Alkaline amylase mutant and its application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to an alkaline amylase mutant and its application. The present invention mutates the amino acids M, V, Y, V, V, Y and D at positions 57, 152, 203, 223, 294, 345 and 392 in the amino acid sequence of alkaline amylase from alkaliphilic Bacillus to I, I, D, I, G, C and N, respectively, to obtain an alkaline amylase mutant, wherein the mutant has the characteristics of alkali resistance and heat resistance, and at the same time, a recombinant expression vector containing the mutant gene is transferred into Bacillus amyloliquefaciens ZD-708 (△GAΔEGL) for expression, and the mutant is expressed at 60 m 3 The average enzyme activity level in the fermentation tank reached 31243U / mL, which can further reduce the production cost of alkaline amylase and promote the widespread application of alkaline amylase in the textile and washing industries.

Description

Technical field:

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to an alkaline amylase mutant and a coding gene thereof, and a preparation method and application thereof. Background technology:

[0002] α-Amylase (EC3.2.1.1) catalyzes the hydrolysis of α-1,4-glycosidic bonds in molecules such as starch and glycogen to produce products such as dextrin, maltooligosaccharide, maltose and glucose. Under alkaline conditions, α-amylase that can efficiently complete the above catalytic reaction is alkaline amylase, which is generally produced by alkaliphilic microorganisms and has a stable pH range of 9.0 to 11.0. The molecular weight of alkaline amylase is mostly concentrated in the range of 53kDa to 66kDa, and a few have a larger molecular weight, reaching 85kDa to 110kDa. Among the alkaline amylases reported, the largest molecular weight is 159kDa.

[0003] Alkaline amylase is widely used in textile desizing, detergent addition, starch processing, pharmaceutical production and other fields. The stable temperature of alkaline amylase reported so far is mostly below 60°C, which is difficult to meet the process requirements. At the same time, since the washing temperature of automatic dishwashers is generally 55°C to 60°C, this requires that alkaline amylase used in the detergent industry must have good thermal stability at around 60°C.

[0004] Alkaline amylase is mainly derived from bacteria and some actinomycetes. The main source of bacterial alkaline amylase genes is Bacillus, such as alkaliphilic Bacillus, Bacillus licheniformis, and Bacillus subtilis; there are relatively few alkaline amylase genes from actinomycetes, such as Streptomyces. At present, the methods for producing alkaline amylase at home and abroad are mostly focused on the screening of alkaline-philic microorganisms producing alkaline amylase, the screening of high-yield mutant strains, the construction of enzyme-producing recombinant microorganisms and the optimization of fermentation conditions, antioxidant and thermal stability modification, etc. For the research team of Japanese scholar Igarashi, who has conducted in-depth research on the thermal stability of alkaline amylase, Arg 124 located in a loop of residues 122-134 in the alkaline amylase domain B was replaced with Pro, and the overall structure of the enzyme was made more compact by reducing the flexibility of the loop. The half-life of the mutant enzyme at 50°C was increased from 4.4min of the wild type to 11.3min. Deng Zhuangmei et al. improved the thermal stability of AmyK based on rational design and computer-aided design, and obtained a mutant H209L / Q226V / P477V with the most significant thermal stability improvement. The half-life of this mutant at 60°C increased by 2.8 times, the optimal reaction temperature increased from 50°C to 55°C, the optimal reaction pH increased from 9.5 to 10.0, the stable pH increased from 8.0-11.0 to 7.0-11.0, and the catalytic efficiency increased from 1.8×10 4 L / g / min increased to 3.5×10 4L / g / min. Ma Yingfang induced and screened the recombinant B.subtilis strain producing alkaline amylase by atmospheric pressure room temperature plasma (ARTP), and directed the modification of the B.subtilis signal peptide and promoter suitable for secretory expression of alkaline amylase, and finally obtained the mutant B.subtilis PM2 with an alkaline amylase activity of 1361.0U / mL. Wang Jingli et al. expressed the alkaline amylase gene amyK from A.amylolytica in B.subtilis WB600. After signal peptide screening and optimization of fermentation conditions for recombinant bacteria enzyme production, the alkaline amylase production reached 640.3U / mL after 72h of fermentation; further optimization was performed in a 3L fermenter, and the final extracellular enzyme activity reached 809.0U / mL. Wang et al. achieved heterologous expression of the amyA gene from A.amylolytica in E.coli DH5α, and the extracellular recombinant enzyme activity was 117.0U / mL. Baysal et al. optimized the solid-state fermentation conditions and culture medium of Bacillus sp., and the alkaline amylase activity reached 216000U / g. Huang Wenjing constructed recombinant bacteria based on B.licheniformis B5102 and its alkaline protease gene, obtained recombinant bacteria with significantly improved alkaline protease production or significantly shortened fermentation cycle, and established a corresponding 25L fermentation tank fermentation process for the recombinant bacteria. In a 25L fermentation tank, the highest enzyme activity of B.licheniformis B5102 / pLY-apr fermentation reached 26500U / mL, which was 80% higher than before optimization, and the fermentation time was 104h. Amory et al. mutated Asn at positions 42, 114, and 115 in the alkaline protease Subtilisin309 to Arg, and the mutant's catalytic activity at temperatures between 46 and 58°C and pH 10 and 11 was improved, and the storage time was extended. The low enzyme activity and yield of alkaline amylase limit its application in many industrial fields. Summary of the invention:

[0005] The main purpose of the present invention is to provide an alkaline amylase mutant and its gene, a recombinant expression vector and a recombinant strain containing the gene, and a method for preparing alkaline amylase by the recombinant bacteria, aiming to solve the problems of poor alkali resistance and high temperature resistance of alkaline amylase in the prior art and low fermentation enzyme activity.

