CN113234743B - Heat-resistant alpha-galactosidase gene and application thereof - Google Patents
Heat-resistant alpha-galactosidase gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering and fermentation engineering, and particularly relates to a heat-resistant alpha-galactosidase gene and application thereof, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:2, respectively. The alpha-galactosidase of the invention has high activity, is derived from paecilomyces thermophilus, and has good heat resistance. The highest expression level of the alpha-galactosidase reaches 10g/L, the enzyme activity of the fermentation broth reaches 10000U/mL, and the highest level is the highest level reported in the open. And the alpha-galactosidase is secreted and expressed in the fermentation supernatant, and the alpha-galactosidase crude enzyme solution with high purity can be obtained by simply filtering and removing thalli without breaking cells, so that the cost of separating and purifying downstream protein is reduced.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and fermentation engineering, and particularly relates to a heat-resistant alpha-galactosidase gene and application thereof.
Background
Alpha-galactosidase (alpha-galactosidase, EC 3.2.1.22), also known as melibiase, is an exoglycosidic hydrolase that catalyzes the hydrolysis of non-reducing terminal alpha-galactoside compounds, such as melibiose, raffinose, stachyose, mannan, and the like. The enzyme has wide application, and is mainly applied to the industries of food, feed, medicine and the like at present. In the food industry, raffinose family oligosaccharides (raffinose, stachyose and the like) which are not digested by the human digestive tract in food can be hydrolyzed, and flatulence is prevented from occurring. In the feed industry, raffinose family oligosaccharide (anti-nutritional factor) which can not be utilized by monogastric animals can be hydrolyzed into galactose and sucrose, diarrhea and flatulence caused by the decomposition of the raffinose family oligosaccharide by intestinal microorganisms are eliminated, and the feed utilization rate is improved. In addition, the research and application of alpha-galactosidase in the medical fields of Fabry disease treatment, blood type conversion, organ transplantation and the like also become research hotspots.
The alpha-galactosidase can be produced in large scale in industrial strains through gene engineering technology, the production process is simplified, and the production cost is reduced. Human tissues, animal organs, coffee beans, cucumbers, and alpha-galactosidase from various microorganisms have been cloned and recombinantly expressed in common industrial strains such as escherichia coli, pichia pastoris, and filamentous fungi. The difference of the physicochemical properties of the alpha-galactosidase from different sources is obvious, and the alpha-galactosidase in the current market has the problems of low activity, poor heat resistance and the like. The low enzyme activity causes high production cost, the granulation process of the feed industry needs high-temperature treatment, and the poor heat resistance causes serious enzyme activity loss, which limits the popularization and application of the feed. Although the alpha-galactosidase realizes heterologous recombinant expression, the enzyme activity is low, the heat resistance is poor, the production cost is high, and the application effect is poor.
Disclosure of Invention
In order to solve these problems, the present invention provides a novel thermostable α -galactosidase and a gene thereof.
The invention aims to provide a thermostable alpha-galactosidase.
It is still another object of the present invention to provide a gene encoding the above thermostable α -galactosidase.
It is still another object of the present invention to provide a recombinant plasmid comprising the above gene.
It is still another object of the present invention to provide a recombinant strain comprising the above gene.
It is still another object of the present invention to provide a method for preparing thermostable a-galactosidase.
It is a further object of the present invention to provide the use of the thermostable α -galactosidase described above.
The amino acid sequence of the heat-resistant alpha-galactosidase is shown as SEQ ID NO:1 is shown.
SEQ ID NO：1
1 MLRSSAAVAA AVGLLTASSQ GPMTLAQSTS GSNAIVVDGT TFALHGAGMS
51 YVFHANTTTG DLITDHYGAS VSGALPSPPE PVVNGWVGMI GRTRREFPDQ
101 GRGDFRIPAV RIRQTAGYAV SDLRYQGHEV RDGKPGLPGL PATFGEAGDV
151 TTLVVHLYDN HSAVAADLSY SVFPEFDAVV RSVNITNKGN GNITIEHLAS
201 MSVDFPFEDL DLLGLRGDWA REAHRMRRRV EYGVQGFGSS TGYSSHLHNP
251 FFVLAHPSTT ESQGEAWGFN LTYTGSFSAQ VEKGSQGLTR ALIGFNPDQL
301 SWTLGPGETL TSPECVSVYS SDGIGGMSRK FHRLYRKHLI RSKYATLDRP
351 PLLNSWEGVY FDYNQTGIER LARQSAALGI RLFVMDDGWF GNKYPRTSDK
401 AGLGDWTPNP DRFPDGLEPV VERITNLPVN GTAGEKLRFG IWVEPEMVNP
451 NSSLYREHPD WVLHAGSYPR TERRNQLVLN LALPEVQDFI IDFMTNLLNS
501 ADISYVKWDN NRGMHEMPST RTYHEYMLGL YRVLDTLSAR FPDVLWEGCA
551 SGGGRFDAGI LHYFPQIWTS DNTDGVDRIT IQFGTSLAYP PSTMGAHLSA
601 VPNHQTSRTV PLEFRAHVAM MGGSFGLELD PATLQDDPEV RRLIKLAEKV
651 NPLVINGDLY RLRLPEESQW PAALFVAEDG SQAVLFYFQV GPNVNHAAPW
701 VRLQGLDPEA RYTVDGNATY KGATLMNLGL QFTFDSEYGS KVVFLEKQ
The alpha-galactosidase gene according to the present invention encodes the above enzyme.
