CN102220301B - Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof - Google Patents

Abstract

The invention relates to an Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof. The invention provides an alpha-galactosidase AgaAJB13 derived from Sphingobium sp., a coding gene agaAJB13 for encoding the alpha-galactosidase AgaAJB13, a recombinant vector containing the gene agaAJB13 and a recombination strain containing the gene agaAJB13, wherein, the amino acid sequence of the alpha-galactosidase AgaAJB13 is shown in SEQ ID No.1. The alpha-galactosidase AgaAJB13 has the following properties that: the optimal PH is 5.0; the optimal temperature is 60 DEG C, the enzyme activity of more than 10% and the enzyme activity of more than 20% can be achieved respectively at the temperature of 10 DEG C and 20 DEG C; the activity of about 40% can be maintained after processing for 1h at the temperature of 37 DEG C by using a buffer with 0.1M and pH 11.0; and the alpha-galactosidase AgaAJB13has good thermal stability, good proteinase resistance and good hydrolysis to various natural supports, thus being capable of being used as a feed or food additive in fields of feed and food.

Description

An alkaline-resistant low-temperature α-galactosidase AgaAJB13 and its gene

technical field

The invention relates to the technical field of genetic engineering, in particular to an alkaline-resistant low-temperature α-galactosidase AgaAJB13 and its gene.

Background technique

α-galactosidase, melibiase (1,6-α-d-galactoside galactohydrolase; α-galactosidase; melibiase; EC 3.2.1.22), removes α-linked terminal non-reducing properties in different substrates D-galactose, including oligosaccharide substrates such as melibiose, raffinose, stachyose and verbascose, and polysaccharide substrates such as galactomannan. These polysaccharides are widely found in food and feed ingredients, especially in the seeds of legume plants, such as soybean meal, cotton meal and rapeseed meal (Karr-Lilienthal et al. Livest Prod Sci, 2005, 97: 1–12.).

α-galactosidase can be used in feed, food, papermaking and medical industries. In the feed industry, α-galactosidase preparations can promote the digestion of nutrients and eliminate or reduce the side effects of anti-nutritional factors (oligosaccharides such as raffinose) in the feed composition on the digestion of nutrients, thereby improving animal health. production performance and reduce feed cost; in the food industry, α-galactosidase preparations can reduce the content of raffinose in soybean milk and increase the absorption of soybean nutrients by humans; α-galactosidase can also improve the quality of paper The bleaching effect and treatment of Fabry disease, etc. (Cao et al. Appl Microbiol Biotechnol, 2009, 83: 875–884.).

α-galactosidase widely exists in bacteria, actinomycetes, fungi and plants. According to amino acid sequence similarity, α-galactosidases are divided into glycoside hydrolase families 4, 27, 36 and 57, and most α-galactosidases belong to glycoside hydrolase families 27 and 36. Most α-galactosidases derived from eukaryotes belong to family 27, while α-galactosidases derived from prokaryotes basically belong to family 36 (Finn et al. Nucleic Acids Res, 2008, 36: D281–D288.). However, most of the α-galactosidases reported so far are mesophilic or high-temperature enzymes and acid-resistant or neutral pH-resistant enzymes. Not reported.

Contents of the invention

The purpose of the present invention is to provide an alkaline-resistant low-temperature alpha-galactosidase.

Another object of the present invention is to provide a gene encoding the above-mentioned α-galactosidase.

Another object of the present invention is to provide a recombinant vector comprising the above gene.

Another object of the present invention is to provide recombinant strains containing the above genes.

The α-galactosidase AgaAJB13 of the present invention can be obtained from Sphingobium sp., such as Sphingobium estrogenivorans ATCC BAA-1367. The amino acid sequence of AgaAJB13 is shown in SEQ ID NO.1.

The enzyme contains a total of 738 amino acids, of which the N-terminal 23 amino acids are its predicted signal peptide sequence "MVMRRWGAALAAATMLAAAPAHA" (SEQ ID NO. 2).

Therefore, the theoretical molecular weight of the mature α-galactosidase AgaAJB13 is 80.0kDa, and its amino acid sequence is shown in SEQ ID NO.3.

