CN101560476A - Gene engineering bacterium of dual-copy beta-mannase and application thereof - Google Patents

Abstract

The invention discloses a gene engineering bacterium of dual-copy beta-mannase and application thereof. The gene engineering bacterium is a recombinant bacterium obtained by introducing two copied beta-mannase genes into yeast. The invention also discloses a method for producing the beta-mannase, which uses the recombinant bacterium to ferment to produce the beta-mannase. The beta-mannase produced by the method has the advantages of high specific activity, good acid stability, wide catalytic pH value range and the like, and is suitable to be used as additives for monogastric animals such as pig, chick and the like.

Description

The genetic engineering bacterium of two copy 'beta '-mannases and application thereof

Technical field

The present invention relates to the genetic engineering bacterium and the application thereof of two copy 'beta '-mannases.

Background technology

Mannosans is the important component of plant hemicellulose, by β-1, and the wire polysaccharide that the 4-D mannopyranose is formed by connecting.Mannosans has high-hydrophilic, absorbs water in a large number in the digestive tube of monogastric animal, has increased the viscosity of alimentary canal content, and the wriggling of opposing stomach and intestine directly influences digestion and the absorption of animal to nutritive substance.In recent years, along with the widespread use of bean products (dregs of beans etc.) in the animal diet, the anti-oxidant action of mannosans also more and more comes into one's own.2005,1,800 ten thousand tons of China's soybean yieldss, import volume is 2,300 ten thousand tons, wherein, is used for feed more than 80%.Content of mannan is more than 2% in the dregs of beans.The main dependence of its degraded added 'beta '-mannase.In recent years, along with the widespread use of bean products (dregs of beans etc.) in diet, the anti-nutrition problem of mannosans also more and more comes into one's own, and adding zymin in feed is the main path that addresses this problem.The complete enzymolysis of mannosans needs the synergy of 'beta '-mannase, beta-Mannosidase, beta-glucosidase enzyme, alpha-galactosidase and deacetylase, and wherein the application of 'beta '-mannase in feed is more extensive.'beta '-mannase is the hemicellulose enzyme of class energy hydrolysis mannosans, extensively is present in animals and plants and the microorganism.Report is arranged, in hog grower feed, add 'beta '-mannase, can improve average daily gain more than 10%; In broiler fodder, add the transformation efficiency 4%～6% that 'beta '-mannase can improve feed, improve the disease resistance of chicken, improve the healthy state of meat chicken.

Summary of the invention

The purpose of this invention is to provide a kind of genetic engineering bacterium and application thereof of two copy 'beta '-mannases.

The genetic engineering bacterium of two copy 'beta '-mannases provided by the present invention, called after GS115-PIC-MANM is the reorganization bacterium that obtains in the beta-mannase gene importing yeast with two copies.

Wherein, the nucleotides sequence of described beta-mannase gene is classified GenBank DQ464114 as.Described yeast is pichia spp GS115.

Pichia pastoris (pichia pastoris) GS115-PIC-MANM has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 04 23rd, 2009, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCCNo.3031.

Another object of the present invention provides a kind of method of producing 'beta '-mannase.

The method of production 'beta '-mannase provided by the present invention is with described reorganization bacterium fermentative production 'beta '-mannase.

Wherein, described fermention medium can be the substratum of any suitable yeast growth, as the BMGY substratum.

Saturation dissolved oxygen is 100% o'clock for the first time in the described fermenting process, and stream adds 25% (quality percentage composition) glycerine.Saturation dissolved oxygen is 100% o'clock for the second time in the described fermenting process, stream glycerol adding and methanol mixture.Saturation dissolved oxygen is 30% with upper reaches glycerol adding and carbinol mixture in the described fermenting process; Saturation dissolved oxygen is below 30% in the described fermenting process, the stream glycerol adding.In described glycerine and the methanol mixture, glycerine and methyl alcohol mass ratio be (6-9): 1.

The 'beta '-mannase that method of the present invention is produced has advantages such as specific activity height, acid acceptance is good, catalytic pH value range is wide, is suitable for use as the additive of monogastric animals such as pig, chicken.

Description of drawings

Fig. 1 is the pcr amplification beta-mannase gene.