According to the specific implementation mode of the invention, the alpha-galactosidase coding gene is subjected to codon optimization according to the codon preference of pichia pastoris, a signal peptide sequence is removed, and a termination codon TAA is added at the tail end of the gene sequence to obtain a mature alpha-galactosidase gene sequence, wherein the gene sequence is shown as SEQ ID NO:2, respectively.
SEQ ID NO:2, respectively.
1 CAGAGTACAT CAGGAAGTAA TGCAATCGTC GTAGATGGAA CGACTTTTGC
51 CTTGCACGGT GCTGGAATGT CATACGTTTT CCACGCAAAT ACGACTACCG
101 GTGATCTAAT CACTGACCAT TATGGAGCCT CTGTAAGTGG CGCTTTGCCA
151 TCCCCCCCTG AGCCCGTTGT GAATGGTTGG GTTGGCATGA TAGGCAGGAC
201 GAGACGTGAA TTCCCAGATC AAGGTCGTGG CGACTTCCGT ATTCCTGCAG
251 TACGTATACG TCAGACCGCT GGTTATGCTG TTTCCGATCT AAGGTATCAA
301 GGCCACGAGG TGAGAGATGG TAAGCCAGGA TTACCTGGTC TGCCAGCAAC
351 GTTCGGTGAG GCTGGTGACG TTACTACCTT GGTCGTACAT CTTTACGATA
401 ATCACTCCGC AGTTGCCGCT GACTTGTCAT ATAGTGTGTT TCCTGAATTC
451 GATGCTGTGG TAAGGAGTGT TAACATCACG AACAAGGGAA ACGGCAACAT
501 TACCATTGAG CACCTGGCTA GTATGTCTGT CGACTTTCCT TTCGAGGATC
551 TTGATCTTCT TGGATTGCGT GGAGACTGGG CAAGAGAGGC ACATAGAATG
601 CGTAGAAGAG TCGAGTACGG CGTGCAGGGA TTCGGTTCTA GTACTGGATA
651 TAGTTCTCAT CTGCACAACC CTTTTTTTGT ACTTGCACAT CCAAGTACAA
701 CCGAAAGTCA AGGCGAGGCC TGGGGCTTCA ACTTGACTTA CACTGGAAGT
751 TTCAGTGCCC AGGTAGAGAA AGGCTCTCAA GGACTTACGC GTGCCCTTAT
801 CGGATTCAAC CCAGACCAAC TTAGTTGGAC TCTGGGACCA GGCGAGACGC
851 TGACCTCCCC TGAATGCGTC AGTGTGTATT CCTCTGACGG CATTGGTGGA
901 ATGAGTCGTA AGTTCCACAG GCTATATAGG AAACATTTAA TACGTTCAAA
951 ATACGCTACG TTGGACAGAC CACCTCTTTT GAATTCTTGG GAAGGTGTGT
1001 ACTTCGATTA CAATCAGACA GGAATCGAAC GTCTAGCCCG TCAAAGTGCC
1051 GCTCTTGGCA TTCGTCTATT CGTCATGGAT GACGGCTGGT TCGGCAATAA
1101 ATACCCCCGT ACCTCTGATA AAGCCGGATT AGGAGATTGG ACTCCCAACC
1151 CCGACCGTTT TCCTGACGGC TTAGAACCCG TGGTGGAGAG GATCACGAAC
1201 CTACCAGTCA ACGGTACTGC CGGAGAGAAA CTGAGATTTG GCATTTGGGT
1251 AGAACCAGAA ATGGTTAATC CCAACTCTTC CCTGTATAGG GAGCACCCCG
1301 ATTGGGTACT TCACGCCGGA TCCTATCCCC GTACCGAAAG GAGGAACCAG
1351 CTGGTACTTA ATCTGGCACT TCCTGAAGTT CAGGACTTCA TTATTGATTT
1401 TATGACGAAC CTTTTGAATT CAGCAGACAT CTCTTATGTA AAGTGGGATA
1451 ACAACAGGGG TATGCATGAA ATGCCATCAA CCAGGACATA CCACGAATAC
1501 ATGTTGGGCC TATACCGTGT TCTAGATACG TTATCCGCAA GATTCCCAGA
1551 TGTCTTGTGG GAGGGATGCG CCTCTGGAGG AGGTAGGTTT GACGCAGGCA
1601 TTCTACATTA TTTTCCCCAA ATATGGACGT CCGATAATAC TGATGGCGTG
1651 GATCGTATAA CGATCCAGTT TGGAACGAGT CTAGCTTACC CCCCATCCAC
1701 AATGGGAGCC CACCTATCCG CCGTGCCTAA CCATCAGACT TCCAGAACCG
1751 TCCCCTTGGA ATTCAGGGCT CACGTCGCCA TGATGGGAGG TTCTTTTGGA
1801 TTAGAACTAG ATCCAGCTAC TTTGCAAGAC GATCCAGAGG TACGTAGGCT
1851 GATCAAGTTA GCCGAGAAAG TTAATCCATT AGTCATAAAC GGAGATTTAT
1901 ATAGGCTAAG ATTACCTGAG GAGAGTCAGT GGCCAGCAGC CCTGTTCGTA
1951 GCAGAGGACG GCTCCCAGGC CGTACTGTTC TATTTCCAAG TTGGACCCAA
2001 TGTAAATCAT GCCGCCCCAT GGGTCCGTCT ACAGGGCCTG GACCCTGAGG
2051 CTAGATATAC CGTCGACGGT AATGCTACAT ATAAAGGTGC AACACTTATG
2101 AACCTTGGCT TACAATTCAC CTTCGACAGT GAGTATGGTA GTAAGGTCGT
2151 CTTTTTGGAG AAACAATAA
