WO2018129984A1 - 活性提高的α-淀粉酶AmyL突变体及其编码基因和应用 - Google Patents

活性提高的α-淀粉酶AmyL突变体及其编码基因和应用 Download PDF

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WO2018129984A1
WO2018129984A1 PCT/CN2017/107817 CN2017107817W WO2018129984A1 WO 2018129984 A1 WO2018129984 A1 WO 2018129984A1 CN 2017107817 W CN2017107817 W CN 2017107817W WO 2018129984 A1 WO2018129984 A1 WO 2018129984A1
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amylase
mutant
amyl
activity
bsaamy6
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French (fr)
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李阳源
黄江
王建荣
聂金梅
陈丽芝
何小梅
杨玲
黄佳乐
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广东溢多利生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

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  • the invention relates to the field of genetic engineering, in particular to an alpha-amylase AmyL mutant with enhanced activity and a gene encoding the same and application.
  • Alpha-amylase the system name is 1,4- ⁇ -D-glucan hydrolase, and is aliased as liquefied amylase, liquefaction enzyme, ⁇ -1,4-dextrinase.
  • Alpha-amylase is an endohydrolase whose main function is to catalyze the production of reduced dextrins and sugars from 1,4- ⁇ -D-glucan of starch in the fields of starch, detergents, beverages and textiles. Has an important role.
  • Bacillus salsus ⁇ -amylase AmyL is a medium-temperature amylase. Our research team has found that AmyL has potential applications in many industrial fields through a series of application evaluation experiments. In the paper industry, the ⁇ -amylase AmyL can improve the viscosity and concentration of paper-coated starch and improve the quality of paper. In the feed industry, alpha-amylase AmyL can help young animals digest and utilize starch, which is very beneficial to their growth performance and feed conversion ratio. Although the ⁇ -amylase AmyL has great application potential, the current ⁇ -amylase AmyL has low production activity and high fermentation cost. In order to make the ⁇ -amylase AmyL of Bacillus salsus widely used in many industrial fields, it is an urgent problem to improve the specific activity and expression level and reduce the production cost.
  • the object of the present invention is to carry out molecular modification of the ⁇ -amylase AmyL derived from Bacillus salsus, and the modified ⁇ -amylase has higher specific activity, lowers production cost, and meets the requirements of large industrial production.
  • a further object of the present invention is to provide a gene encoding the above ⁇ -amylase AmyL mutant.
  • a further object of the present invention is to provide a recombinant vector comprising the above-described activity-enhanced ⁇ -amylase AmyL mutant gene.
  • a further object of the present invention is to provide an ⁇ -amylase AmyL mutant gene comprising the above-described activity-enhanced ⁇ -amylase Recombinant strain.
  • the amino acid sequence of the ⁇ -amylase AmyL of Bacillus salsus is shown in SEQ ID NO.
  • the invention adopts error-prone PCR and site-directed saturation mutation to molecularly modify the ⁇ -amylase AmyL shown in SEQ ID NO. 1, and obtains high specific activity ⁇ -amylase BsaAmy6 through high-throughput screening, and the high ratio of the present invention.
  • the live ⁇ -amylase BsaAmy6 has 7 amino acid differences, and the mutation sites are +18N, +39S, +159Y, +220T, +281N, +363S.
  • +474Y is mutated to +18D, +39N, +159D, +220K, +281D, +363C, +474K.
  • the mutated amino acid sequence is shown in SEQ ID NO. 2:
  • the present invention also provides the gene sequence of the above mutant ⁇ -amylase BsaAmy6, the base sequence of which is shown in SEQ ID NO.
  • the present invention also provides a recombinant vector comprising the above-described activity-enhancing alpha-amylase, which ligates the activity-enhancing ⁇ -amylase gene BsaAmy6 of the present invention between the EcoR I and Not I restriction sites on the yeast expression vector pPICz ⁇ A.
  • the nucleotide sequence was located downstream of and regulated by the AOX1 promoter, resulting in a recombinant yeast expression plasmid pPICz ⁇ A-BsaAmy6.