Fig. 2 is the 'beta '-mannase SDS-PAGE electrophoresis that GS115-PIC-MANM expresses through the methanol induction different time in the 10L fermentor tank.

Fig. 3 is cell concentration and a 'beta '-mannase activity change in the 10L fermentor tank.

Embodiment

Among the following embodiment if no special instructions method therefor be ordinary method, agents useful for same all can obtain from commercial channels.

Embodiment 1, GS115-PIC-MANM produce 'beta '-mannase

One, expresses the structure of the GS115-PIC-MANM of 'beta '-mannase

1, the medicine of sulfuraspergillus is joined and the extraction of RNA always

With sulfuraspergillus (Aspergillus Sulphureus, preserving number: CGMCC No.0608) be inoculated into its growth medium (with 400mL tap water dissolving 85g wheat bran, 10g soybean cake powder, 0.2g KH ₂PO ₄, 0.3g NaCl and 10g sucrose, 121 ℃ of boiling 30min cross the leaching supernatant, autoclaving again after the packing) in, 30 ℃ of shaking tables are cultivated 48h.After cultivating end, the centrifugal 1min of 5000r/min collects thalline, places liquid N ₂The middle grinding.Get the thalline after 100mg grinds, place the 1.5mL centrifuge tube, add 500 μ LRNA extraction buffer (20mmol/LTris-HCl, pH8.0,1%SDS, 200mmol/L NaCl, 5mmol/L EDTA), add 500 μ L phenol/imitative (1: 1) again, 4 ℃, the centrifugal 5min of 12000r/min, supernatant adds equal-volume 4mol/L LiCl, 4 ℃ of precipitations 4h, the centrifugal 15min of 12000r/min then, abandon supernatant, precipitation is washed twice with 70% ethanol, and vacuum is drained, and is dissolved in the ddH that 20 μ l DEPC handle ₂Among the O, obtain total RNA of purpose bacterial strain.

2, the segmental clone of beta-mannase gene cDNA

To the aspergillus echinulatus announced among the GenBank (the GenBank accession number: L35487) and Rui Shi wood mould (the GenBank accession number: L25310) the mRNA sequence of beta-mannase gene is analyzed, analytical results shows at interlude one section height homologous nucleotide sequence, according to the nucleotide sequence design pair of degenerate primers of this high conservative, primer sequence is as follows:

P1 (upstream primer): 5 '-TTCAACGACGTCAACAC-3 ';

P2 (downstream primer): 5 '-A (T/C) CCAGCC (G/A) TTGCCCCA-3 '

The total RNA of sulfuraspergillus that extracts with step 1 is a template, with synthetic its first chain cDNA of M-MLV ThermoScript II (Promega).Be template with the reverse transcription synthetic first chain cDNA again, carry out pcr amplification under the guiding of primer 1 and primer 2,50 μ L PCR reaction systems are: 10 * PCR damping fluid, 5 μ L, primer P1 and P2 each 1 μ L (20 μ M), Taq enzyme 1 μ L (5 μ/μ L), dNTPs 1 μ L (2.5mM), template cDNA 1 μ L, H ₂O 40 μ L; The PCR reaction conditions is: 94 ℃ of pre-sex change 3min at first; 94 ℃ of pre-sex change 45s then, 49 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are extended 10min.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, reclaim the purpose fragment of the about 600bp of length and it is carried out purifying, to reclaim fragment cloning in carrier pUCm-T (worker's biotechnology company limited is given birth in Shanghai), obtain containing the segmental recombinant plasmid of recovery, it is checked order, and sequencing result shows that amplified fragments contains the nucleotide sequence of above-mentioned high conservative, and length is 608bp.

3, the clone of the 3 ' UTR (non-translational region) of beta-mannase gene cDNA

The Partial cDNA Sequence of the beta-mannase gene that obtains according to step 2 is designed 3 '-RACE special primer MAN-3-2:5 '-TACCCGTACCAATTCGC-3 ' that increases.Be template with total RNA then, with 3 ' terminal fragment, reaction system and the reaction conditions reference reagent box specification sheets of 3 '-FullRACE test kit (TaKaRa company) amplification beta-mannase gene cDNA.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, reclaim the also amplified fragments of the about 600bp of purifying length, it is cloned into carrier pUCm-T, obtain containing the segmental recombinant plasmid of recovery, it is checked order, and sequencing result shows that 3 ' the UTR fragment length of the beta-mannase gene cDNA of amplification is 151bp.