The invention also provides a recombinant vector containing the gene.
The invention also provides a recombinant strain containing the gene.
The method for preparing alpha-galactosidase according to the present invention comprises the steps of:
constructing a recombinant vector containing a gene encoding the above-mentioned alpha-galactosidase;
introducing the recombinant vector into a host cell;
the alpha-galactosidase is expressed inducibly and isolated.
The alpha-galactosidase has high activity, is derived from paecilomyces thermophilus, and has good heat resistance. The pichia pastoris expression system is adopted to carry out the secretory expression of the alpha-galactosidase, so that the high-efficiency expression of the alpha-galactosidase is realized, and the production cost is reduced. The highest expression level of alpha-galactosidase of a pichia pastoris expression system used in the invention reaches 10g/L, and the enzyme activity of fermentation liquor reaches 10000U/mL, which is the highest level of public reports. And the alpha-galactosidase is secreted and expressed in the fermentation supernatant, and the crude alpha-galactosidase enzyme solution with high purity can be obtained by simply filtering and removing thalli without breaking cells, so that the cost of separating and purifying downstream protein is reduced.
Drawings
FIG. 1 shows the results of colony PCR electrophoresis verification of Top 10/pPICz. Alpha.A-AGA, wherein M: a standard molecular weight; 1: a positive control; 2: negative control, top10 strain; 3-5: top 10/pPICz. Alpha.A-AGA transformants);
FIG. 2 shows the alpha-galactosidase fermentation curve;
fig. 3 shows the results of electrophoresis of α -galactosidase protein, wherein M: a standard molecular weight; AGA: fermenting supernate with alpha-galactosidase, and carrying out deglycosylation treatment;
FIG. 4 shows the results of the α -galactosidase thermostability assay, in which, untreated: AGA samples were not heat treated, AGA: AGA samples were heat treated at 70 ℃ for 5min, aspergillus niger: carrying out heat treatment on an Aspergillus niger gene alpha-galactosidase sample (pichia pastoris expression) at 70 ℃ for 5min; aspergillus oryzae: carrying out heat treatment on an Aspergillus oryzae gene alpha-galactosidase sample (pichia pastoris expression) at 70 ℃ for 5min;
FIG. 5 shows the optimum catalytic temperature for the alpha-galactosidase of the invention;
FIG. 6 shows the optimum pH of the alpha-galactosidase of the present invention.
Detailed Description
The experimental procedures without specifying the conditions in the following examples were carried out under the conditions described in molecular Cloning, A Laboratory Manual (2002), or according to the kit and instructions. DNA polymerase, restriction enzyme, DNA ligase were purchased from New England Biolabs, and Pichia pastoris X33 and its expression vector were purchased from Invitrogen. Other materials are commercially available unless otherwise specified.
