCTCGC

2751 CCTCGAGGCC CTGGAGGCGG AGCTTTGGGC CTTTGCCGAG GAGGTGGCCG

2801 ACCACCTCCA CGCCGCCTTC CTCCAAGCCT ACCGCTCCGC CCTCCCCGAG

2851 GAGGCCCTGG AGGAGGCGGG CTGGACGCGG CACATGGCCG AGGTGGCGGC

2901 GGAGCACCTC CACCGGGAGG AAAGGCCCGC CCGCAAGCGC ATCCACGAGC

2951 GCTGGCAGGC CAAGGCCGGA AAAGCCTAGC CGCGG

The DNA fragmentation of synthesis is carried out being connected with T4 ligase enzyme after double digestion with restriction enzyme BamH I and Sac II with plasmid pMLK83-amyM, be transformed in bacillus coli DH 5 ɑ competent cell, obtain recombinant plasmid pMLK83-amyM-taTreS. through Screening and Identification

Embodiment 4

Integrative plasmid pMLK83-p43-cgTreS, pMLK83-p43-tcTreS and the pMLK83-amyM-taTreS conversion in subtilis

Get a full ring subtilis 1A751 glycerol stock and draw LB flat board (flat board adds 1.5%Agar for LB substratum: peptone 1%, yeast extract paste 0.5%, NaCl1%), 37 DEG C of incubator overnight incubation.Transform and choose single bacterium colony in 3ml LB substratum night the day before yesterday, 37 DEG C, 250rpm overnight incubation, the next morning gets 160 μ l nutrient solutions and is forwarded to (SPI substratum: it is 50%(W/V that SP salt adds 1% volumetric concentration) glucose solution in 8ml SPI substratum, 1% volume 100 × CAYE solution; SP salts solution: containing 1.96g/L (NH ₂) 2SO ₄, 13.72g/L K ₂hPO ₄, 5.88g/L KH ₂pO ₄, 0.196g/L MgSO ₄.7H ₂the independent sterilizing of O() and 0.98g/L Trisodium Citrate; 100 × CAYE solution: containing 20g/L casamino acids and 100g/L yeast extract), 37 DEG C, 250rpm is cultured to logarithmic growth latter stage (about 4 ~ 5 hours); Get 0.2ml and grow to the nutrient solution of late log phase to (SPII substratum: SPI substratum adds 1% volume 50mmol/L CaCl in 2ml SPII substratum ₂solution, 1% volume 250mmol/L MgCl ₂solution), 37 DEG C, 100 rpm cultivate 90 minutes; In the thalline of above-mentioned SPII substratum, add 20ul 10mmol/L EGTA, then in 37 DEG C, 100rpm cultivates 10 minutes; Bacterium liquid after above-mentioned process is distributed into 0.5ml often manage, add 5ulpMLK83-p43-cgTreS plasmid (50ng/ul), again in 37 DEG C, 250rpm cultivates 90 minutes, get bacterium liquid coating Liu Suanyan NEOMYCIN SULPHATE (20ug/ml) LB dull and stereotyped, namely screening amylase disappearance transformant is Bacillus subtilis genes engineering strain 1A751 [p43-cgTreS].

With same method by pMLK83-p43-cgTreS, pMLK83-p43-tcTreS or pMLK83-amyM-taTreS Plastid transformation subtilis WB600 and WB800, WB600 [p43-cgTreS] can be obtained, WB600 [p43-tcTreS], WB600 [amyM-taTreS], WB800 [p43-cgTreS], WB800 [p43-tcTreS] or WB800 [amyM-taTreS].

Embodiment 5

The production of TreP, operation steps is as follows:

1) preparation of one-level kind: connect above-mentioned Bacillus subtilis genes engineering strain list bacterium colony in 4ml LB liquid nutrient medium 37 DEG C, 220rpm overnight incubation from the LB flat board containing Liu Suanyan NEOMYCIN SULPHATE 20ug/ml, the bacterial classification of gained is one-level kind.