4, the clone of the 5 ' UTR of beta-mannase gene cDNA

The Partial cDNA Sequence of the beta-mannase gene that obtains according to step 2 is designed 5 '-RACE increase special primer Race-1, Race-2, Race-3, Race-4 and RT-P, and primer sequence is as follows:

Race-1：5’-GTATTTGCGTGGGAGTTGGC-3’；

Race-2：5’-TACCCGTACCAATTCGCCGA-3’；

Race-3：5’-TGCAATCTGCGTATCAGACG-3’；

Race-4：5’-ACGAGACGGATTTCTATACC-3’；

RT-P：5’-CCAACTCCCACGCAA-3’。

Be template with the total RNA of sulfuraspergillus then, with 5 ' terminal fragment, reaction system and the reaction conditions reference reagent box specification sheets of 5 '-Full RACE test kit (TaKaRa company) amplification beta-mannase gene cDNA.After should finishing, the PCR product is carried out 1% agarose gel electrophoresis to be detected, reclaim the also amplified fragments of the about 500bp of purifying length, it is cloned on the carrier pUCm-T, obtain containing the segmental recombinant plasmid of recovery, it is checked order, and sequencing result shows that 5 ' the UTR fragment length of the beta-mannase gene cDNA of amplification is 24bp.

5, the acquisition of beta-mannase gene full length cDNA sequence

5 ' and 3 ' end sequence according to step 3 and 4 beta-mannase genes that obtain designs a pair of primer, and primer sequence is as follows:

F1 (upstream primer): 5-cggaattcatgaagctctccagctc-3;

F2 (downstream primer): 5-cccaagcttttaggcgctatcaatagc-3

With the total RNA of sulfuraspergillus is template, under the guiding of primers F 1 and F2, the full-length cDNA of pcr amplification beta-mannase gene, 10 * PCR buffer, 5 μ L, primer (F1/F2) each 1 μ L (20 μ M), Taq enzyme 1 μ L (5 μ/μ L), dNTPs 1 μ L (2.5mM), template cDNA 1 μ L, H ₂O 40 μ L, total reaction system 50 μ L, response procedures are 94 ℃ of pre-sex change 3min, carry out amplified reaction by following loop parameter then: 94 ℃ of pre-sex change 45s, 49 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, reclaim the also amplified fragments of the about 1152bp of purifying length, it is cloned on the carrier pUCm-T, obtain containing the segmental recombinant plasmid of recovery, with M13+/-primer checks order to it, sequencing result shows that sulfuraspergillus beta-mannase gene full-length cDNA has SEQ ID № in the sequence table: 2 nucleotide sequence, SEQ ID № in the sequence table: 2 by 1327 based compositions, encoding sequence is from 5 ' end 25-1176 bit base, coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence, from 5 ' end 1-24 bit base is 5 ' end non-translational region, from 5 ' end 25-87 bit base is signal coding sequence, from 5 ' end 88-1176 bit base is mature protein coding sequence, from 5 ' the end 613-621 bit base typical mannase conserved sequence of encoding, be 3 ' end non-translational region from 5 ' end 1177-1327 bit base.SEQ ID № in the sequence table: 1 is made up of 383 amino-acid residues, from aminoterminal (N end) 1-21 amino acids residue is signal peptide, from aminoterminal 22-383 amino acids residue is maturation protein, is typical mannase conserved sequence from aminoterminal 205-207 amino acids residue.The molecular weight of sulfuraspergillus 'beta '-mannase maturation protein is 41kDa.The aminoacid sequence of clone's the nucleotide sequence of sulfuraspergillus beta-mannase gene and proteins encoded thereof is carried out homology with the sequence among the GenBank to be compared, comparison result shows that the sibship of this enzyme and aspergillus echinulatus 'beta '-mannase is nearest, the nucleotide sequence similarity reaches 71%, and amino acid sequence similarity reaches 72%.