[0006] To achieve the above object, the present invention obtains an alkaline amylase mutant by mutating methionine, valine, tyrosine, valine, valine, tyrosine and aspartic acid at positions 57, 152, 203, 223, 294, 345 and 392 in the amino acid sequence of alkaline amylase derived from Bacillus alkaliphilus Ap180 (Bacillus alcalophilus) CGMCC No.7545 to isoleucine, isoleucine, aspartic acid, isoleucine, glycine, cysteine ​​and asparagine, respectively. The mutant has the amino acid sequence shown in SEQ ID NO.2 in the sequence table.

[0007] The present invention also provides a gene encoding the mutant;

[0008] Furthermore, the coding gene of the mutant has the nucleotide sequence described in SEQ ID NO.4 in the sequence listing.

[0009] The present application also provides a recombinant expression vector, comprising the above-mentioned alkaline amylase mutant encoding gene;

[0010] Preferably, the expression plasmid used in the recombinant expression vector is pKS1;

[0011] The present application also provides a recombinant strain, comprising the above-mentioned alkaline amylase mutant encoding gene or recombinant expression vector;

[0012] Preferably, the host cell of the recombinant strain is Bacillus amyloliquefaciens ZD-708 (ΔGAΔEGL) (the host bacteria has been disclosed in Chinese invention patent CN 201911119213.5);

[0013] The present application also provides the application of the above-mentioned recombinant expression vector or recombinant strain, especially in the preparation of alkaline amylase;

[0014] Furthermore, the method for preparing alkaline amylase using the above recombinant strain is as follows:

[0015] At 60m 3 Culture in fermenter: Inoculate 2-4% of the volume of the fermenter medium, culture temperature 34-37°C, tank pressure 0.04-0.08MPa, initial speed 100rpm, initial air volume 200m 3 / h, the dissolved oxygen is controlled to be greater than 10% by increasing the speed and ventilation volume, the maximum speed is 600rpm, and the maximum ventilation volume is 4000m 3 / h; after about 6 hours of fermentation, 40% maltose syrup was added to maintain the reducing sugar content in the culture medium at 12-15g / L; during the entire fermentation stage, the pH was controlled at 7.0-7.2 by adding ammonia, and the fermentation was terminated when the autolysis of the bacteria was serious. The entire fermentation cycle was 100-110h.

[0016] Furthermore, in 3m 3 The culture conditions in the seed tank are: temperature 34-37℃, tank pressure 0.04-0.08Mpa, initial speed 100rpm, initial air volume 20m 3 / h, the dissolved oxygen is controlled to be greater than 10% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, ammonia was added to control pH at 7.0-7.2, and culture was carried out for 40h;

[0017] Further, the seed culture medium is composed of: tryptone 10-15g / L, yeast extract powder 2-5g / L, sodium chloride 0.2-0.5g / L, potassium chloride 0.10-0.12g / L, calcium chloride 0.08-0.15g / L, magnesium sulfate 1.5-2.2g / L, glucose 2.5-3.6g / L, skim milk powder 1.0-1.7g / L, potassium dihydrogen phosphate 1.0-1.2g / L, disodium hydrogen phosphate 0.5-0.96g / L, pH 7.0-7.2;

[0018] Furthermore, the fermentation medium is composed of: 2-5 g / L corn starch, 1-3 g / L fish peptone, 2-4 g / L soybean cake powder, 0.2-0.6 g / L yeast powder, 1-1.5 g / L corn steep liquor, 3.5-4.0 g / L dipotassium hydrogen phosphate, 1.2-1.4 g / L potassium dihydrogen phosphate, and pH is 7.0-7.2.

[0019] The present invention also provides the application of the alkaline amylase mutant, especially in the textile and washing industries.

[0020] Beneficial effects:

[0021] The wild-type alkaline amylase derived from Bacillus alcalophilus Ap180 CGMCC No.7545 has a pH range of pH7-8.5, an optimal reaction pH of 7.5, and can retain more than 80% of the enzyme activity after being stored at a temperature below 55°C for 90 minutes. The optimal reaction temperature is 50°C.

[0022] The alkaline amylase mutant provided by the present invention has a pH range of pH 8-10, an optimal reaction pH of 9.5, can retain more than 80% of the enzyme activity after being stored at a temperature below 65° C. for 90 minutes, and the optimal reaction temperature is 60° C. Its enzymatic properties are particularly suitable for deep processing in the textile and washing industries.