The optimal pH value of the α-galactosidase AgaAJB13 of the present invention is 5.0, and more than 80% of the enzyme activity is maintained in the range of pH 4.5-6.0; after being treated with a buffer solution of pH 4.0-11.0 for 1 hour, the enzyme activity remains More than 35%; the optimum temperature is 60°C, and it has more than 10% and 20% of the enzyme activity at 10°C and 20°C respectively, which has the low-temperature catalytic characteristics of low-temperature enzymes; the half-life at 37°C and 60°C is >60min, and has Good thermal stability; it can hydrolyze soybean meal, cotton meal and raffinose; AgaAJB13 can still maintain 102.1% and 114.0% of the enzyme activity after being treated with trypsin and proteinase K for 1 hour, respectively.

The present invention provides the gene agaAJB13 encoding the above-mentioned α-galactosidase, the gene sequence of which is shown in SEQ ID NO.4.

The results of DNA sequence structure analysis showed that the nucleotide sequence of the signal peptide encoded by the α-galactosidase gene agaAJB13 is shown in SEQ ID NO. 5.

The nucleotide sequence of the mature peptide encoded by the α-galactosidase gene agaAJB13 is shown in SEQ ID NO.

The present invention isolates and clones the coding gene agaAJB13 of α-galactosidase AgaAJB13 by PCR method, its full length is 2217bp, the start codon is ATG, and the stop codon is TAG. According to BLAST comparison, the amino acid sequence encoded by the α-galactosidase gene agaAJB13 has the highest identity of 59.2% with the potential α-galactosidase (EFI56085) derived from Acidobacterium sp. MP5ACTX8 in GenBank; The identity of the Lichtheimia corymbifera IFO 8084-derived α-galactosidase (AAF68953) was 40.1%. It shows that α-galactosidase AgaAJB13 is a new α-galactosidase.

The present invention also provides a recombinant vector comprising the above-mentioned α-galactosidase gene agaAJB13 , preferably pET- agaAJB13 . The α-galactosidase gene of the present invention is inserted between suitable restriction enzyme cutting sites of the expression vector, and its nucleotide sequence is connected with the expression control sequence. As a most preferred embodiment of the present invention, the α-galactosidase gene of the present invention is inserted between the Eco RI and Hind III restriction enzyme sites on the plasmid pET-28a (+), to obtain the recombinant large intestine Bacillus expression plasmid pET- agaAJB13 .

The present invention also provides a recombinant strain comprising the above-mentioned α-galactosidase gene agaAJB13 , preferably the strain is Escherichia coli, yeast, Bacillus or Lactobacillus, preferably the recombinant strain BL21(DE3) / agaAJB13 .

The method for preparing α-galactosidase AgaAJB13 of the present invention is carried out according to the following steps:

1) Transform host cells with the above-mentioned recombinant vectors to obtain recombinant strains;

2) Cultivate recombinant strains to induce the expression of recombinant α-galactosidase;

3) Recover and purify the expressed α-galactosidase AgaAJB13.

Wherein, the host cell is preferably an E. coli cell, and the recombinant E. coli expression plasmid is preferably transformed into an E. coli cell BL21(DE3) to obtain a recombinant strain BL21(DE3) / agaAJB13 .

The present invention provides a new α-galactosidase gene, the optimal pH of the α-galactosidase encoded by it is 5.0; at 10°C and 20°C, the enzyme activity is above 10% and 20%; 0.1M pH11.0 buffer solution can maintain about 40% of its activity after being treated at 37°C for 1 hour. Widely used in feed and food industries.

Description of drawings

Figure 1: SDS-PAGE analysis of recombinant α-galactosidase expressed in Escherichia coli, wherein, M: low molecular weight protein marker; 1: purified recombinant α-galactosidase.

Figure 2: Optimal pH of recombinant α-galactosidase.

Figure 3: pH stability of recombinant α-galactosidase.

Figure 4: Optimal temperature for recombinant α-galactosidase.

Figure 5: Thermostability of recombinant α-galactosidase.

Detailed ways

Test materials and reagents

1. Bacterial strains and vectors: Sphingobium sp. is the same as that reported in the literature, such as Sphingobium estrogenivorans ATCC BAA-1367; Escherichia coli BL21 (DE3) and expression vector pET-28a (+) can be Purchased from Novagen.

2. Enzymes and other biochemical reagents: restriction endonucleases, DNA polymerases, ligases and dNTPs were purchased from TaKaRa Company; p NPG ( p -nitrophenyl-α-d-galactopyranoside) was purchased from Sigma Company; others were domestically produced Reagents (both can be purchased from common biochemical reagent companies).