6, sulfuraspergillus 'beta '-mannase mature protein gene is synthetic

Under the prerequisite that does not change aminoacid sequence, only the proteic codon of encoding mature in the sulfuraspergillus beta-mannase gene is replaced with the preference codon of pichia spp, thereby obtain the nucleotide sequence of an other sulfuraspergillus beta-mannase gene, be the SEQ ID № in the sequence table: 3, SEQ ID № in the sequence table: 3 by 1327 based compositions, encoding sequence is from 5 ' end 25-1176 bit base, coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence, from 5 ' end 1-24 bit base is 5 ' end non-translational region, from 5 ' end 25-87 bit base is signal coding sequence, from 5 ' end 88-1176 bit base is mature protein coding sequence, from 5 ' the end 613-621 bit base typical mannase conserved sequence of encoding, be 3 ' end non-translational region from 5 ' end 1177-1327 bit base.Synthetic SEQ ID № then: the gene fragment of coding sulfuraspergillus 'beta '-mannase maturation protein in 3 (SEQ ID №: in 3 from 5 ' end 88-1176 bit base), and add restriction enzyme EcoR I and Xba I restriction enzyme site respectively at 5 ' and 3 ' end.

7, the acquisition of sulfuraspergillus beta-mannase gene yeast expression vector

After with restriction enzyme EcoR I and Xba I step 1 synthetic gene being carried out double digestion, it is connected into the downstream of alpha factor sequence in the carrier pPICz of same enzyme double digestion α A (Invitrogen), obtain the yeast expression vector of sulfuraspergillus beta-mannase gene, called after pPIC-Mann.

8, express the structure of the GS115-PIC-MANM of 'beta '-mannase

Nucleotide sequence design primer according to the sulfuraspergillus beta-mannase gene

Mann-opt-F (5 '-CCG GAATTCTTGCCAAAGGCTTC-3 ', underscore are the EcoRI restriction enzyme site) and mann-opt-Rn:(5 '-ATAAGAAT GCGGCCGCTTAAGCAGAATCAATAGCAG-3 ', underscore are Not I restriction enzyme site), be template with pPIC-manM, carry out the PCR reaction by following condition: 95 ℃ of 5min, 35 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min), last 72 ℃ are extended 10min.Reaction is got PCR product (5 μ L) and is carried out 1% agarose gel electrophoresis after finishing, and confirms pcr amplification product (Fig. 1).M among Fig. 1: molecular weight standard; 1 and the 2:PCR amplified production.Reclaim the PCR product, use the EcoRI/NotI double digestion, the expression vector pPIC9k with same double digestion behind the purifying is connected, and connects product chemical method transformed into escherichia coli Top10 competent cell, and screening obtains recombinant expression vector pPIC9k-mann through kantlex.

Picking yeast list bacterium colony is seeded to and contains in the 5mL YPD substratum test tube, and 28-30 ℃, the 250-300rpm overnight incubation.The culture of getting 50 μ L is seeded in the 500mL triangular flask that contains the 50mL fresh culture, and 28-30 ℃, the 250-300rpm overnight incubation reaches 1.3-1.5 to the OD600 value.With cell culture in 4 ℃, the centrifugal 5min of 1500g, with the aqua sterilisa of 500mL ice precooling that bacterial sediment is resuspended.4 ℃, the centrifugal 5min of 1500g, with the aqua sterilisa of 250mL ice precooling that bacterial sediment is resuspended.4 ℃, the centrifugal 5min of 1500g, with the Sorbitol Solution USP of the 1mol/L of 20mL ice precooling that bacterial sediment is resuspended.4 ℃, the centrifugal 5min of 1500g, with the Sorbitol Solution USP of the 1mol/L of 1mL ice precooling that bacterial sediment is resuspended, its final volume is about 1.5mL.