2) preparation of secondary kind: one-level kind is inoculated in 800ml LB liquid nutrient medium, in 37 DEG C, it is about 0.6 (about 4 ~ 5 hours) that 220rpm is cultured to OD600.

3) preparation of three grades of kinds: secondary kind be inoculated in 80L LB liquid fermentation tank, controls pH about 7.0 with citric acid, NaOH by 37 DEG C, and ventilate and stir, dissolved oxygen controls 20 ~ 30%, and being cultured to OD600 is about 0.6 (about 5 ~ 6 hours).

4) tank fermentation is produced: be inoculated in 3T fermentor tank by three grades of kinds, LB liquid nutrient medium, 36 ~ 38 DEG C, ventilate and stir, dissolved oxygen controls 20 ~ 30%, controls pH 6 ~ 8 with citric acid, NaOH, cultivate about 26 hours, 10000g centrifugal force is degerming, after molecular weight cut-off 5000 ~ 10000 ultra-filtration membrane concentrated supernatant, obtains TreP concentration stock solution.

The mensuration that TreP enzyme is lived is carried out as follows.Get enzyme liquid+0.6ml 10% maltose substrate (with the preparation of 50mM pH6.5 phosphoric acid buffer) that 0.6ml suitably dilutes, at 37 DEG C of reaction 30min after mixing, reaction solution 10000rpm is centrifugal, and 3min gets supernatant, reducing sugar content is measured with pressing Determination of Reducing Sugars (3,5-dinitrosalicylic Acid Colorimetry).0.6ml10% maltose substrate+0.6ml 50mM pH6.5 phosphoric acid buffer is then got in contrast, and 37 DEG C of centrifugal 3min of reaction 30min, 10000rpm get supernatant, and the same method measures reducing sugar content.

Unit of activity (U) is defined as 1ml enzyme liquid at pH6.5, and under 37 DEG C of conditions, per minute conversion 1ug maltose is non-reducing sugar is a TreP unit of activity.Method of calculation are as follows: U(μ g/ml.min)=(V ₁× M × (OD ₁-OD ₂)/OD ₁)/(T × V ₂)=(0.6ml × 100mg/ml × (OD ₁-OD ₂)/OD ₁× 1000)/(30min × 0.6ml)

(V ₁: substrate volume (ml), V ₂: the enzyme liquid long-pending (ml) participating in reaction, M: concentration of substrate (mg/ml), OD ₁: control sample surveys light absorption value during reducing sugar, OD ₂: the light absorption value of reducing sugar surveyed by sample, and 1000:mg converts the coefficient of μ g to, T: the enzyme reaction time (min)).

After measured, after fermenation raw liquid is centrifugal, the highest enzyme of each Bacillus subtilis genes engineering strain fermentation expression is lived in table 1.

Table 1

Bacterial strain	Total enzyme lives (U)	Enzymatic activities (U)
			1A751[p43-cgTreS]	542	428
WB600[p43-cgTreS]	1085	923
			WB600[p43-tcTreS]	1426	980
WB600[amyM-taTreS]	1240	1010
			WB800[p43-cgTreS]	968	812
WB800[p43-tcTreS]	1522	1028
			WB800[amyM-taTreS]	1434	1225

Embodiment 6

The preparation of trehalose

1, malt syrup is converted into trehalose

Getting the solution of 100L containing 30mg/ml maltose, is 5mM with the final concentration that phosphoric acid buffer adjustment pH is 6.9(damping fluid).The enzyme liquid that WB800 [amyM-taTreS] bacterial strain adding 10L 1000U is expressed, reacts 32 hours under 25 DEG C of conditions.Measuring with high performance liquid chromatograph the content of the trehalose of principal reaction after 32 hours in reaction solution to be 17.2mg/ml, and maltose is 8.6mg/ml, and to be the transformation efficiency that the total maltose of 2.9mg/ml. is converted into trehalose be glucose: 17.2X1.1/30=63.1%.