3. Medium:

LB medium: Peptone 10g, Yeast extract 5g, NaCl 10g, add distilled water to 1000ml, pH natural (about 7). On the basis of solid medium, add 2.0% (w/v) agar.

Note: For the molecular biology experiment methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kit and product manual.

Example 1: Cloning of α-galactosidase gene agaAJB13

Extract the genomic DNA of Sphingomonas: centrifuge the 2-day cultured bacteria liquid to get the bacteria, add 1mL lysozyme, treat at 37°C for 60min, then add the lysate, lyse in a water bath at 70°C for 60min, and mix every 10min. Centrifuge at 10000 rpm for 5 min at 4°C. The supernatant was extracted in phenol/chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuo, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

According to the conserved sequence of the 36th family of glycoside hydrolases ([F/L/V]-[L/V]-[L/M/V]-D-D-G-W-F and E-P-E-M-[V/I]-[N/S]-[ P/E]) designed and synthesized degenerate primers GH36F and GH36R (Table 1).

PCR amplification was performed using the total DNA of Sphingomonas as template. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 43°C for 30 sec, extension at 72°C for 30 sec, and after 30 cycles, incubation at 72°C for 10 min. A fragment of about 172bp was obtained, which was recovered and connected to the pMD 18-T vector, and then sent to Guangzhou Branch of Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing. the

According to the nucleotide sequence obtained by sequencing, two upstream and downstream TAIL-PCR specific primers were respectively designed: the design direction was the direction of the unknown region to be amplified, and the position of sp2 was designed inside of sp1. The distance between each two primers is not strictly regulated, the primer length is generally 20–30nt, and the annealing temperature is 65–70°C. And they were named as usp1 and usp2 (upstream specific primers) and dsp1 and dsp2 (downstream specific primers; Table 1).

The flanking sequences of known gene sequences were obtained by TAIL-PCR, and the amplified products were sent to Guangzhou Branch of Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing. The sequencing results were spliced with known gene sequence fragments to obtain the α-galactosidase gene agaAJB13 , the gene sequence of which is shown in SEQ ID NO. 4.

Embodiment 2: Preparation of recombinant α-galactosidase

The expression vector pET-28a (+) was subjected to double enzyme digestion ( Eco RI and Hind III), and the gene agaAJB13 encoding α-galactosidase was subjected to double enzyme digestion ( Eco RI and Hind III). The α-galactosidase agaAJB13 was connected with the expression vector pET-28a (+), and the recombinant plasmid pET- agaAJB13 containing the α-galactosidase gene agaAJB13 was obtained and transformed into Escherichia coli BL21 (DE3), and the recombinant Escherichia coli strain BL21 was obtained (DE3) / agaAJB13 .

Take the E. coli BL21 (DE3) strain containing the recombinant plasmid pET- agaAJB13 and the E. coli BL21 (DE3) strain containing only the pET-28a (+) empty plasmid, and inoculate it in LB (containing 50 μg/ mL Kan) culture medium, shake rapidly at 37°C for 16h. Then inoculate the activated bacterial solution into fresh LB (50 μg/mL Kan) culture solution with 1% inoculum, and after rapid shaking culture for about 2–3 hours (OD ₆₀₀ reaches 0.6–1.0), add a final concentration of 0.7mM For IPTG induction, continue shaking culture at 20°C for about 20h or shake culture at 26°C for about 8h. Centrifuge at 12000rpm for 5min to collect the bacteria. After suspending the cells with an appropriate amount of pH7.0 Tris-Hcl buffer, the cells were ultrasonically disrupted in a low-temperature water bath. The above intracellular concentrated primary enzyme solution was centrifuged at 13,000rpm for 10min, the supernatant was aspirated and the target protein was purified with Nickel-NTA Agarose. The results of SDS-PAGE (Figure 1) showed that the recombinant α-galactosidase was expressed in Escherichia coli, and it was a single band after being purified by Nickel-NTA Agarose.