EXAMPLE 1 obtaining of galactosidase Gene
Amino acid sequence information of alpha-galactosidase from paecilomyces thermophilus, as shown in SEQ ID NO:1 is shown. Carrying out codon optimization on alpha-galactosidase according to codon preference of Pichia pastoris (Pichia pastoris), removing a signal peptide sequence, and adding a termination codon TAA at the tail end of a gene sequence to obtain a mature alpha-galactosidase gene sequence, wherein the gene sequence is shown as SEQ ID NO:2, respectively. The gene sequence was synthesized by Shanghai Bioengineering Co., ltd.
EXAMPLE 2 construction of alpha-galactosidase expressing strains
Using the synthetic gene as a template, amplifying an alpha-galactosidase gene by Polymerase Chain Reaction (PCR), adding an EcoRI enzyme digestion site (GAATTC) at the 5 'end of an upstream primer, adding a SalI enzyme digestion site (GTCGAC) at the 5' end of a downstream primer, amplifying alpha-galactosidase mature genes containing enzyme digestion sites at two ends, wherein the primer information is as follows, and the underlining is the enzyme digestion sites:
an upstream primer: 5' -GCTGAATTCCAGAGTACATCAGGAAGTAATGCAA-3’(SEQ ID NO：3)
A downstream primer: 5' -ATGGTCGACTTATTGTTTCTCCAAAAAGACGACC-3’(SEQ ID NO：4)
Amplifying a target gene band of about 2.1kb of alpha-galactosidase, separating a target gene by agarose gel electrophoresis, purifying and recovering the target gene according to the instruction of a gene recovery kit, carrying out double digestion on the alpha-galactosidase gene by restriction enzymes EcoRI and SalI, carrying out enzyme digestion for 2h in an incubator at 37 ℃, separating the target gene by agarose gel electrophoresis, and purifying and recovering the enzyme digested alpha-galactosidase gene according to the instruction of the gene recovery kit. The enzyme-cut alpha-galactosidase gene and a pichia pastoris expression vector pPICz alpha A which is also subjected to double enzyme cutting by restriction enzymes EcoRI and SalI are subjected to ligation reaction, and T4 DNA ligase is subjected to overnight reaction at 16 ℃. The ligation products were transformed into E.coli Top10 competent cells by heat shock method, spread on LB plates containing 100. Mu.g/mL bleomycin resistance, and cultured overnight at 37 ℃ until single colonies grew. Colony PCR was performed using universal primers (5 AOX and 3 AOX) of pPICz. Alpha.A vector to verify positive transformants, the amplified products were verified by agarose gel electrophoresis, the band of interest was about 2.7kb, and the verification results are shown in FIG. 1.
5AOX：5’-GACTGGTTCCAATTGACAAGC-3’(SEQ ID NO.5)
3AOX：5’-GCAAATGGCATTCTGACATCC-3’(SEQ ID NO.6)
Sequencing the positive transformant to obtain the alpha-galactosidase Pichia pastoris expression vector pPICz alpha A-AGA. The expression of the alpha-galactosidase in the expression vector is regulated and controlled by the strongest promoter AOX1 transcription of pichia pastoris, and meanwhile, the alpha-signal peptide is contained, so that the normal secretion of protein to the outside of cells can be ensured. The expression vector is cut for 2h by restriction enzyme PmeI, the linearized expression vector is recovered by agarose gel electrophoresis, and is electrotransferred (1.5 kv-2.5 kv) to pichia pastoris competent cells, such as X33, GS115, SMD1168, KM71 and the like, and X33 is preferred. The bacterial liquid is coated on YPD plates containing bleomycin resistance and cultured for 3-5 days at 30 ℃ until single colonies grow. And (3) selecting a single colony for culture fermentation, detecting the alpha-galactosidase enzyme activity of the fermentation supernatant, and screening to obtain the optimal expression strain.
EXAMPLE 3 expression and detection of recombinant alpha-galactosidase
And (3) shake flask culture: the Pichia alpha-galactosidase expression strain constructed in example 2 was selected, inoculated into 5mL YPD medium, and cultured at 30 ℃ and 220rpm for 24 hours to prepare seed liquid. Inoculating the strain into 50mL BMGY medium (250 mL triangular flask) according to the inoculation amount of 1%, culturing at 30 ℃,220rpm for 24 hours, centrifuging at 4000Xg for 5min, collecting the thallus, suspending the thallus by 50mL BMMY medium, culturing at 30 ℃,220rpm, adding 1% methanol (final concentration) every 24 hours for induction, inducing for 3 days, centrifuging at 4000Xg for 5min, and taking the supernatant for detection.