  • the present invention also provides a recombinant strain comprising the above-described activity-enhancing ⁇ -amylase gene BsaAmy6, and preferably the recombinant strain is Pichia pastoris strain X33.
  • the present invention also provides a method for expressing the above-described activity-enhanced ⁇ -amylase gene BsaAmy6, comprising the steps of:
  • the recombinant expression plasmid pPICz ⁇ A-BsaAmy6 was linearized, transformed into Pichia pastoris X33, and the transformants were screened with a high concentration antibiotic plate, and the selected transformants were first subjected to comparative analysis under shake flask culture conditions.
  • the high-enzyme live transformants were selected from the shake flask culture, and then fermented in a 50-liter fermenter. During the fermentation, the fermentation broth was taken every 24 hours to measure the OD 600 and the wet weight of the cells, and the supernatant was taken for ⁇ - Amylase activity assay. At the end of the fermentation, the average average fermentation enzyme activity reached 36900 U/mL, which was 41.5% higher than that of the starting bacteria, and the high expression of the recombinant ⁇ -amylase BsaAmy6 was achieved.
  • the invention molecularly transforms the ⁇ -amylase AmyL of Bacillus salsus Bacillus salsus by combining error-prone PCR technology and high-throughput screening technology.
  • the average fermentation enzyme activity of the recombinant engineering strain containing the mutant gene BsaAmy6 in the culture condition of 50L fermenter was 36900 U/mL, and the fermentation enzyme of the mutant ⁇ -amylase BsaAmy6 Live is 41.5% better than AmyL. Therefore, the mutant ⁇ -amylase BsaAmy6 and the recombinant engineered bacteria of the present invention greatly reduce the production cost of fermentation, and exhibit great application potential in many industrial fields.
  • Figure 1 is a graph comparing enzyme activity of a yeast strain of ⁇ -amylase AmyL and its mutant BsaAmy6 in a 50 liter fermentor.
  • E. coli strain Top10 Pichia pastoris X33, vector pPICz ⁇ A, pGAPz ⁇ A, Zeocin were purchased from Invitrogen.
  • PCR enzyme plasmid extraction, gel purification, restriction endonuclease, and kit were purchased from Shanghai Shenggong Company.
  • the E. coli medium was LB, and the formula was: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0. LBZ was added to 25 ⁇ g/mL Zeocin in LB medium.
  • the yeast medium is YPD, formulated as 1% yeast extract, 2% peptone, 2% glucose.
  • the yeast screening medium was YPDZ and the formula was: YPD + 100 mg/L zeocin.
  • BMGY Yeast induction medium
  • BMGY formulated as 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V), BMMY, divided by 0.5% methanol instead of glycerol, the remaining ingredients are BMGY is the same.
  • Recombinant yeast fermentation medium This salt medium: 5% diammonium phosphate, 0.5% potassium dihydrogen phosphate, 1.5% magnesium sulfate heptahydrate, 1.95% potassium sulfate, 0.1% calcium sulfate, 0.1% potassium hydroxide, defoamer 0.03 %. After the high pressure, add 4.35 ml of PTM1 per liter.
  • PTM1 minimum salt solution: copper sulfate 0.6%, potassium iodide 0.018%, manganese sulfate monohydrate 0.3%, sodium molybdate dihydrate 0.02%, boric acid 0.002%, cobalt chloride hexahydrate 0.05%, zinc chloride 2%, seven Water iron sulfate 6.5%, concentrated sulfuric acid 0.5%, biotin 0.02%.
  • Example 1 Synthesis and cloning of ⁇ -amylase AmyL gene of Bacillus salsus
  • PCR primers were designed to contain EcoRI and NotI restriction enzyme sites at the 5' and 3' ends according to the synthesized genes, and the primer sequences were as follows:
  • 5'-end primer amyl-F1 5'-GTAGAATTCATGAGACAGGTTAGAATTGCTTTTG-3'
  • the synthetic gene was used as a template, and the above primers were used for PCR amplification, and the amplified fragment was cloned into the vector pGAPz ⁇ A to obtain a recombinant vector pGAPz ⁇ A-AMYL.