Example 3: Activity analysis of recombinant α-galactosidase

Enzyme activity was determined by the p NPG method. pNPG was dissolved in 0.1 M buffer to a final concentration of 2 mM. The reaction system contains 50 μL of appropriate enzyme solution and 450 μL of 2 mM substrate. After the substrate was preheated at the reaction temperature for 5 minutes, the enzyme solution was added to react for another 10 minutes, and then 1.5 mL of 1M Na ₂ CO ₃ was added to terminate the reaction. After cooling to room temperature, the released p NP was measured at a wavelength of 405 nm. 1 enzyme activity unit (U) is defined as the amount of enzyme required to decompose pNPG to produce 1 μmol pNP per minute. The method for determining the activity of the substrate raffinose, soybean meal and cotton meal is 3,5-dinitrosalicylic acid (DNS) method: the substrate is dissolved in 0.1M buffer to make the final concentration 0.5% (w /v); the reaction system contains 100 μL of appropriate enzyme solution and 900 μL of substrate; after the substrate is preheated at the reaction temperature for 5 minutes, add the enzyme solution and react for 120 minutes, then add 1.5mL DNS to terminate the reaction, boil in water for 5 minutes, and cool to room temperature Then measure the OD value at a wavelength of 540nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol of galactose per minute under the given conditions.

Example 4: Determination of the properties of recombinant α-galactosidase AgaAJB13

1. The optimum pH and pH stability determination methods of recombinant α-galactosidase AgaAJB13 are as follows:

Determination of the optimal pH of the enzyme: The α-galactosidase AgaAJB13 purified in Example 2 was subjected to an enzymatic reaction at 37° C. and a buffer solution of pH 3.0–8.0. Determination of the pH stability of the enzyme: put the purified enzyme solution in a 0.1M buffer solution with a pH of 2.0–12.0, treat it at 37°C for more than 1 hour, and then carry out an enzymatic reaction at a pH of 5.0 and 37°C to ensure the stability of the enzyme. Treated enzyme solution was used as a control. The buffers are: 0.1M McIlvaine buffer (pH2.0–8.0) and 0.1M glycine-NaOH (pH9.0–12.0). Using pNPG as the substrate, reacted for 10min, and measured the enzymatic properties of the purified AgaAJB13. The results showed that the optimum pH of AgaAJB13 was 5.0, and more than 80% of the enzyme activity was maintained in the range of pH 4.5–6.0 (Figure 2); after being treated with pH 4.0–11.0 buffer for 1 hour, the remaining enzyme activity was ~40% Or more than 40% (Figure 3).

2. The optimum temperature and thermostability determination methods of recombinant α-galactosidase AgaAJB13 are as follows:

Optimum temperature determination of enzymes: Enzyme-catalyzed reactions were carried out at 0–70°C in pH 5.0 buffer. Determination of thermal stability of enzyme: put the same amount of enzyme solution at the set temperature (37°C, 60°C or 70°C) for 0-60min, then carry out the enzymatic reaction at pH 5.0 and 37°C, Untreated enzyme solution was used as a control. Using pNPG as the substrate, reacted for 10min, and measured the enzymatic properties of the purified AgaAJB13. The results showed that the optimum temperature of AgaAJB13 was 60°C, and the enzyme activity was over 10% and 20% at 10°C and 20°C respectively (Figure 4); the half-life of AgaAJB13 at 60°C was >60min, and it was inactivated quickly at 70°C (Figure 5).

3. The kinetic parameter determination method of recombinant α-galactosidase AgaAJB13 is as follows:

Kinetic parameters of enzyme First-order reaction time determination: at pH 5.0 and 60°C, with 0.5mM pNPG as substrate, stop the reaction within 1-30min of the enzymatic reaction and measure the enzyme activity, calculate the enzyme The ratio of activity to reaction time, if the ratio remains stable within a certain period of time, this time is the first-order reaction time. Km , Vmax and kcat were determined according to the Lineweaver-Burk method using 0.05–0.5 mM pNPG as substrate at pH 5.0, 60°C and first order reaction time. It was determined that the K _m , V _max and k _cat of AgaAJB13 to pNPG were 2.75 mM ⁻¹ , 1250.00 μmol min ⁻¹ mg ⁻¹ and 1792.08 s ⁻¹ at 60°C and pH 5.0, respectively.