[0023] The alkaline amylase mutant provided by the present invention is integrated into the genome of the host bacteria for expression, thereby preventing the mutant gene from being lost in the host bacteria and enabling the mutant gene to be stably expressed in the host bacteria at 60 m3 The average fermentation enzyme activity in the fermentation tank is 31243U / mL, which can further reduce the production cost of alkaline amylase, promote the widespread application of alkaline amylase in the textile and washing industries, and actively respond to the dilemma of rising raw material prices caused by the new crown epidemic.

[0024] In addition, the present invention confirms seven key alkali-resistant and heat-resistant amino acid sites in alkaline amylase, and proves the importance of these sites to the alkali-resistant and heat-resistant properties of the enzyme, providing important clues for studying the alkali-resistant and heat-resistant mechanism of alkaline amylase, and providing a reliable reference for the alkali-resistant and heat-resistant properties of other alkaline amylases. Description of the drawings:

[0025] Figure 1 PCR verification of the recombinant strain ZD-708(△GAΔEGL)-AMYM;

[0026] Figure 2 Comparison of the optimal reaction pH curves of alkaline amylase before and after mutation;

[0027] Figure 3 Comparison of pH stability range curves before and after mutation of alkaline amylase;

[0028] Figure 4 Comparison of the optimal reaction temperature curves of alkaline amylase before and after mutation;

[0029] Figure 5 Comparison of temperature stability range curves of alkaline amylase before and after mutation. Specific implementation method:

[0030] The present invention is described in detail below through specific implementation cases. The specific implementation cases are only for illustration and are not intended to limit the scope of implementation of the present invention.

[0031] According to the technical solution of the present invention, the amino acid sequence of the wild-type alkaline amylase derived from Bacillus alcalophilus Ap180 CGMCC No.7545 is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.3.

[0032] The alkali-resistant and heat-resistant alkaline amylase mutant of the present invention is obtained by subjecting the amino acid sequence (SEQ ID NO.1) of the wild-type alkaline amylase to point mutations M57I, V152I, Y203D, V223I, V294G, Y345C, and D392N, that is, the 57th methionine (M), the 152nd valine (V), and the 223rd valine (V) are all mutated to isoleucine (I), the 203rd tyrosine (Y) is mutated to aspartic acid (D), the 294th valine (V) is mutated to glycine (G), the 345th tyrosine (Y) is mutated to cysteine ​​(C), and the 392nd aspartic acid (D) site is mutated to asparagine (N), and the mutant of the present invention is obtained, and its amino acid sequence is shown in SEQ ID NO.2, and the gene sequence is shown in SEQ ID NO.4. The specific sequence is as follows:

[0033] SEQ ID No.1:

[0034] MKLFWLLFTIGFCWAQYSSNTQQGRTSIVHLFEWRWVDIASYKLCTRSGNEDEFRNMVTR

[0035] CNNVGVRIYVDAVINHMCGNLECERYLAPKGFGGVQVSPPFPAVPYSAVSAGTSSTCGSYF

[0036] NPGSRDGWDFNDGKCKTGSGDIENYNDATQVRDCRLLSGLAGTYCDVISGDKINDIKAILDK

[0037] LHNLNSNWFPEGLDLALGKDYVRSKIAEYMNHSKPFIYQEVIDLGLIDIGVAGFRIDASKHM

[0038] WPGFMPSDRALVFVDNHDNQRGHGEPIKSSDYFGNGRVTEFKYGAKLGTVGFMLAHPYG

[0039] FTRGAGGASILTFWdarLYKMAVVMSSYRWPIRKWNGEKMSYLKNWGEGWGFIVFNND

[0040] DWTFSTLQTGLPHRWRQIRRYFENGKDVNDWVGPPNDNGNMVNFRNVVDGQPFTNWY

[0041] DNGSNQVAFGRGNRGVTKEVTINPDTTCGNDWVCEGNCTGIKIYVSDDGKAHFSISNSAED

[0042] PFIAIHAESKL

[0043] SEQ ID No.2:

[0044] MKLFWLLFTIGFCWAQYSSNTQQGRTSIVHLFEWRWVDIASYKLCTRSGNEDEFRNIVTRC

[0045] NNVGVRIYVDAVINHMCGNLECERYLAPKGFGGVQVSPPFPAVPYSAVSAGTSSTCGSYFN

[0046] PGSRDGWDFNDGKCKTGSGDIENYNDATQIRDCRLSGLAGTYCDVISGDKINDIKAILDKL

[0047] HNLNSNWFPEGLDLALGKDDVRSKIAEYMNHSKPFIYQEIIDLGLIDIGVAGFRIDASKHMW

[0048] PGFMPSDRALVFVDNHDNQRGHGEPIKSSDYFGNGRVTEFKYGAKLGTGGFMLAHPYGFT

[0049] RGAGGASILTFWDARLYKMAVVMSSYRWPIRKWNGEKMSCLKNWGEGWGFIVFNNDDW

[0050] TFSLTLQTGLPHRWRQIRRYFENGKDVNNWVGPPNDNGNMVNFRNVVDGQPFTNWYDN

[0051] GSNQVAFGRGNRGVTKEVTINPDTTCGNDWVCEGNCTGIKIYVSDDGKAHFSISNSAEDPFI

[0052] AIHAESKL

[0053] SEQ ID No.3:

[0054] ATGAAGCTCTTTTGGTTGCTTTTCACCATTGGGTTCTGCTGGGCTCAGTATTCCTCAAATACACAACAA

[0055] GGACGAACATCTATTGTTCATCTGTTTGAATGGCGATGGGTTGATATTGCTAGCTATAAATTATGCACAA

[0056] GATTCGGAAATGAAGATGAATTTAGAAACATGGTGACTAGATGCAACAATGTTGGGGTTCGTATTTATG

[0057] TGGATGCTGTAATTAATCATATGTGTGGTAATCTTGAATGTGAGCGATATTTAGCTCCCAAGGGATTTGG

[0058] AGGGGTTCAGGTCTCTCCACCATTTCCAGCAGTCCCATATTCTGCTGTGAGTGCAGGAACAAGCAGTA

[0059] CCTGTGGAAGTTACTTCAACCCTGGAAGTAGGGACGGATGGGATTTTAATGATGGTAAATGTAAAACT

[0060] GGAAGTGGAGATATCGAGAACTATAATGATGCTACTCAGGTCAGAGATTGTCGTGTCTGGTCTTGC

[0061] TGGCACATACTGTGATGTCATTTCTGGAGATAAAATTAATGACATAAAGGCAATTTTGGACAAACTGCA

[0062] TAATCTAAACAGTAACTGGTTCCCGGAAGGTCTCGATCTTGCACTGGGGAAGGATTATGTGCGTTCTA

[0063] AGATTGCCGAATATATGAACCATAGTAAACCTTTCATTTACCAGGAGGTAATTGATCTGGGTCTCATTG

[0064] ACATTGGTGTTGCAGGGTTCAGAATTGATGCTTCCAAGCACATGTGGCCTGGATTCATGCCTTCTGAC

[0065] AGAGCGCTTGTCTTTGTGGATAACCATGACAATCAACGAGGACATGGTGAGCCAATTAAAAGCAGTG

[0066] ACTACTTTGGTAATGGCCGGGTGACAGAATTCAAGTATGGTGCAAAACTCGGCACAGTTGGATTTATG

[0067] CTTGCTCATCCTTATGGATTTACACGAGGGCGCTGGAGGAGCCTCTATACTTACCTTCTGGGATGCTAGG

[0068] CTGTACAAAATGGCAGTTGTAATGTCAAGCTACCGTTGGCCAATTCGCAAGTGGAATGGAGAGAAGA

[0069] TGTCTTACTTAAAGAACTGGGGAGAAGGTTGGGGTTTCATTGTTTTCAACAATGATGACTGGACATTT

[0070] TCTTTAACTTTGCAAACTGGTCTTCCTCATCGATGGCGCCAAATAAGGAGATATTTTGAAATGGAAA

[0071] AGATGTTAATGATTGGGTTGGGCCACCAAATGATAATGGAAACATGGTTAATTTCCGCAATGTAGTGGA

[0072] TGGCCAGCCTTTTACAAACTGGTATGATAATGGGAGCAACCAAGTGGCTTTTGGGAGAGGAAACAGA

[0073] GGAGTAACTAAAGAAGTTACTATTAATCCAGACACTACTTGTGGCAATGACTGGGTCTGTGAAGGCAA

[0074] CTGCACAGGCATTAAAATCTACGTTTCTGATGATGGCAAAGCTCATTTTTCTATTAGTAACTCTGCTGA

[0075] AGATCCATTTATTGCAATTCATGCTGAATCTAAATTGTAA

[0076] SEQ ID No.4:

[0077] ATGAAGCTCTTTTGGTTGCTTTTCACCATTGGGTTCTGCTGGGCTCAGTATTCCTCAAATACACAACAA

[0078] GGACGAACATCTATTGTTCATCTGTTTGAATGGCGATGGGTTGATATTGCTAGCTATAAATTATGCACAA

[0079] GATCTGGAAATGAAGATGAATTTAGAAACATTGTGACTAGATGCAACAATGTTGGGGTTCGTATTTATG

[0080] TGGATGCTGTAATTAATCATATGTGTGGTAATCTTGAATGTGAGCGATATTTAGCTCCCAAGGGATTTGG

[0081] AGGGGTTCAGGTCTCTCCACCATTTCCAGCAGTCCCATATTCTGCTGTGAGTGCAGGAACAAGCAGTA

[0082] CCTGTGGAAGTTACTTCAACCCTGGAAGTAGGGACGGATGGGATTTTAATGATGGTAAATGTAAAACT

[0083] GGAAGTGGAGATATCGAGAACTATAATGATGCTACTCAGATCAGAGATTGTCGTCTGTCTGGTCTTGCT

[0084] GGCACATACTGTGATGTCATTTCTGGAGATAAAATTAATGACATAAAGGCAATTTTGGACAAACTGCAT

[0085] AATCTAAACAGTAACTGGTTCCCGGAAGGTCTCGATCTTGCACTGGGGAAGGATGATGTGCGTTCTAA

[0086] GATTGCCGAATATATGAACCATAGTAAACCTTTCATTTACCAGGAGATAATTGATCTGGGTCTCATTGAC

[0087] ATTGGTGTTGCAGGGTTCAGAATTGATGCTTCCAAGCACATGTGGCCTGGATTCATGCCTTCTGACAG

[0088] AGCGCTTGTCTTTGTGGATAACCATGACAATCAACGAGGACATGGTGAGCCAATTAAAAGCAGTGACT

[0089] ACTTTGGTAATGGCCGGGTGACAGAATTCAAGTATGGTGCAAAACTCGGCACAGGTGGATTTATGCTT

[0090] GCTCATCCTTATGGATTTACACGAGGCGCTGGAGGAGCCTCTATACTTACCTTCTGGGATGCTAGGCTG

[0091] TACAAAATGGCAGTTGTAATGTCAAGCTACCGTTGGCCAATTCGCAAGTGGAATGGAGAGAAGATGT

[0092] CTTGCTTAAAGAACTGGGGAGAAGGTTGGGGTTTCATTGTTTTCAACAATGATGACTGGACATTTTCT

[0093] TTAACTTTGCAAACTGGTCTTCCTCATCGATGGCGCCAAATAAGGAGATATTTTGAAAATGGAAAAGA

[0094] TGTTAATAATTGGGTTGGGCCACCAAATGATAATGGAAACATGGTTAATTTCCGCAATGTAGTGGATGG

[0095] CCAGCCTTTTACAAACTGGTATGATAATGGGAGCAACCAAGTGGCTTTTGGGAGAGGAAACAGAGGA

[0096] GTAACTAAAGAAGTTACTATTAATCCAGACACTACTTGTGGCAATGACTGGGTCTGTGAAGGCAACTG

[0097] CACAGGCATTAAAATCTACGTTTCTGATGATGGCAAAGCTCATTTTTCTATTAGTAACTCTGCTGAAGA

[0098] TCCATTTATTGCAATTCATGCTGAATCTAAATTGTAA

[0099] Identification of the alkaline amylase mutant used in the present invention:

[0100] The mutated amino acid in the mutant is represented by "amino acid replaced by the original amino acid position". For example, M57I means that the amino acid at position 57 is replaced by Ile from the wild-type Met, and the position number corresponds to the amino acid sequence number of the wild-type alkaline amylase in SEQ ID No. 1, as shown in the following table.

[0101]

[0102] The present invention will be further explained below in conjunction with specific implementation modes.

[0103] Example 1 Obtaining the Alkaline Amylase Mutant Gene, Recombinant Expression Vector and Recombinant Strain

[0104] 1.1 Acquisition of alkaline amylase mutant gene

[0105] The invention obtains an alkali-resistant and heat-resistant alkaline amylase mutant by mutating methionine, valine, tyrosine, valine, valine, tyrosine and aspartic acid at positions 57, 152, 203, 223, 294, 345 and 392 in the amino acid sequence of alkaline amylase derived from alkaliphilic bacillus Ap180 (Bacillus alcalophilus) CGMCC No. 7545 to isoleucine, isoleucine, aspartic acid, isoleucine, glycine, cysteine ​​and asparagine, respectively.

[0106] Take 300mL of the logarithmic seed solution of Bacillus alcalophilus Ap180 (Bacillus alcalophilus) CGMCC No.7545 in 6 50mL centrifuge tubes, centrifuge at 8000rpm for 5min, discard the supernatant, wash the precipitated bacteria twice with 600mL of physiological saline, and send them to Shanghai Biotech for genome sequencing to obtain the gene sequence and upstream and downstream sequence information. According to the obtained genome, primers Y1 (containing HindⅢ restriction site) and Y2 (containing PstⅠ restriction site) were used to amplify the alkaline amylase gene AMY, and connected to the T vector for storage, and this recombinant vector was named T-0. The site-directed mutagenesis kit used the kit of Shanghai Biotech, model B639281, and the specific operation steps are as follows:

[0107] (1) Primers for amplifying the original alkaline amylase gene and various point mutations.

[0108] Table 1 The primers used for the above point mutation are as follows

[0109]

[0110]

[0111] (2) Mutation PCR verification: Taking the point mutation of M57I as an example, use the two primers M57I-1 and M57I-2, and the recombinant T vector T-0 containing the original alkaline amylase gene AMY as a template. According to the system in the kit, add other reagents and perform PCR amplification. After the amplification, take 10 μL of the product of the above mutation PCR amplification and perform 0.8% agarose gel electrophoresis detection. After the band is correct, add 1 μL of digestion enzyme to the mutation product, mix well, and incubate at 37°C for 60 minutes.

[0112] (3) Transformation: Add 5 μL of the mutated product to 50 μL of DH5α competent cells, mix well, and place on ice for 30 min; heat shock at 42°C for 45 s and immediately cool on ice for 10 min; add 500 μL of LB medium, culture at 180 r / min, and 37°C for 1 h; then centrifuge the culture at 10,000 r / min for 5 min, discard part of the supernatant, retain 100-150 μL of the supernatant, flick to suspend the bacteria, take all the bacterial solution to plate, and culture at 37°C overnight.