  • the first round of amplification PCR amplification using the vector promoter primers amyl-F1 and amyl-R1 as primers, the reaction system is as follows:
  • the first round of PCR product was recovered and diluted 50-100 fold to 1 ⁇ L for use as a template for the second round of PCR.
  • second round Error-prone PCR also carried out PCR reactions with specific primers amyl-F1 and amyl-R1.
  • the second round of the product was double digested with EcoRI and NotI and ligated into the pGAPz ⁇ A vector.
  • the ligation product was transformed into Pichia pastoris X33, and the mutant strain was screened on YPDZ agarose plate.
  • the mutant single colonies were picked from the error-prone PCR plate of Example 2.
  • the recombinant transformants were picked one by one with a toothpick to a 24-well plate, and 1 mL of YPD-containing medium was added to each well, and cultured at 30 ° C, 220 rpm for 48 or so, and centrifuged. clear.
  • the supernatant was taken out from 200 ⁇ L to a 96-well plate, and the ⁇ -amylase activity assay was performed.
  • the ⁇ -amylase enzyme activity assay was carried out in accordance with the National Standard of the People's Republic of China, GB/T24401-2009. Eight positive mutant clones with increased enzyme activity were extracted, and genomic DNA was extracted one by one, and the target gene was amplified by PCR to determine the mutation site.
  • the amino acid mutation site was determined by sequencing.
  • the mutation site of clone 1 was replaced by +18N with +18D;
  • the mutation site of clone 2 was replaced by +39N with +39N;
  • the mutation point of clone 3 was replaced by +141G with +141K;
  • the mutation point of 4 is +159Y replaced by +159D;
  • the mutation point of clone 5 is +220T replaced by +220K;
  • the mutation point of clone 6 is +363S replaced by +363C;
  • the mutation point of clone 7 is +474Y replaced by +474K .
  • the high specific activity ⁇ -amylase BsaAmy6 of the present invention has 7 amino acid differences, and the mutation sites are +18N, +39S, +159Y, +220T, + 281N, +363S, +474Y is mutated to +18D, +39N, +159D, +220K, +281D, +363C, +474K.
  • the DNA fragment containing the BsaAmy6 gene was digested with restriction endonucleases EcoRI and NotI, and ligated into the pPICzaA vector to obtain the expression vector pPICzaA-BsaAmy6.
  • the expression vector pPICzaA-BsaAmy6 was linearized, electroporated into yeast X33, and the transformed product was separately coated with a solid culture plate and cultured at 30 ° C for 2-3 d.
  • Example 6 shake flask and 50L fermentor culture
  • the yeast transformant on the plate was inoculated into a 500 mL flask containing 50 mL of BMGY medium, and cultured at 30 ° C, 250 r / min overnight until the OD 600 reached 2-6.
  • the cells were collected by centrifugation, resuspended in BMMY medium, diluted to an OD600 of 1.0, and cultured continuously.
  • the methanol was added to the BMMY medium every 24 hours to a final concentration of 0.75% for induction and simultaneous determination of the enzyme. live.
  • the recombinant engineering bacteria selected by shake flask culture were inoculated into 100 mL BMGY medium, and cultured at 30 ° C, 240 rpm for 20 h.
  • the domestic 50L fermenter was added with 20L fermentation base medium, sterilized at 121 °C for 20 min, the temperature was adjusted to 30 ° C, the pH was adjusted to 5.0 with ammonia water, PTMl (4.35 mL / L) was added, and the seed bacteria (1:10) were added.
  • the temperature is controlled at 30 ° C
  • the ventilation is maintained at 2 vvm
  • the speed is controlled. It is made between 500-800 rpm to maintain dissolved oxygen of 20% or more.
  • Fermentation is divided into three stages: growth period, from the addition of seed bacteria, culture for about 16-24h, until the glycerin in the fermenter is exhausted, which shows a sudden increase in dissolved oxygen; then enters the glycerol growth phase, supplemented with 50% glycerol ( Contains PTMl, 12mL/L), feed rate is 18mL/L ⁇ h, lasts for 4-6h; finally enters the induction period, adjusts the pH to the desired value with ammonia or phosphoric acid, and adds 100% methanol (containing PTMl, 12mL/ L), the flow rate was linearly increased from 1 mL/L ⁇ h to 15 mL/L ⁇ h over 15 h for 120 h.