4. The method for determining the effect of different metal ions and chemical reagents on the enzyme activity of AgaAJB13 is as follows:

Add 10mM metal ions and chemical reagents to the enzymatic reaction system to study their influence on the enzyme activity. Enzyme activity was measured at 37°C, pH 5.0. The results (Table 2) showed that Ag ⁺ and Hg ²⁺ at 10 mM could completely inhibit AgaAJB13; SDS inhibited AgaAJB13 strongly (remaining enzyme activity ~7%); Fe ²⁺ inhibited AgaAJB13 weakly (remaining enzyme activity ~ 70%); Ca ²⁺ and Pb ²⁺ can increase the enzyme activity of AgaAJB13 by about 0.2 times and about 0.3 times, respectively; other metal ions and chemical reagents have little effect on AgaAJB13.

Table 2. Effects of metal ions and chemical reagents on the activity of recombinant α-galactosidase AgaAJB13

5. Anti-protease ability of α-galactosidase AgaAJB13

Protease resistance of the enzyme: Treat the recombinant enzyme with trypsin (pH7.5) and proteinase K (pH7.5) equivalent to 10 times (w/w) of the recombinant enzyme at 37 ° C for 1 h, then at pH 5.0 and 37 The enzymatic reaction was carried out at ℃, and the enzyme solution in the pH buffer corresponding to the protease but without protease was used as a control. After being treated with trypsin and proteinase K for 1 h, AgaAJB13 could still retain 102.1% and 114.0% of the enzyme activities, respectively.

6. Degradation of natural substrates by α-galactosidase AgaAJB13

At 37℃ and pH5.0, the specific activities of AgaAJB13 to 1.0% (w/v) soybean meal and cotton meal were 6.99 ± 0.21 and 2.80 ± 0.21 U mg ⁻¹ , respectively, and the specific activities to 0.5% raffinose were 1.67 ± 0.01 U mg ⁻¹

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the

the

Val Leu Phe Ala Phe Leu His Ser Ser Gln Glu Leu Asp Arg Leu Ser

645 650 655

the

the

Ala Ile Arg Leu Arg Gly Leu Ala Pro Lys Lys Asn Tyr Arg Val Ala

660 665 670

the

the

Arg Ile Asp Gly Arg Pro Leu Ala Asp Asp Thr Pro Ala Lys Ala Ser

675 680 685

the

the

Gly Ala Tyr Trp Met Ala Arg Gly Ile Asp Val Pro Leu Ile Gly Asp

690 695 700

the

the

Phe Asp Ala Ala Gly Tyr Ile Phe Gln Ala Ile

705 710 715

the

the

<210> 4

<211> 2217

<212> DNA

<213> Sphingobium sp.