10 μ g are dissolved in the 5-10 μ L sterilization distilled water with the linearizing pPIC9K-mann of SacI, pichia spp GS115 competent cell mixing with 80 μ L, the electricity that goes to the precooling of 0.2cm ice transforms in the cup, will shock by electricity behind the electricity conversion cup ice bath 5min, and the electric shock condition is: 2.0kV, 5ms.After electric shock finished, the Sorbitol Solution USP of the 1mol/L of adding 1mL ice precooling went in the 50mL centrifuge tube 28 ℃ of incubation 2h with the thalline mixing.The thalline suspension is coated MDS (0.67g/100ml YNB, 1g/100ml glucose, 1mol/L sorbyl alcohol, 2g/100ml agar) on the flat board, every 100-200 μ L coating one flat plate is cultivated 2-3d in 30 ℃, until growing bacterium colony clearly, the bacterial strain called after GS115/PICmann of acquisition.

The linearizing pPIC-manM of 10 μ g is dissolved in the 5-10 μ L sterilization distilled water, and with the GS115/PICmann competent cell mixing of 80 μ L, the electricity that goes to the precooling of 0.2cm ice transforms in the cup, will shock by electricity behind the electricity conversion cup ice bath 5min.After electric shock finished, the Sorbitol Solution USP of the 1mol/L of adding 1mL ice precooling went in the 50mL centrifuge tube 28 ℃ of incubation 2h with the thalline mixing.The thalline suspension is coated (1g/100ml yeast extract on the YPDS flat board, the 2g/100ml peptone, 2g/100ml glucose, the 1mol/L sorbyl alcohol, 2g/100ml agar, 100 μ g/mL Zeocin), every 100-200 μ L coating one flat plate is cultivated 2-3d in 30 ℃, until growing bacterium colony clearly, the bacterial strain called after GS115-PIC-MANM of acquisition.

GS115-PIC-MANM has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 04 23rd, 2009, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.3031.

Two, 10 liters of ferment tanks are produced 'beta '-mannase

Preparation 3L BMGY substratum (1g/100ml yeast extract, 2g/100ml peptone, 1.34g/100mlYNB, 4 * 10 ^-5G/100mlbiotin, 1ml/100ml glycerine, 100mM pH6.0 phosphate buffered saline buffer), control automatically in the fermentor tank after the sterilization at 10L, be cooled to 28.5 ℃.By the 10ml/100ml inoculum size seed liquor (seed liquor: picking GS115-PIC-MANM CGMCC No.3031 mono-clonal inserts the BMGY culture media shaking vase and cultivates) is joined in the 10L fermentor tank of dress 3L fermention medium BMGY, carrying out the stage cultivates, transfer pH to 5.0 with ammoniacal liquor and phosphoric acid, by regulating rotating speed and air flow control dissolved oxygen greater than 30%, leavening temperature is 28.0 ℃.After inserting seed liquor 24h, glycerine consumption is (dissolved oxygen is labeled as 100%) fully, enters stream and adds for 25% (quality percentage composition) glycerine stage, and flow velocity is 60mL/h.After stream adds 4-6h,, treat to add methyl alcohol when wet bacterium weighs 200g/L, begin to induce after the glycerine consumption fully (dissolved oxygen is labeled as 100%).Enter glycerine and methanol mixed stream adds the stage, blending ratio is 9: 1, and flow velocity is 12mL/h.By regulating rotating speed, air flow quantity and methyl alcohol speed control dissolved oxygen is more than 30%, then stops the methyl alcohol interpolation more than 30% as not remaining on, and gos up until dissolved oxygen.Behind the 12h, glycerine and methyl alcohol ratio were transferred to 8: 1.After 12h, ratio transferred to 6: 1, and kept to the final, and the control dissolved oxygen is more than 30%.Live and protein content at different time sampling and measuring biomass, enzyme, and expressing protein is carried out the 12%SDS-PAGE electrophoretic analysis.The measuring method that the 'beta '-mannase enzyme is lived adopts the DNS method, under pH2.4 and 50 ℃ of conditions, (Sigma company, article No.: the required enzyme amount that hydrolysis produces the seminose of 1 μ mol/L G0753) is defined as 1 enzyme unit that lives to per minute, represents with U from the substrate polygalactomannan.Quantification of protein adopts BCA analysis of protein test kit (U.S. Pierce company) to carry out.

12%SDS-PAGE electrophoretic analysis result as shown in Figure 2, M among Fig. 2: protein molecular weight standard; 1-8: recombinant bacterial strain is induced the expression product of 0h, 12h, 24h, 36h, 48h, 60h, 72h, 78h and 84h.