  • the fermentation broth was taken every 24 hours to measure the OD 600 and the wet weight of the cells, and the supernatant was taken for the detection of ⁇ -amylase activity.
  • the average fermentation enzyme activity of the recombinant engineering strain containing the mutant gene BsaAmy6 in the culture condition of 50L fermenter was 36900 U/mL, and the fermentation enzyme activity of the mutant ⁇ -amylase BsaAmy6 was increased by 41.5% compared with AmyL.
  • the fermentation process curve is shown in Figure 1. Show.
  • the optimum pH of the original AmyL and the ⁇ -amylase mutant BsaAmy6 was determined by reference to the national standard method.
  • the optimum pH of the original alpha-amylase AmyL and the alpha-amylase mutant BsaAmy6 is shown in Figure 2. As can be seen from Fig. 2, the optimum pH of the mutant BsaAmy6 did not change much, almost the same as the original ⁇ -amylase.
  • the original ⁇ -amylase AmyL and the ⁇ -amylase mutant BsaAmy6 were each treated at room temperature for 3 hours under the conditions of pH 4-8, and then the enzyme activity was measured by the method of the national standard.
  • the pH stability of the original alpha-amylase AmyL and the alpha-amylase mutant BsaAmy6 is shown in Figure 3. As can be seen from Figure 3, the mutant BsaAmy6 is more stable under acidic conditions than the original alpha-amylase AmyL.
  • the residual enzyme activities of the mutant BsaAmy6 were 95% and 98%, respectively, at pH 4 and 5, and the original ⁇ -amylase AmyL was 81% and 87%, respectively.
  • the optimum reaction temperature of the original AmyL and the ⁇ -amylase mutant BsaAmy6 was determined by the national standard method.
  • the optimum reaction temperature of the original ⁇ -amylase AmyL and the ⁇ -amylase mutant BsaAmy6 is shown in FIG. 4 .
  • the optimum reaction temperature of the mutant BsaAmy6 was 65 ° C
  • the optimum reaction temperature of the original Amy L was 60 °C.
  • the original ⁇ -amylase AmyL and the ⁇ -amylase mutant BsaAmy6 were respectively treated in a water bath at 50° C. to 90° C. for 30 minutes, and then the enzyme activity was measured by the method of the national standard.
  • the thermal stability of the original alpha-amylase AmyL and the alpha-amylase mutant BsaAmy6 is shown in Figure 5. As can be seen from Figure 5, the mutant BsaAmy6 is more thermodynamically stable than the original alpha-amylase AmyL. After 30 minutes of water bath treatment at 80 ° C and 90 ° C, the residual enzyme activity of the mutant BsaAmy6 was 80% and 70%, while the residual enzyme activity of the ⁇ -amylase AmyL was 50% and 40%.