[0113] (4) Verification of positive clones: Pick a single colony in 500 μL LB medium containing ampicillin resistance, culture at 200 r / min for 2-3 h, and then identify by PCR. Send the screened positive clones for sequencing. Compare the sequencing results and select the positive clones that have the mutation of M to I at position 57 in the sequence shown in nucleotide sequence SEQ ID NO.3. The positive clone is the mutant containing the recombinant plasmid with the correct mutation.

[0114] (5) Mutation of other sites: Using the mutants screened correctly as above as templates, primer pairs corresponding to the mutation sites in Table 1 were used one by one, and the same method as above was used to mutate each site, and finally the alkaline amylase mutant gene shown in SEQ ID NO.4 was screened out.

[0115] The alkaline amylase gene AMYM (SEQ ID NO. 4) obtained after the above point mutations was connected to a T vector for storage and named as T-AMYM vector.

[0116] The present invention achieves the expression of the alkaline amylase mutant by replacing the neutral protease gene APR gene on the genome of the host bacteria ZD-708 (ΔGAΔEGL) with the alkaline amylase mutant encoding gene AMYM of the present invention, as follows:

[0117] 1.2 Obtaining the recombinant expression vector

[0118] 1.2.1 Design of homology arm primers

[0119] According to the 700bp-100bp range of the upstream and downstream sequences of the neutral protease gene APR sequence in the genome sequence of strain ZD-708 (ΔGAΔEGL), primers for amplifying the upstream and downstream homology arms were designed. At the same time, KpnⅠ and HindⅢ restriction sites and protective bases were added to the upstream primer, and PstⅠ and NotⅠ restriction sites and protective bases were added to the downstream primer. The designed primers are named as follows:

[0120] Two pairs of primers APR-S1 / APR-X1 and APR-S2 / APR-X2 were used to amplify the upstream and downstream homologous arm fragments of the neutral protease gene APR. The names of the amplified upstream and downstream homologous arm fragments were APR-S (upstream homologous arm) and APR-X (downstream homologous arm), respectively. Primers YZ-5 and YZ-6 were used to verify whether the APR gene was replaced by the alkaline amylase mutant gene AMY. If the APR gene fragment could be amplified, the gene was not replaced. If the AMY gene fragment could be amplified, the gene was replaced successfully.

[0121] The specific primer sequences are shown in the following table:

[0122]

[0123] 1.2.2 Amplification of homology arms and construction of recombinant plasmid pKS1-APR-SX carrying upstream and downstream homology arms of APR gene

[0124] The above two pairs of primers were used to amplify different homology arms using the genome of strain ZD-708 (ΔGAΔEGL) extracted by the kit as a template. After the amplified homology arm fragments were verified by nucleic acid electrophoresis, the two homology arm fragments APR-S and APR-X were recovered using a gel excision recovery kit. The homology arm APR-S and plasmid pKS1 were double-digested with KpnⅠ and HindⅢ, and then connected with DNA ligase to obtain the recombinant plasmid pKS1-APR-S. The homology arm APR-X and plasmid pKS1-APR-S were double-digested with PstⅠ and NotⅠ, and then connected with DNA ligase to obtain the recombinant plasmid pKS1-APR-SX.

[0125] 1.2.3 Construction of recombinant expression plasmid pKS1-APR-S-AMYM-X

[0126] The T-AMYM vector connected with the alkaline amylase mutant gene sequence and the recombinant plasmid pKS1-APR-SX were double-digested with HindⅢ and PstⅠ, and after the nucleic acid was recovered from the electrophoresis gel, they were connected together with DNA ligase to form the recombinant expression plasmid pKS1-APR-S-AMYM-X.

[0127] 1.3 Obtaining recombinant strains

[0128] After preparing strain ZD-708 (ΔGAΔEGL) into electrocompetent cells, the recombinant expression plasmid pKS1-APR-S-AMYM-X was electrotransformed into strain ZD-708 (ΔGAΔEGL) by electrotransformation, and then 100 μL was spread on an erythromycin (100 μg / mL) resistant LB plate, placed in a 30°C incubator, and cultured for 15 hours. Pick a single colony grown on the resistant plate, inoculate it into 50 mL of LB liquid culture medium without resistance, place it in a shaker at 37°C, 200 rpm and culture it for 15 hours, take 100 μL and spread it on an erythromycin (100 μg / mL) resistant LB plate, place it in a 37°C incubator, and culture it for 15 hours. The single colony that grew is the transformant that has undergone homologous recombination single exchange. Then transfer the single exchange transformant to LB medium without resistance, place it in a shaker at 30°C and 200rpm for 38h, take 100μL and spread it on a LB plate without resistance, place it in a 37°C incubator, and culture it for 15h. Randomly select about 100 single colonies and transfer them to LB plates with erythromycin (100μg / mL) resistance. Select single colonies that do not grow on the resistance plate for colony PCR identification. After PCR amplification with primers YZ-5 and YZ-6, if the APR gene is successfully replaced by the AMYM gene, the PCR product length is 3150bp, and if the APR gene is not successfully replaced, the PCR product length is 3530bp. The strain PCR verification electrophoresis after successful replacement is as follows Figure 1 As shown, the strain with the AMYM gene successfully integrated into the genome was named ZD-708 (ΔGAΔEGL)-AMYM.