In the 10L fermentor tank GS115-PIC-MANM CGMCC No.3031 cell concentration and 'beta '-mannase activity change as shown in Figure 3, among Fig. 3, a: glycerine cultivation stage; B: glycerine feed supplement cultivation stage; C: glycerine and methanol mixed induction period; (■): wet bacterium is heavy; (▲): the beta-mannase enzyme activity, show that the fermentative activity of 10 liters of fermentor tanks of reorganization bacterium GS115-PIC-MANM CGMCC No.3031 can reach 2300U/mL, expressing quantity reaches 6mg/mL, is suitable for animal feedstuff additive and uses.

Sequence table

＜110〉China Agricultural University

＜120〉genetic engineering bacterium of two copy 'beta '-mannases and application thereof

<160>3

<210>1

<211>383

<212>PRT

＜213〉sulfuraspergillus (Aspergillus Sulphureus)

<400>1

Met?Lys?Leu?Ser?Ser?Ser?Leu?Leu?Thr?Leu?Ala?Ser?Leu?Ala?Leu?Ala

1 5 10 15

Asn?Leu?Ser?Thr?Ala?Leu?Pro?Lys?Ala?Ser?Pro?Ala?Pro?Ser?Thr?Ser

20 25 30

Ser?Ser?Ser?Ala?Ser?Thr?Ser?Phe?Ala?Ser?Thr?Ser?Gly?Leu?Gln?Phe

35 40 45

Thr?Ile?Asp?Gly?Glu?Thr?Gly?Tyr?Phe?Ala?Gly?Thr?Asn?Ser?Tyr?Trp

50 55 60

Ile?Gly?Phe?Leu?Thr?Asp?Asp?Ser?Asp?Val?Asp?Leu?Val?Met?Ser?His

65 70 75 80

Leu?Lys?Ser?Ser?Gly?Leu?Lys?Ile?Leu?Arg?Val?Trp?Gly?Phe?Asn?Asp

85 90 95

Val?Thr?Thr?Gln?Pro?Ser?Ser?Gly?Thr?Val?Trp?Tyr?Gln?Leu?His?Gln

100 105 110

Asp?Gly?Lys?Ser?Thr?Ile?Asn?Thr?Gly?Ala?Asp?Gly?Leu?Gln?Arg?Leu

115 120 125

Asp?Tyr?Val?Val?Ser?Ser?Ala?Glu?Gln?His?Gly?Ile?Lys?Leu?Ile?Ile

130 135 140

Asn?Phe?Val?Asn?Tyr?Trp?Thr?Asp?Tyr?Gly?Gly?Met?Ser?Ala?Tyr?Val

145 150 155 160

Ser?Ala?Tyr?Gly?Gly?Ser?Asp?Glu?Thr?Asp?Phe?Tyr?Thr?Ser?Asp?Thr

165 170 175

Met?Gln?Ser?Ala?Tyr?Gln?Thr?Tyr?Ile?Lys?Thr?Val?Val?Glu?Arg?Tyr

180 185 190

Ser?Asn?Ser?Ser?Ala?Val?Phe?Ala?Trp?Glu?Leu?Ala?Asn?Glu?Pro?Arg

195 200 205

Cys?Pro?Ser?Cys?Asp?Thr?Thr?Val?Leu?Tyr?Asp?Trp?Ile?Glu?Lys?Thr

210 215 220

Ser?Lys?Phe?Ile?Lys?Gly?Leu?Asp?Ala?Asp?His?Met?Val?Cys?Ile?Gly

225 230 235 240

Asp?Glu?Gly?Phe?Gly?Leu?Asn?Thr?Asp?Ser?Asp?Gly?Ser?Tyr?Pro?Tyr

245 250 255

Gln?Phe?Ala?Glu?Gly?Leu?Asn?Phe?Thr?Met?Asn?Leu?Gly?Ile?Asp?Thr

260 265 270

Ile?Asp?Phe?Ala?Thr?Leu?His?Leu?Tyr?Pro?Asp?Ser?Trp?Gly?Thr?Ser

275 280 285