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Abstract

提供一种活性提高的α-淀粉酶AmyL突变体及其编码基因和应用。突变体的氨基酸序列如SEQ ID NO.2所示。经过改良的α-淀粉酶菌株在180小时的发酵酶活达到36900U/mL，比改造前的α-淀粉酶生产菌提高41.5％。

Description

活性提高的α-淀粉酶AmyL突变体及其编码基因和应用 技术领域
本发明涉及基因工程领域，具体涉及活性提高的α-淀粉酶AmyL突变体及其编码基因和应用。
背景技术
α-淀粉酶，系统名称为1,4-α-D-葡聚糖水解酶，别名为液化型淀粉酶、液化酶、α-1，4-糊精酶。α-淀粉酶是一种内切水解酶，其主要作用是催化淀粉的1,4-α-D-葡聚糖生成还原性糊精和糖类，在淀粉、清洁剂、饮料和纺织等领域具有重要作用。
由于从自然界筛选得到的野生菌产的α-淀粉酶的活力一般都比较低，不能直接运用于工业化的发酵生产。现有技术一般是通过对野生菌株进行诱变、杂交育种等技术手段来提高菌株的产酶能力。随着分子生物学的迅速发展，基因工程育种技术越来越受到国内外研究者的青睐。
好盐芽孢杆菌Bacillus salsusα-淀粉酶AmyL是一种中温淀粉酶，本课题组通过一系列应用评价实验发现AmyL在许多工业领域具有应用潜力。在造纸工业中，α-淀粉酶AmyL可以很好的改良纸张涂层淀粉的黏度和浓度，提高纸张的质量。饲料工业中，α-淀粉酶AmyL可以帮助幼龄动物消化利用淀粉，对其生长性能与饲料转化率十分有益。虽然α-淀粉酶AmyL具有很大的应用潜力，但目前α-淀粉酶AmyL的生产活力低，发酵成本高。为了使好盐芽孢杆菌Bacillus salsus的α-淀粉酶AmyL广泛应用到众多工业领域中，提高其比活力及表达水平，降低生产成本是急需解决的问题。
发明内容
本发明的目的是通过对来源于好盐芽孢杆菌Bacillus salsus的α-淀粉酶AmyL进行分子改造，改造后的α-淀粉酶具有更高的比活，降低生产成本，符合工业化大生产的要求。
本发明的目的是提供活性提高的α-淀粉酶AmyL突变体。
本发明的再一目的是提供编码上述α-淀粉酶AmyL突变体的基因。
本发明的再一目的是提供包含上述的活性提高的α-淀粉酶AmyL突变体基因的重组载体。
本发明的再一目的是提供包含上述的活性提高的α-淀粉酶AmyL突变体基因的 重组菌株。
好盐芽孢杆菌Bacillus salsus的α-淀粉酶AmyL的氨基酸序列如SEQ ID NO.1所示：
Figure PCTCN2017107817-appb-000001
本发明采用易错PCR及定点饱和突变的方法对SEQ ID NO.1所示的α-淀粉酶AmyL进行分子改造，经过高通量筛选得到高比活α-淀粉酶BsaAmy6，本发明的高比活α-淀粉酶BsaAmy6和原有的Bacillus salsus的α-淀粉酶AmyL相比，有7个氨基酸的差异，突变位点由+18N,+39S,+159Y,+220T,+281N,+363S,+474Y突变成+18D,+39N,+159D,+220K,+281D,+363C,+474K。突变后的氨基酸序列如SEQ ID NO.2所示：
Figure PCTCN2017107817-appb-000002
Figure PCTCN2017107817-appb-000003
本发明还提供了上述突变α-淀粉酶BsaAmy6的基因序列，其碱基序列如SEQ ID NO.3所示：
本发明还提供了包含上述活性提高α-淀粉酶的重组载体，将本发明的活性提高α-淀粉酶基因BsaAmy6连接到酵母表达载体pPICzαA上的EcoR I和Not I限制性酶切位点之间，使该核苷酸序列位于AOX1启动子的下游并受其调控，得到重组酵母表达质粒pPICzαA-BsaAmy6。
本发明还提供了包含上述活性提高α-淀粉酶基因BsaAmy6的重组菌株，优选重组菌株是毕赤酵母菌株X33。
本发明还提供了表达上述活性提高的α-淀粉酶基因BsaAmy6的方法，包括以下步骤：
1)用上述的重组载体转化宿主细胞，得重组菌株；