2, tapioca (flour) is converted into trehalose

First tapioca (flour) is generated maltose by after beta-amylase process again through α-amylaseliquefied, then the maltose of acquisition method as above is converted into trehalose.Experiment shows that 100 kg of starch can obtain about 40 kilograms of trehaloses.

Claims (2)

1. can express a recombined bacillus subtilis bacterial strain for TreP in integrated mode, it is characterized in that, its preparation method is:

TreP Expression element is cloned on subtilis integrated plasmid, the recombinant plasmid transformed host subtilis obtained, selects foreign gene and be incorporated into recombined bacillus subtilis in subtilis karyomit(e) by double exchange;

Described TreP Expression element comprises following component: can efficient promotor gene is expressed in subtilis promotor, can in subtilis the signal peptide DNA segment of efficient secretory expression albumen and trehalose synthesize enzyme gene;

Described can efficient promotor gene is expressed in subtilis promotor for deriving from subtilis (Bacillus subtilis) overlapping Promoter P43, or derive from the promotor of Bacillus licheniformis (Baclicus lincheniformis) maltogenic amylase gene amyM;

Described can produce the beta-amylase gene signal peptide DNA segment of hydrogen sulfide clostridium (Clostridium thermosulfurogenes) by the signal peptide DNA segment of efficient secretory expression albumen in subtilis for deriving from high temperature, or derives from the ɑ-acetolactate decarboxylase gene signal peptide DNA segment of bacillus brevis (Bacillus brevis);

Described trehalose synthesize enzyme gene is for deriving from the trehalose synthesize enzyme gene of Corynebacterium glutamicum (Corynebacterium glutamicum), derive from the trehalose synthesize enzyme gene of bending thermomonospora fusca (Thermomonospora curvata), or derive from the trehalose synthesize enzyme gene of aquatic thermophile bacteria (Thermus aquaticus);

Described subtilis integrated plasmid is integrated plasmid pMLK83 and plasmid thereof;

Described host subtilis is subtilis 168 derivative strain, comprises 1A751, WB600, and WB800.

2. utilize described in claim 1 recombined bacillus subtilis bacterial strain to produce TreP and to prepare the method for trehalose, it is characterized in that, processing step is as follows:

1) preparation of one-level kind: connect above-mentioned Bacillus subtilis genes engineering strain list bacterium colony in 4ml LB liquid nutrient medium 37 DEG C, 220rpm overnight incubation from the LB flat board containing Liu Suanyan NEOMYCIN SULPHATE 20ug/ml, the bacterial classification of gained is one-level kind;

2) preparation of secondary kind: one-level kind is inoculated in 800ml LB liquid nutrient medium, in 37 DEG C, 220rpm cultivate 4 ~ 5 little up to OD600 be 0.6;

3) preparation of three grades of kinds: secondary kind be inoculated in 80L LB liquid fermentation tank, controls pH 7.0 with citric acid, NaOH by 37 DEG C, ventilate stir, dissolved oxygen controls 20 ~ 30%, cultivate 5 ~ 6 little up to OD600 be 0.6;

4) tank fermentation is produced: be inoculated in 3T fermentor tank by three grades of kinds, LB liquid nutrient medium, 36 ~ 38 DEG C, ventilate and stir, dissolved oxygen controls 20 ~ 30%, controls pH 6 ~ 8 with citric acid, NaOH, cultivate 26 hours, 10000g centrifugal force is degerming, after molecular weight cut-off 5000 ~ 10000 ultra-filtration membrane concentrated supernatant, obtains TreP concentration stock solution;

5) with malt syrup or starch slurry for raw material, transform the method for producing trehalose by standard enzymatic and utilize above-mentioned restructuring TreP solution to manufacture trehalose.