Asp?Asp?Trp?Gly?Asn?Gly?Trp?Ile?Ser?Ala?His?Gly?Ala?Ala?Cys?Lys

290 295 300

Ala?Ala?Gly?Lys?Pro?Cys?Leu?Leu?Glu?Glu?Tyr?Gly?Val?Thr?Ser?Asn

305 310 315 320

His?Cys?Ser?Val?Glu?Ser?Pro?Trp?Gln?Gln?Thr?Ala?Leu?Asn?Thr?Thr

325 330 335

Gly?Val?Ser?Ala?Asp?Leu?Phe?Trp?Gln?Tyr?Gly?Asp?Asp?Leu?Ser?Thr

340 345 350

Gly?Glu?Ser?Pro?Asp?Asp?Gly?Asn?Thr?Ile?Tyr?Tyr?Gly?Thr?Ser?Asp

355 360 365

Tyr?Glu?Cys?Leu?Val?Thr?Asp?His?Val?Ala?Ala?Ile?Asp?Ser?Ala

370 375 380

<210>2

<211>1327

<212>DNA

＜213〉sulfuraspergillus (Aspergillus Sulphureus)

<400>2

aatacagcca?agcaaaccac?cacaatgaag?ctctccagct?ccctcctcac?cctggccagc 60

ctggcgctgg?ccaacctctc?cacggccctg?cccaaagcct?ctcctgcacc?aagcaccagc 120

agcagcagcg?cctccacctc?cttcgccagc?acctcgggcc?tccaattcac?catcgacggc 180

gaaaccggct?acttcgccgg?aacgaacagt?tactggatcg?gtttcctgac?cgacgactcc 240

gatgtcgacc?tggtgatgag?ccacctgaag?tcatccggcc?tcaaaatcct?ccgcgtatgg 300

ggcttcaacg?acgtcaccac?gcagccctct?tccggcacag?tctggtacca?actgcaccag 360

gacggcaaat?caaccatcaa?cactggcgcc?gacggtctcc?agcgcctcga?ctacgtcgtc 420

tcctccgccg?aacagcacgg?catcaaactc?atcatcaact?tcgtcaacta?ctggaccgac 480

tacggcggta?tgtccgcgta?cgtgagcgca?tatggcggat?ccgacgagac?ggatttctat 540

accagtgata?cgatgcaatc?tgcgtatcag?acgtatatta?agacggtcgt?ggagcggtat 600

agtaactcct?ctgcggtatt?tgcgtgggag?ttggcgaatg?agccgagatg?tcctagttgt 660

gatactaccg?tgttgtatga?ttggattgag?aagacgagta?aatttattaa?ggggttggat 720

gccgatcata?tggtttgtat?tggtgatgag?ggcttcggcc?tcaacaccga?ctcggacggc 780

agctacccgt?accaattcgc?cgagggtctc?aacttcacca?tgaacctcgg?tatcgatact 840

attgactttg?ctaccctcca?cttgtatcct?gatagctggg?gcacctccga?cgactggggc 900

aacggatgga?tcagcgcaca?cggcgcggca?tgcaaagcgg?ccggcaagcc?ctgtctactg 960

gaggaatacg?gagtcacctc?gaaccactgt?agtgtggaga?gcccgtggca?gcagacggcc 1020

ctcaacacga?ctggcgtcag?cgcagatctc?ttctggcagt?atggtgatga?tttgagcacg 1080

ggcgagtcgc?cggatgatgg?aaataccatt?tactacggga?cgagcgatta?tgagtgtttg 1140

gtcacggatc?atgtggctgc?tattgatagc?gcctaaggga?tagggagatg?tggatatatc 1200

tagttgatac?attatgaggg?gtgctgtaca?taagaatgcg?ccggagggag?ggtgtgaagg 1260

atgataactg?atctttgagt?ttggatagga?tacaattcga?gaggcaaatc?atcccttcga 1320

ttaccat 1327

<210>3

<211>1327

<212>DNA

＜213〉sulfuraspergillus (Aspergillus Sulphureus)