2)重组菌株进行发酵，诱导重组α-淀粉酶的表达；
3)发酵结束后，回收并纯化所表达的α-淀粉酶BsaAmy6。
具体地，将重组表达质粒pPICzαA-BsaAmy6线性化后，转化到毕赤酵母X33中，用高浓度的抗生素平板筛选转化子，将筛选到的转化子，首先在摇瓶培养条件下进行比较分析。将摇瓶培养筛选出来的的高酶活转化子，再在50L的发酵罐中进行发酵，发酵过程中，每隔24h取发酵液测定OD600以及菌体湿重，取上清液进行α-淀粉酶活性检测。发酵结束最终平均发酵酶活达到36900U/mL，比出发菌的酶活提高41.5％，实现了重组α-淀粉酶BsaAmy6的高效表达。
本发明通过结合易错PCR技术和高通量筛选技术对好盐芽孢杆菌Bacillus salsus的α-淀粉酶AmyL进行分子改造。含有突变基因BsaAmy6的重组工程菌在50L发酵罐培养条件下的平均发酵酶活为36900U/mL，突变后的α-淀粉酶BsaAmy6的发酵酶 活比AmyL提高41.5％。因此，本发明的突变α-淀粉酶BsaAmy6及重组工程菌，大大降低了发酵生产成本，使其在众多工业领域中显示出巨大的应用潜力。
附图说明
图1为α-淀粉酶AmyL和其突变体BsaAmy6的酵母菌株在50升发酵罐中酶活比较图。
图2突变体BsaAmy6和原始AmyL的最适反应pH
图3突变体BsaAmy6和原始AmyL的pH稳定性
图4突变体BsaAmy6和原始AmyL的最适反应温度
图5突变体BsaAmy6和原始AmyL的热稳定性
具体实施方式
以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
实验材料和试剂：
1、菌株与载体
大肠杆菌菌株Topl0、毕赤酵母X33、载体pPICzαA，pGAPzαA,Zeocin购自Invitrogen公司。
2、酶与试剂盒
PCR酶，质粒提取，胶纯化，限制性内切酶、试剂盒购自上海生工公司。
3、培养基
大肠杆菌培养基为LB，配方为：1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0。LBZ为LB培养基加25μg/mL Zeocin。
酵母培养基为YPD，配方为1％酵母提取物，2％蛋白胨，2％葡萄糖。酵母筛选培养基为YPDZ，配方为：YPD+100mg/L zeocin。
酵母诱导培养基BMGY，配方为1％酵母提取物、2％蛋白胨、1.34％YNB、0.00004％Biotin、1％甘油(V/V))，BMMY，除以0.5％甲醇代替甘油，其余成份相与BMGY相同。
重组酵母发酵培养基本盐培养基：磷酸氢二铵5％、磷酸二氢钾0.5％、七水硫酸镁1.5％、硫酸钾1.95％、硫酸钙0.1％、氢氧化钾0.1％、消泡剂0.03％。高压后每升加4.35毫升PTM1。
PTM1(微量盐溶液)：硫酸铜0.6％、碘化钾0.018％、一水硫酸锰0.3％、二水钼酸钠0.02％、硼酸0.002％、六水氯化钴0.05％、氯化锌2％、七水硫酸铁6.5％、浓硫酸0.5％、生物素0.02％。
实施例1、好盐芽孢杆菌Bacillus salsus的α-淀粉酶AmyL基因合成及克隆
将已公布的好盐芽孢杆菌Bacillus salsusα-淀粉酶AmyL氨基酸序列(Genebank：SDP85898)，根据毕赤酵母密码子优化后进行合成。
根据合成的基因分别在5’端和3’端设计PCR引物含EcoRI和NotI酶切酶位点，起引物序列如下：
5’端引物amyl-F1：5'-GTAGAATTCATGAGACAGGTTAGAATTGCTTTTG-3'
3’端引物amyl-R1：5'-ACTGCGGCCGCTTATTTTTGTACATAAACTGAAACT-3'
以合成基因为模板，用上述引物进行PCR扩增，将扩增得到的片段克隆到载体pGAPzαA上，得到重组载体pGAPzαA-AMYL。
实施例2、基因易错PCR随机突变
以上述pGAPzαA-AMYL为模板，进行易错PCR随机突变扩增，具体地扩增方法是：
第一轮扩增：以载体启动子引物amyl-F1和amyl-R1为引物进行PCR扩增，反应体系如下：