the

<400> 4

atggtgatga ggcgatgggg ggcagccctt gcggccgcga cgatgctggc ggcggcgccg 60

the

gcgcatgcgt cggcgggcta cgacgcgaag acccgcatgt tccggctcga cggcggcggc 120

the

accacctacg cgttcggggt gaccgacgac ggctatctcc aggccgccta ttggggcggg 180

the

cgactcggcg ccgacgaccc gatccggctg accaaggcgc aagggctgag cggcttcgat 240

the

ctggtcaact cgatcctgcc gcaggaattt cccgggcaag gcgccggcct ctataccgag 300

the

ccggcgctca aggtcgcctg gcccgacggc aaccgcgatc tcgtgctcaa atacgtctcg 360

the

cacaagatgt ccagggacca tgttgagatc gtgctcaagg atatcgagcg accgttgttc 420

the

gtcacgctcg actacagcat cgatcccgat accggcgtgg tcggccgctc ggcgcgtatc 480

the

gaaaaccgca gcgataccga cgtgcggatc gatcaggccg aggcgggcgc gctcaccctg 540

the

cccgtcgcgc acgattaccg gctgcactat ctcaccggcc gctgggccgc cgagtggacg 600

the

ctgcaggatc gcccgctgac cccgggcgcg accgtcctcg aaagccgccg cggctcgacc 660

the

ggctcggaaa acaacccctg gttcgcgatc acccgcgatc acgatgccgg cgaggagtac 720

the

gggcccgtct ggttcggcgc gctggcgtgg agcggatcgt ggcggatcac ggtcgaccag 780

the

gatccggccg gcgaggtccg cgtcgtcggc gggttcaacc cgttcgactt cgcctatcgc 840

the

ctcaagcccg gcgaatcgct cgacacgccg accttctacg ccggctattc ggatcacggc 900

the

atgggcggcg cctcgcggct gctccaccgc ttcgagcgcg acacgatcct gccccacgat 960

the

gccgacggca agctgccgct gcgccccgtc ctctacaaca gctgggaagc gaccgggttc 1020

the

gatgtcgacg aggccggcca gatcgcgctt gccgaaaagg cggcgaagat tggcgtcgag 1080

the

cgcttcgtga tggacgacgg ctggttcggc gcgcgcaacg acgatcatgc cgggctcggc 1140

the

gactggaccg tcaaccgcac caaattcccc aacggcctca aaccgctgat cgacaaggtc 1200

the

cacggcctcg gcatgcagtt cgggctgtgg gtcgagcccg agatgaccaa tcccgacagc 1260

the

gatctctatc gcgcgcatcc cgattgggtg atgaactata ccggccgccc gcgcaccgag 1320

the

gggcgtaacc agctcgtcct caatctcgcg cgaaccgacg tgcgcgatta catcttcaag 1380

the

gtgctcgacg acctgctcga cgagaacgac atccagttcc tcaaatggga ttacaaccgc 1440

the

aactggagcg agcccggctg gcccgaggcc gatgtcgccg accagcagca gatctacgtc 1500

the

aaatacgtcc gcaacctcta ttggatcatc gacaagctgc gcgccaggca tcccaagctc 1560

the

gagatcgaat cgtgctcggg cggcggcggc cgcgtcgatc tcggcattat gagccgcacc 1620

the

gacgaggtgt ggccgtcgga caataccgat ccgttcgatc ggctgacgat ccagaacggc 1680

the

tttacttacg cctatccgcc ggccgcgatg atggcgtggg tgacggcgtc gcccaattgg 1740

the

gtcaataatc gcgctacctc gctcgattat cgcttcctgt cggcgatgca aggcgggctc 1800

the

ggtattggcg ccgacctcaa taaatggagc gatgcagaat ttgcggaggc gagtcgcatg 1860

the

gtggcggcct ataagcgtgt ccgagcgacg gtgcagcaag gcgacctgta tcggttgatt 1920

the

atcccgaacg gaatcgatcg tgacgaccgc gtcgccaatc tctcggtatc tccagacaag 1980

the

cagcaggcgg tgctgttcgc gtttctgcac agcagccagg agctcgatcg gctttctgct 2040

the

atccgactgc gcgggctcgc tcctaagaag aactaccgcg tcgcccggat cgatggccgc 2100

the

ccgctggccg acgacacccc agctaaggcg agcggcgctt attggatggc gcgtggcatc 2160

the

gacgttccat taatcggcga cttcgacgcc gctggctata tctttcaggc catctag 2217

the

the

<210> 5

<211> 69

<212> DNA

<213> Sphingobium sp.

the

<400> 5

atggtgatga ggcgatgggg ggcagccctt gcggccgcga cgatgctggc ggcggcgccg 60

the

gcgcatgcg 69

the

the

<210> 6

<211> 2145

<212> DNA

<213> Sphingobium sp.