Fermentation in a fermentation tank: the Pichia α -galactosidase expression strain constructed in example 2 was selected and inoculated into 25mLYPD medium and cultured at 30 ℃ and 220rpm for 24 hours to prepare a first seed solution. 20mL of the primary seed solution was inoculated into 200mLBMGY medium to prepare a secondary seed solution, which was cultured at 30 ℃ and 220rpm for 24 hours. The second-stage seed liquid is completely inoculated in a 5L fermentation tank (2L BSM culture medium), the temperature is controlled to be 30 +/-0.5 ℃, the dissolved oxygen is 20% +/-5%, and the pH is =5.0 +/-0.5. After basic glycerin is exhausted, 10% of glycerin is fed in, after the glycerin is exhausted, until dissolved oxygen suddenly rises to 80% or above, starving for half an hour, feeding methanol to induce alpha-galactosidase expression, inducing expression for 180-200 hours, and sampling at different time points for detection.
And (3) detecting the protein concentration: the total protein concentration of the fermentation supernatant was determined by the Bradford method. The fermentation curve of alpha-galactosidase in the fermenter is shown in FIG. 2, and the highest expression level reaches 10g/L.
Detection of alpha-galactosidase activity: the enzyme activity of alpha-galactosidase is measured by a p-nitrophenol colorimetric method. Definition of enzyme activity: the enzyme amount required for degrading and releasing 1 mu mol of p-nitrophenol from a p-nitrophenol-alpha-D-galactopyranose solution with the concentration of 5mmol/L per minute at the temperature of 37 ℃ and the pH value of 5.50 is one enzyme activity unit U.
The detection method comprises the following steps: the fermentation supernatant was diluted by an appropriate factor, and the substrate p-nitrophenol-alpha-D-galactopyranose solution (10mM, prepared in 0.1M acetic acid buffer, pH 5.50) was preheated at 37 ℃ for 10 minutes by shaking. Then 0.4mL of each solution was aspirated, mixed well, reacted at 37 ℃ for 10 minutes, added with 3.2mL of sodium carbonate solution (0.2M), shaken and mixed well, the enzyme reaction was stopped, adjusted to zero with water, and the absorbance value A was measured at 400nm E . Preheating enzyme solution 0.4mL, adding sodium carbonate solution 3.2mL, shaking, mixing, adding preheated substrate p-nitrophenol-alpha-D-galactopyranose solution 0.4mL, standing at 37 deg.C for 10 min, adjusting zero with water, and measuring absorbance A at 400nm B 。
And (3) calculating:
in the formula:
x: activity of alpha-galactosidase, U/mL;
A E : the absorbance OD value of the enzyme reaction solution;
A B : blank absorbance OD value of the enzyme reaction solution;
k: slope of the p-nitrophenol standard curve;
C 0 : intercept of p-nitrophenol standard curve;
m: amount of enzyme preparation, mL;
t: enzymolysis reaction time, min;
n: dilution factor of the sample;
the fermentation enzyme activity curve is shown in figure 2, and the maximum enzyme activity reaches 10000U/mL after 185h of fermentation.
Protein electrophoresis (SDS-PAGE): after the fermentation liquor is diluted by a proper time, the fermentation liquor is deglycosylated by endoglycosidase, and protein electrophoresis of alpha-galactosidase is carried out by adopting protein electrophoresis preformed gel. As shown in FIG. 3, a protein band of a target size is present between the standard molecular weights of 70-100kDa, which is close to the theoretical molecular weight of alpha-galactosidase and is approximately 79.9kDa. Through software analysis, the content of alpha-galactosidase in the fermentation liquor reaches over 80 percent, the content of hybrid protein is very low, and the separation and purification of the protein are facilitated.
EXAMPLE 4 evaluation of the enzymatic Properties of alpha-galactosidase
Heat resistance:
taking 0.2mL of fermentation supernatant, heating in a water bath at 70 ℃ for 5min, and observing that the fermentation liquor after heat treatment is still clear, which indicates that the alpha-galactosidase does not generate denaturation and precipitation when being heated. 10000Xg for 5min, and taking supernatant to carry out enzyme activity detection according to the method of the embodiment 3. The results are shown in fig. 4, the alpha-galactosidase of the invention can tolerate 70 ℃, the enzyme activity retention rate after heat treatment is 65%, and the heat-resistant effect is better than that of alpha-galactosidase from other sources. The novel heat-resistant alpha-galactosidase has good heat resistance, and is particularly suitable for the feed industry requiring high-temperature treatment in the granulation process.
Optimum temperature:
reaction solutions were prepared according to the method of example 3, and were reacted in water baths at 37, 55, 60, and 65 ℃ respectively, and the enzyme activity was detected, and the results are shown in fig. 5, where the optimum catalytic temperature of α -galactosidase of the present invention was 60 ℃ and the enzyme activity retention rate at 55-60 ℃ was 80% or more.