[0129] Example 2 Preparation of alkaline amylase

[0130] The alkaline amylase mutant of the present invention was prepared by fermentation using the recombinant strain ZD-708 (ΔGAΔEGL)-AMYM constructed in Example 1, and the method is as follows:

[0131] 3m 3 Seed cultivation in seed tank: temperature 35℃, tank pressure 0.06Mpa, initial speed 100rpm, initial air volume 20m 3 / h, the dissolved oxygen is controlled to be greater than 10% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, ammonia was added to control the pH at 7.1, and the culture was carried out for 40h;

[0132] Seed culture medium: tryptone 12g / L, yeast extract powder 4g / L, sodium chloride 0.4g / L, potassium chloride 0.11g / L, calcium chloride 0.11 / L, magnesium sulfate 1.8g / L, glucose 2.9g / L, skim milk powder 1.6g / L, potassium dihydrogen phosphate 1.1g / L, disodium hydrogen phosphate 0.80g / L, pH 7.1;

[0133] 60m 3 Fermentation in the fermenter: Inoculate 2% of the volume of the fermenter medium, culture temperature 35°C, tank pressure 0.06MPa, initial speed 100rpm, initial air volume 200m 3 / h, the dissolved oxygen is controlled to be greater than 10% by increasing the speed and ventilation volume, the maximum speed is 600rpm, and the maximum ventilation volume is 4000m 3 / h; after fermentation for about 6 hours, start to add 40% maltose syrup to maintain the reducing sugar content in the culture medium at 13g / L, add ammonia to control the pH at 7.1, and end the fermentation when the bacteria autolyze seriously. The culture period is 100-110h.

[0134] The fermentation tank medium composition is: corn starch 4g / L, fish peptone 2g / L, soybean meal powder 3g / L, yeast powder 0.4g / L, corn steep liquor 1.3g / L, dipotassium hydrogen phosphate 3.8g / L, potassium dihydrogen phosphate 1.3g / L, pH is 7.1;

[0135] The results of three batches of fermentation are as follows, with an average fermentation enzyme activity of 31243 U / mL.

[0136] batch Fermentation cycle (h) Fermentation enzyme activity (U / mL) 1 105 31250 2 100 31220 3 110 31260

[0137] Example 3 Determination of alkaline amylase activity

[0138] The present invention adopts DNS method to determine the activity of alkaline amylase:

[0139] 1) Preparation of DNS reagent: Weigh 1 g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of water. Add 0.2 g of phenol, and then dissolve 0.05 g of sodium sulfite, 1 g of sodium hydroxide, and 20 g of potassium sodium tartrate. Transfer the mixture into a 100 mL volumetric flask, shake well and set to volume. Store the mixture in a brown bottle in a refrigerator at 4°C until use.

[0140] 2) Preparation of glucose standard curve: Prepare 1 mg / mL glucose solution. Add the following amount:

[0141] Sample No. Glucose solution volume / mL Distilled water volume / mL DNS solution volume / mL 1 0 0.5 1.5 2 0.1 0.4 1.5 3 0.2 0.3 1.5 4 0.3 0.2 1.5 5 0.4 0.1 1.5 6 0.5 0 1.5

[0142] Place in a boiling water bath for 10 minutes. Cool with cold water to 10 mL and measure the absorbance by A550. Use the absorbance as the horizontal axis and the glucose concentration as the vertical axis to create a standard curve: Y (glucose concentration) = 0.8172 * absorbance measured by A550 + 0.0179

[0143] 3) Add 2mL of 1% soluble starch to a test tube, add 1mL of glycine-sodium hydroxide buffer (pH9.5, 20mmol / L), mix well, preheat at 55℃ for 5min, add 1mL of diluted enzyme solution, and react for 5min. Take 0.5mL of reaction solution and mix well with 1.5mL of DNS reagent, boil in a boiling water bath for 10min, cool with cold water, and dilute to 10mL. After mixing well, use the reaction system without enzyme solution but with an equivalent amount of deionized water as a control, measure the A550 absorbance value, and obtain it from the standard curve in step 2. Different glucose concentrations are converted to calculate the enzyme activity of alkaline amylase.

[0144] Enzyme activity = (0.8172*absorbance value determined by A550+0.0179)*n(dilution factor)*4(total reaction volume)

[0145] Definition of alkaline amylase activity unit: Under certain conditions (temperature is 55°C and pH is 9.5 if not otherwise specified), 1 mL (or 1 g enzyme powder) of alkaline amylase degrades substrate (1% soluble starch) per minute to generate mg of reducing sugar (calculated as glucose), which is one enzyme activity unit, expressed as U / mL.