the

<400> 6

tcggcgggct acgacgcgaa gacccgcatg ttccggctcg acggcggcgg caccacctac 60

the

gcgttcgggg tgaccgacga cggctatctc caggccgcct attggggcgg gcgactcggc 120

the

gccgacgacc cgatccggct gaccaaggcg caagggctga gcggcttcga tctggtcaac 180

the

tcgatcctgc cgcaggaatt tcccgggcaa ggcgccggcc tctataccga gccggcgctc 240

the

aaggtcgcct ggcccgacgg caaccgcgat ctcgtgctca aatacgtctc gcacaagatg 300

the

tccagggacc atgttgagat cgtgctcaag gatatcgagc gaccgttgtt cgtcacgctc 360

the

gactacagca tcgatcccga taccggcgtg gtcggccgct cggcgcgtat cgaaaaccgc 420

the

agcgataccg acgtgcggat cgatcaggcc gaggcgggcg cgctcaccct gcccgtcgcg 480

the

cacgattacc ggctgcacta tctcaccggc cgctgggccg ccgagtggac gctgcaggat 540

the

cgcccgctga ccccgggcgc gaccgtcctc gaaagccgcc gcggctcgac cggctcggaa 600

the

aacaacccct ggttcgcgat cacccgcgat cacgatgccg gcgaggagta cgggcccgtc 660

the

tggttcggcg cgctggcgtg gagcggatcg tggcggatca cggtcgacca ggatccggcc 720

the

ggcgaggtcc gcgtcgtcgg cgggttcaac ccgttcgact tcgcctatcg cctcaagccc 780

the

ggcgaatcgc tcgacacgcc gaccttctac gccggctatt cggatcacgg catgggcggc 840

the

gcctcgcggc tgctccaccg cttcgagcgc gacacgatcc tgccccacga tgccgacggc 900

the

aagctgccgc tgcgccccgt cctctacaac agctgggaag cgaccgggtt cgatgtcgac 960

the

gaggccggcc agatcgcgct tgccgaaaag gcggcgaaga ttggcgtcga gcgcttcgtg 1020

the

atggacgacg gctggttcgg cgcgcgcaac gacgatcatg ccgggctcgg cgactggacc 1080

the

gtcaaccgca ccaaattccc caacggcctc aaaccgctga tcgacaaggt ccacggcctc 1140

the

ggcatgcagt tcgggctgtg ggtcgagccc gagatgacca atcccgacag cgatctctat 1200

the

cgcgcgcatc ccgattgggt gatgaactat accggccgcc cgcgcaccga ggggcgtaac 1260

the

cagctcgtcc tcaatctcgc gcgaaccgac gtgcgcgatt acatcttcaa ggtgctcgac 1320

the

gacctgctcg acgagaacga catccagttc ctcaaatggg attacaaccg caactggagc 1380

the

gagcccggct ggcccgaggc cgatgtcgcc gaccagcagc agatctacgt caaatacgtc 1440

the

cgcaacctct attggatcat cgacaagctg cgcgccaggc atcccaagct cgagatcgaa 1500

the

tcgtgctcgg gcggcggcgg ccgcgtcgat ctcggcatta tgagccgcac cgacgaggtg 1560

the

tggccgtcgg acaataccga tccgttcgat cggctgacga tccagaacgg ctttacttac 1620

the

gcctatccgc cggccgcgat gatggcgtgg gtgacggcgt cgcccaattg ggtcaataat 1680

the

cgcgctacct cgctcgatta tcgcttcctg tcggcgatgc aaggcgggct cggtattggc 1740

the

gccgacctca ataaatggag cgatgcagaa tttgcggagg cgagtcgcat ggtggcggcc 1800

the

tataagcgtg tccgagcgac ggtgcagcaa ggcgacctgt atcggttgat tatcccgaac 1860

the

ggaatcgatc gtgacgaccg cgtcgccaat ctctcggtat ctccagacaa gcagcaggcg 1920

the

gtgctgttcg cgtttctgca cagcagccag gagctcgatc ggctttctgc tatccgactg 1980

the

cgcgggctcg ctcctaagaa gaactaccgc gtcgcccgga tcgatggccg cccgctggcc 2040

the

gacgacaccc cagctaaggc gagcggcgct tattggatgg cgcgtggcat cgacgttcca 2100

the

ttaatcggcg acttcgacgc cgctggctat atctttcagg ccatc 2145

the

the

Claims (8)

1. An alkali-resistant low-temperature alpha-galactosidase AgaAJB13 is characterized in that its amino acid sequence is as shown in SEQ ID NO.1. 2. Alkali-resistant low temperature α-galactosidase AgaAJB13 as claimed in claim 1, is characterized in that the signal peptide sequence of described α-galactosidase AgaAJB13 is as shown in SEQ ID NO.2. 3. The alkaline-resistant low-temperature α-galactosidase AgaAJB13 as claimed in claim 1, characterized in that the mature peptide sequence of the α-galactosidase AgaAJB13 is as shown in SEQ ID NO. 3. 4. A α-galactosidase gene agaAJB13 encoding the alkaline-resistant low-temperature α-galactosidase AgaAJB13 of claim 1, characterized in that its nucleotide sequence is as shown in SEQ ID NO. 4. 5. The α-galactosidase gene agaAJB13 according to claim 4, characterized in that the nucleotide sequence of the signal peptide encoded by the α-galactosidase gene agaAJB13 is shown in SEQ ID NO. 5. 6. The α-galactosidase gene agaAJB13 according to claim 4, characterized in that the nucleotide sequence of the mature peptide encoded by the α-galactosidase gene agaAJB13 is shown in SEQ ID NO. 6. 7. A recombinant vector comprising the α-galactosidase gene agaAJB13 according to claim 4. 8. A recombinant strain comprising the α-galactosidase gene agaAJB13 according to claim 4.

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