Optimum pH:
the reaction solution was prepared according to the method of example 3, pH was 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, pH4.0-5.5, respectively, and was prepared using a citric acid buffer, pH5.5-7.0 was prepared using an acetic acid buffer, and the enzyme activity was examined, and the results are shown in FIG. 6, where the optimum pH of the α -galactosidase of the present invention was 5.0, and the enzyme activity retention rate was 80% between pH 4.0-6.0. The alpha-galactosidase of the invention has higher enzyme activity under the acidic condition, is similar to the pH condition of animal intestinal tracts, and is suitable for animal husbandry and breeding industry.
Sequence listing
<110> Guangdong overflow Multi-interest Biotech Ltd
<120> heat-resistant alpha-galactosidase gene and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 748
<212> PRT
<213> Paecilomyces thermophilus (Paecilomyces thermophila)
<400> 1
Met Leu Arg Ser Ser Ala Ala Val Ala Ala Ala Val Gly Leu Leu Thr
1 5 10 15
Ala Ser Ser Gln Gly Pro Met Thr Leu Ala Gln Ser Thr Ser Gly Ser
20 25 30
Asn Ala Ile Val Val Asp Gly Thr Thr Phe Ala Leu His Gly Ala Gly
35 40 45
Met Ser Tyr Val Phe His Ala Asn Thr Thr Thr Gly Asp Leu Ile Thr
50 55 60
Asp His Tyr Gly Ala Ser Val Ser Gly Ala Leu Pro Ser Pro Pro Glu
65 70 75 80
Pro Val Val Asn Gly Trp Val Gly Met Ile Gly Arg Thr Arg Arg Glu
85 90 95
Phe Pro Asp Gln Gly Arg Gly Asp Phe Arg Ile Pro Ala Val Arg Ile
100 105 110
Arg Gln Thr Ala Gly Tyr Ala Val Ser Asp Leu Arg Tyr Gln Gly His
115 120 125
Glu Val Arg Asp Gly Lys Pro Gly Leu Pro Gly Leu Pro Ala Thr Phe
130 135 140
Gly Glu Ala Gly Asp Val Thr Thr Leu Val Val His Leu Tyr Asp Asn
145 150 155 160
His Ser Ala Val Ala Ala Asp Leu Ser Tyr Ser Val Phe Pro Glu Phe
165 170 175
Asp Ala Val Val Arg Ser Val Asn Ile Thr Asn Lys Gly Asn Gly Asn
180 185 190
Ile Thr Ile Glu His Leu Ala Ser Met Ser Val Asp Phe Pro Phe Glu
195 200 205
Asp Leu Asp Leu Leu Gly Leu Arg Gly Asp Trp Ala Arg Glu Ala His
210 215 220
Arg Met Arg Arg Arg Val Glu Tyr Gly Val Gln Gly Phe Gly Ser Ser
225 230 235 240
Thr Gly Tyr Ser Ser His Leu His Asn Pro Phe Phe Val Leu Ala His
245 250 255
Pro Ser Thr Thr Glu Ser Gln Gly Glu Ala Trp Gly Phe Asn Leu Thr
260 265 270
Tyr Thr Gly Ser Phe Ser Ala Gln Val Glu Lys Gly Ser Gln Gly Leu
275 280 285
Thr Arg Ala Leu Ile Gly Phe Asn Pro Asp Gln Leu Ser Trp Thr Leu
290 295 300
Gly Pro Gly Glu Thr Leu Thr Ser Pro Glu Cys Val Ser Val Tyr Ser
305 310 315 320
Ser Asp Gly Ile Gly Gly Met Ser Arg Lys Phe His Arg Leu Tyr Arg
325 330 335
Lys His Leu Ile Arg Ser Lys Tyr Ala Thr Leu Asp Arg Pro Pro Leu
340 345 350
Leu Asn Ser Trp Glu Gly Val Tyr Phe Asp Tyr Asn Gln Thr Gly Ile
355 360 365
Glu Arg Leu Ala Arg Gln Ser Ala Ala Leu Gly Ile Arg Leu Phe Val
370 375 380
Met Asp Asp Gly Trp Phe Gly Asn Lys Tyr Pro Arg Thr Ser Asp Lys
385 390 395 400
Ala Gly Leu Gly Asp Trp Thr Pro Asn Pro Asp Arg Phe Pro Asp Gly
405 410 415
Leu Glu Pro Val Val Glu Arg Ile Thr Asn Leu Pro Val Asn Gly Thr
420 425 430
Ala Gly Glu Lys Leu Arg Phe Gly Ile Trp Val Glu Pro Glu Met Val
435 440 445
Asn Pro Asn Ser Ser Leu Tyr Arg Glu His Pro Asp Trp Val Leu His
450 455 460
Ala Gly Ser Tyr Pro Arg Thr Glu Arg Arg Asn Gln Leu Val Leu Asn
465 470 475 480
Leu Ala Leu Pro Glu Val Gln Asp Phe Ile Ile Asp Phe Met Thr Asn
485 490 495
Leu Leu Asn Ser Ala Asp Ile Ser Tyr Val Lys Trp Asp Asn Asn Arg
500 505 510
Gly Met His Glu Met Pro Ser Thr Arg Thr Tyr His Glu Tyr Met Leu