[0146] Example 4 Basic properties of the alkaline amylase produced

[0147] The fermentation broth supernatant prepared in batch 3 of Example 2 was used as the sample for measuring the enzyme activity of the alkaline amylase mutant of the present invention;

[0148] The fermentation supernatant of Bacillus alcalophilus Ap180 CGMCC No.7545 was used as the wild-type sample, and the seed culture medium and control conditions were consistent with those of the mutant strain; the fermentation conditions of the wild-type were: temperature 36°C, tank pressure 0.06Mpa, initial speed 100rpm, initial air volume 20m 3 / h, the dissolved oxygen is controlled to be greater than 30% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, pH was controlled at 7.5 by ammonia supplement, DE value was controlled at 11 by feed supplement, and cultured for 82h; the fermentation medium formula of wild type was: corn flour 3g / L, peptone 2g / L, soybean meal 6g / L, yeast powder 1g / L, corn steep liquor 2.3g / L, dipotassium hydrogen phosphate 3.1g / L, sodium dihydrogen phosphate 1.9g / L, pH was 7.6; the feed medium formula of wild type was: 40% liquefied starch;

[0149] Changes in enzyme activity of alkaline starch: the average fermentation enzyme activity of the wild type was 10322 U / mL, and the average fermentation enzyme activity after mutation was 31243 U / mL, and the enzyme activity increased by 2 times.

[0150] The enzymatic properties were determined as follows:

[0151] Determination of the optimal reaction pH: Glycine-sodium hydroxide buffer was used to prepare buffers of different pH values ​​(8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0). The buffer was mixed with soluble starch according to the method of step 3) of Example 3, and then amylase was added. The optimal reaction pH of alkaline amylase was determined at 55°C. Figure 2 As shown, the optimal reaction pH of the alkaline amylase mutant of the present invention is 9.5, and the optimal reaction pH of the wild-type alkaline amylase is 7.5. Compared with the wild-type, the mutant of the present invention is more suitable for alkaline conditions.

[0152] Determination of pH stability: Use glycine-sodium hydroxide buffer to prepare different gradient pH buffers (7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0). Mix the same concentration of enzyme solution (500U / mL) with buffers of different pH in test tubes, keep them in a 25°C water bath for 24 hours, and then determine the enzyme activity according to the method of Example 3. Based on the original enzyme activity of each enzyme without incubation in different pH buffers, divide all enzyme activities by the original enzyme activity and multiply by 100% to obtain the residual activity of alkaline amylase as the pH changes. Figure 3 As shown, the alkaline amylase mutant of the present invention is stable in the pH range of 8-10. Compared with the wild type, the mutant has higher enzyme activity stability under alkaline conditions.

[0153] Optimum reaction temperature: Under pH 9.5, the enzyme activity of the fermentation supernatant prepared in Batch 3 of Example 2 of the present invention was measured at 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C, respectively, and the relative enzyme activity was calculated with the highest enzyme activity as 100%. The results are shown in FIG. Figure 4 As shown, the optimum action temperature of the alkaline amylase of the present invention is 60°C, and compared with the wild type, the mutant has higher enzyme activity at high temperature.

[0154] Determination of temperature stability: The pH of the enzyme solution to be tested was adjusted to 9.5, and the residual enzyme activity was determined by the method of Example 3 after being treated at 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C for 90 min. The relative enzyme activity was calculated with the enzyme activity of the untreated sample as 100%. The results are as follows: Figure 5 As shown, the mutant can retain more than 80% of the enzyme activity after being stored at a temperature below 65°C for 90 minutes.

[0155] In summary, the alkaline amylase mutant provided by the present invention has a pH usage range of pH 8-10, an optimal reaction pH of 9.5, can retain more than 80% of the enzyme activity after being stored at a temperature below 65°C for 90 minutes, and the optimal reaction temperature is 60°C.

[0156] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. An alkaline amylase mutant, characterized in that: The amino acid sequence of the alkaline amylase mutant is shown in SEQ ID NO:

2.

2. A gene encoding the alkaline amylase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:

4.

4. A recombinant expression vector or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant expression vector according to claim 4, characterized in that The pKS1 plasmid was used as the expression vector.

6. The recombinant strain according to claim 4, characterized in that Using Bacillus amyloliquefaciens ZD-708 As a host.

7. Use of the recombinant expression vector or recombinant strain according to claim 4 in producing the alkaline amylase according to claim 1.

8. The use according to claim 7, characterized in that At 60m 3 Culture in fermenter: Inoculate 2-4% of the volume of the fermenter medium, culture temperature 34-37°C, tank pressure 0.04-0.08MPa, initial speed 100rpm, initial air volume 200m 3 / h, the dissolved oxygen is controlled to be greater than 10% by increasing the speed and ventilation volume, the maximum speed is 600rpm, and the maximum ventilation volume is 4000m 3 / h; after about 6 hours of fermentation, 40% maltose syrup was added to maintain the reducing sugar content in the culture medium at 12-15g / L; during the entire fermentation stage, the pH was controlled at 7.0-7.2 by adding ammonia, and the fermentation was terminated when the autolysis of the bacteria was serious. The whole fermentation cycle was 100-110h.

9. The use according to claim 8, characterized in that The fermentation medium consists of 2-5 g / L corn starch, 1-3 g / L fish peptone, 2-4 g / L soybean cake powder, 0.2-0.6 g / L yeast powder, 1-1.5 g / L corn syrup, 3.5-4.0 g / L dipotassium hydrogen phosphate, 1.2-1.4 g / L potassium dihydrogen phosphate, and a pH of 7.0-7.

2.

10. Use of the alkaline amylase according to claim 1 in the textile and washing industries.

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