<400>3

aatacagcca?agcaaaccac?cacaatgaag?ctctccagct?ccctcctcac?cctggccagc 60

ctggcgctgg?ccaacctctc?cacggccttg?ccaaaggctt?ctccagctcc?atctacttct 120

tcttcttctg?cttctacttc?ttttgcttct?acttctggtt?tgcaatttac?tattgatggt 180

gaaactggtt?actttgctgg?tactaactct?tactggattg?gttttttgac?tgatgattct 240

gatgttgatt?tggttatgtc?tcatttgaag?tcttctggtt?tgaagatttt?gagagtttgg 300

ggttttaacg?atgttactac?tcaaccatct?tctggtactg?tttggtacca?attgcatcaa 360

gatggtaagt?ctactattaa?cactggtgct?gatggtttgc?aaagattgga?ttacgttgtt 420

tcttctgctg?aacaacatgg?tattaagttg?attattaact?ttgttaacta?ctggactgat 480

tacggtggta?tgtctgctta?cgtttctgct?tacggtggtt?ctgatgaaac?tgatttttac 540

acttctgata?ctatgcaatc?tgcttaccaa?acttacatta?agactgttgt?tgaaagatac 600

tctaactctt?ctgctgtttt?tgcttgggaa?ttggctaacg?aaccaagatg?tccatcttgt 660

gatactactg?ttttgtacga?ttggattgaa?aagacttcta?agtttattaa?gggtttggat 720

gctgatcata?tggtttgtat?tggtgatgaa?ggttttggtt?tgaacactga?ttctgatggt 780

tcttacccat?accaatttgc?tgaaggtttg?aactttacta?tgaacttggg?tattgatact 840

attgattttg?ctactttgca?tttgtaccca?gattcttggg?gtacttctga?tgattggggt 900

aacggttgga?tttctgctca?tggtgctgct?tgtaaggctg?ctggtaagcc?atgtttgttg 960

gaagaatacg?gtgttacttc?taaccattgt?tctgttgaat?ctccatggca?acaaactgct 1020

ttgaacacta?ctggtgtttc?tgctgatttg?ttttggcaat?acggtgatga?tttgtctact 1080

ggtgaatctc?cagatgatgg?taacactatt?tactacggta?cttctgatta?cgaatgtttg 1140

gttactgatc?atgttgctgc?tattgattct?gcttaaggga?tagggagatg?tggatatatc 1200

tagttgatac?attatgaggg?gtgctgtaca?taagaatgcg?ccggagggag?ggtgtgaagg 1260

atgataactg?atctttgagt?ttggatagga?tacaattcga?gaggcaaatc?atcccttcga 1320

ttaccat 1327

Claims (10)

1, reorganization bacterium is the reorganization bacterium that obtains in the beta-mannase gene importing yeast with two copies.

2, reorganization bacterium according to claim 1, it is characterized in that: the nucleotides sequence of described beta-mannase gene is classified GenBank DQ464114 as.

3, reorganization bacterium according to claim 1 and 2, it is characterized in that: described yeast is pichia spp GS115.

4, reorganization bacterium according to claim 3 is characterized in that: the deposit number of described reorganization bacterium is CGMCCNo.3031.

5, a kind of method of producing 'beta '-mannase is with arbitrary described reorganization bacterium fermentative production 'beta '-mannase in the claim 1 to 4.

6, method according to claim 5 is characterized in that: described fermention medium is the BMGY substratum.

7, according to claim 5 or 6 described methods, it is characterized in that: saturation dissolved oxygen is 100% o'clock for the first time in the described fermenting process, the stream glycerol adding; Saturation dissolved oxygen is 100% o'clock for the second time in the described fermenting process, stream glycerol adding and methanol mixture.

8, method according to claim 7 is characterized in that: described glycerine is the glycerine of quality percentage composition 25%; Described glycerine flow acceleration is 6ml/hL; The flow acceleration of described glycerine and methanol mixture is 1.2ml/hL.

9, method according to claim 8 is characterized in that: saturation dissolved oxygen is 30% with upper reaches glycerol adding and carbinol mixture in the described fermenting process; Saturation dissolved oxygen is below 30% in the described fermenting process, the stream glycerol adding.

10, method according to claim 9 is characterized in that: in described glycerine and the methanol mixture, glycerine and methyl alcohol mass ratio be (6-9): 1.

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