515 520 525
Gly Leu Tyr Arg Val Leu Asp Thr Leu Ser Ala Arg Phe Pro Asp Val
530 535 540
Leu Trp Glu Gly Cys Ala Ser Gly Gly Gly Arg Phe Asp Ala Gly Ile
545 550 555 560
Leu His Tyr Phe Pro Gln Ile Trp Thr Ser Asp Asn Thr Asp Gly Val
565 570 575
Asp Arg Ile Thr Ile Gln Phe Gly Thr Ser Leu Ala Tyr Pro Pro Ser
580 585 590
Thr Met Gly Ala His Leu Ser Ala Val Pro Asn His Gln Thr Ser Arg
595 600 605
Thr Val Pro Leu Glu Phe Arg Ala His Val Ala Met Met Gly Gly Ser
610 615 620
Phe Gly Leu Glu Leu Asp Pro Ala Thr Leu Gln Asp Asp Pro Glu Val
625 630 635 640
Arg Arg Leu Ile Lys Leu Ala Glu Lys Val Asn Pro Leu Val Ile Asn
645 650 655
Gly Asp Leu Tyr Arg Leu Arg Leu Pro Glu Glu Ser Gln Trp Pro Ala
660 665 670
Ala Leu Phe Val Ala Glu Asp Gly Ser Gln Ala Val Leu Phe Tyr Phe
675 680 685
Gln Val Gly Pro Asn Val Asn His Ala Ala Pro Trp Val Arg Leu Gln
690 695 700
Gly Leu Asp Pro Glu Ala Arg Tyr Thr Val Asp Gly Asn Ala Thr Tyr
705 710 715 720
Lys Gly Ala Thr Leu Met Asn Leu Gly Leu Gln Phe Thr Phe Asp Ser
725 730 735
Glu Tyr Gly Ser Lys Val Val Phe Leu Glu Lys Gln
740 745
<210> 2
<211> 2169
<212> DNA
<213> Paecilomyces thermophilus (Artificial Sequence)
<400> 2
cagagtacat caggaagtaa tgcaatcgtc gtagatggaa cgacttttgc cttgcacggt 60
gctggaatgt catacgtttt ccacgcaaat acgactaccg gtgatctaat cactgaccat 120
tatggagcct ctgtaagtgg cgctttgcca tccccccctg agcccgttgt gaatggttgg 180
gttggcatga taggcaggac gagacgtgaa ttcccagatc aaggtcgtgg cgacttccgt 240
attcctgcag tacgtatacg tcagaccgct ggttatgctg tttccgatct aaggtatcaa 300
ggccacgagg tgagagatgg taagccagga ttacctggtc tgccagcaac gttcggtgag 360
gctggtgacg ttactacctt ggtcgtacat ctttacgata atcactccgc agttgccgct 420
gacttgtcat atagtgtgtt tcctgaattc gatgctgtgg taaggagtgt taacatcacg 480
aacaagggaa acggcaacat taccattgag cacctggcta gtatgtctgt cgactttcct 540
ttcgaggatc ttgatcttct tggattgcgt ggagactggg caagagaggc acatagaatg 600
cgtagaagag tcgagtacgg cgtgcaggga ttcggttcta gtactggata tagttctcat 660
ctgcacaacc ctttttttgt acttgcacat ccaagtacaa ccgaaagtca aggcgaggcc 720
tggggcttca acttgactta cactggaagt ttcagtgccc aggtagagaa aggctctcaa 780
ggacttacgc gtgcccttat cggattcaac ccagaccaac ttagttggac tctgggacca 840
ggcgagacgc tgacctcccc tgaatgcgtc agtgtgtatt cctctgacgg cattggtgga 900
atgagtcgta agttccacag gctatatagg aaacatttaa tacgttcaaa atacgctacg 960
ttggacagac cacctctttt gaattcttgg gaaggtgtgt acttcgatta caatcagaca 1020
ggaatcgaac gtctagcccg tcaaagtgcc gctcttggca ttcgtctatt cgtcatggat 1080
gacggctggt tcggcaataa atacccccgt acctctgata aagccggatt aggagattgg 1140
actcccaacc ccgaccgttt tcctgacggc ttagaacccg tggtggagag gatcacgaac 1200
ctaccagtca acggtactgc cggagagaaa ctgagatttg gcatttgggt agaaccagaa 1260
atggttaatc ccaactcttc cctgtatagg gagcaccccg attgggtact tcacgccgga 1320
tcctatcccc gtaccgaaag gaggaaccag ctggtactta atctggcact tcctgaagtt 1380
caggacttca ttattgattt tatgacgaac cttttgaatt cagcagacat ctcttatgta 1440
aagtgggata acaacagggg tatgcatgaa atgccatcaa ccaggacata ccacgaatac 1500
atgttgggcc tataccgtgt tctagatacg ttatccgcaa gattcccaga tgtcttgtgg 1560
gagggatgcg cctctggagg aggtaggttt gacgcaggca ttctacatta ttttccccaa 1620
atatggacgt ccgataatac tgatggcgtg gatcgtataa cgatccagtt tggaacgagt 1680
ctagcttacc ccccatccac aatgggagcc cacctatccg ccgtgcctaa ccatcagact 1740
tccagaaccg tccccttgga attcagggct cacgtcgcca tgatgggagg ttcttttgga 1800
ttagaactag atccagctac tttgcaagac gatccagagg tacgtaggct gatcaagtta 1860
gccgagaaag ttaatccatt agtcataaac ggagatttat ataggctaag attacctgag 1920
gagagtcagt ggccagcagc cctgttcgta gcagaggacg gctcccaggc cgtactgttc 1980
tatttccaag ttggacccaa tgtaaatcat gccgccccat gggtccgtct acagggcctg 2040
gaccctgagg ctagatatac cgtcgacggt aatgctacat ataaaggtgc aacacttatg 2100
aaccttggct tacaattcac cttcgacagt gagtatggta gtaaggtcgt ctttttggag 2160
aaacaataa 2169
Claims (1)
1. A method of preparing a thermostable α -galactosidase, said method comprising the steps of:
constructing a polypeptide containing a nucleotide sequence shown as SEQ ID NO: 2;
transforming pichia pastoris cells by using the plasmids to obtain an alpha-galactosidase pichia pastoris expression strain;
shake flask culture or fermentation in a fermenter, wherein,
and (3) shake flask culture: selecting constructed Pichia alpha-galactosidase expression strain, inoculating the strain in 5mL YPD culture medium, culturing at 30 ℃ and 220rpm for 24 hours to prepare seed solution, inoculating the strain in 50mL BMGY culture medium according to the inoculum size of 1%, culturing at 30 ℃ and 220rpm for 24 hours, centrifuging at 4000Xg for 5min, collecting thalli, suspending thalli on 50mL BMMY culture medium, culturing at 30 ℃ and 220rpm, adding 1% methanol every 24 hours for induction, inducing for 3 days, centrifuging at 4000Xg for 5min, taking supernatant for detection,
fermentation in a fermentation tank: selecting a constructed pichia alpha-galactosidase expression strain, inoculating the pichia alpha-galactosidase expression strain into a 25mL YPD culture medium, culturing at 30 ℃ and 220rpm for 24 hours to prepare a primary seed solution, inoculating 20mL of the primary seed solution into a 200mL BMGY culture medium to prepare a secondary seed solution, culturing at 30 ℃ and 220rpm for 24 hours, completely inoculating the secondary seed solution into a 5L fermentation tank, controlling the temperature to be 30 ℃ plus or minus 0.5 ℃, dissolving oxygen to be 20 percent plus or minus 5 percent, controlling the pH to be 5.0 plus or minus 0.5, feeding 10 percent of glycerol after basic glycerol is exhausted, feeding the glycerol until the dissolved oxygen is suddenly increased to be more than 80 percent after the glycerol is exhausted, starving for half an hour, feeding methanol to induce the alpha-galactosidase expression, inducing the expression for 180-200 hours, and sampling at different time points for detection;
isolating the alpha-galactosidase.
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| CN101074435A (en) * | 2006-05-16 | 2007-11-21 | 中国农业科学院饲料研究所 | Alpha-galactosidase gene, its coding protein, production and use |
| CN101544985A (en) * | 2009-01-22 | 2009-09-30 | 北京德宝群兴科技有限公司 | Optimized sequence of alpha-galactosidase gene and efficient expression strain |
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| CN108841740A (en) * | 2018-07-10 | 2018-11-20 | 青岛蔚蓝生物集团有限公司 | A kind of Pichi strain of high yield alpha-galactosidase |
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| CN101074435A (en) * | 2006-05-16 | 2007-11-21 | 中国农业科学院饲料研究所 | Alpha-galactosidase gene, its coding protein, production and use |
| CN101544985A (en) * | 2009-01-22 | 2009-09-30 | 北京德宝群兴科技有限公司 | Optimized sequence of alpha-galactosidase gene and efficient expression strain |
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| CN108841740A (en) * | 2018-07-10 | 2018-11-20 | 青岛蔚蓝生物集团有限公司 | A kind of Pichi strain of high yield alpha-galactosidase |
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