Figure PCTCN2017107817-appb-000004
反应程序如下：
Figure PCTCN2017107817-appb-000005
回收第一轮PCR产物，去1μL稀释50-100倍用作第二轮PCR的模板。第二轮 易错PCR同样以特异性引物amyl-F1及amyl-R1进行PCR反应。
取第二轮的产物用EcoRI和NotI进行双酶切，连接至pGAPzαA载体上的。连接产物转化毕赤酵母X33，在YPDZ琼脂糖平板培养筛选突变菌株。
实施例3、高通量筛选高酶活突变菌株
从实施例2易错PCR平板上挑取突变单菌落，将重组转化子用牙签逐个挑至24孔板，每个孔中加入1mL含有YPD培养基，30℃，220rpm培养48左右，离心取上清。将上述上清液分别取出200μL至96孔板，进行α-淀粉酶酶活测定。α-淀粉酶酶活检测参照中华人民共和国国家标准《GB/T24401-2009》进行测定。将酶活提高的8个阳性突变克隆，逐一提取基因组DNA，进行目的基因PCR扩增，确定突变位点。
测序结果确定氨基酸突变位点，克隆1的突变位点为+18N替换为+18D；克隆2的突变位点为+39S替换为+39N；克隆3的突变点为+141G替换为+141K；克隆4的突变点为+159Y替换为+159D；克隆5的突变点为+220T替换为+220K；克隆6的突变点为+363S替换为+363C；克隆7的突变点为+474Y替换为+474K。通过定点突变将这些突变位点一一进行结合，通过筛选最终得到一条高比活的α-淀粉酶突变基因BsaAmy6。本发明的高比活α-淀粉酶BsaAmy6和原有的Bacillus salsus的α-淀粉酶AmyL相比，有7个氨基酸的差异，突变位点由+18N,+39S,+159Y,+220T,+281N,+363S,+474Y突变成+18D,+39N,+159D,+220K,+281D,+363C,+474K。
实施例4、α-淀粉酶BsaAmy6表达载体的构建及工程菌株的筛选
用限制性内切酶EcoRI和NotI双酶切纯化含有BsaAmy6基因的DNA片段，连接到pPICzaA载体得到表达载体pPICzaA-BsaAmy6。将表达载体pPICzaA-BsaAmy6线性化，电击转入酵母X33，将转化产物分别涂布固体培养平板，30℃培养2-3d。
实施例6、摇瓶及50L发酵罐培养
将平板上的酵母转化子，接种于含有50mL BMGY培养基的500mL三角瓶中，30℃，250r/min振荡过夜培养至OD600达到2～6。离心收集菌体，再将其重悬浮于BMMY培养基中，稀释至OD600为1.0，继续振荡培养，每隔24h向BMMY培养基中补加甲醇至终浓度为0.75％进行诱导表达，同时测定酶活。
将摇瓶培养筛选出来的重组工程菌，接种于100mL BMGY培养基中，30℃、240rpm培养20h。以1：50的比例接种到300mL BMGY培养基中，30℃、240rpm培养至OD600＝5，用以接种发酵罐。国产50L发酵罐，加入20L发酵基础培养基，121℃灭菌20min，调节温度至30℃，用氨水调节pH至5.0，加入PTMl(4.35mL/L)，接入种子菌(1：10)。发酵过程中，温度控制在30℃，通气量维持在2vvm，转速控 制在500-800rpm之间以维持溶氧20％以上。
发酵分为三个阶段：生长期，从加入种子菌，培养约16-24h，直到将发酵罐中甘油耗尽，表现为溶氧突然上升；之后进入甘油促生长期，补加50％甘油(含有PTMl，12mL/L)，补料速度为18mL/L·h，持续4-6h；最后进入诱导期，用氨水或磷酸调节pH至所需值，流加100％甲醇(含有PTMl，12mL/L)，流速从1mL/L·h经15h线性升至4mL/L·h，持续120h。
发酵过程中，每隔24h取发酵液测定OD600以及菌体湿重，取上清液进行α-淀粉酶活性检测。含有突变基因BsaAmy6的重组工程菌在50L发酵罐培养条件下的平均发酵酶活为36900U/mL，突变后的α-淀粉酶BsaAmy6的发酵酶活比AmyL提高41.5％，发酵过程曲线如图1所示。
实施例7、突变体BsaAmy6和原始AmyL的最适反应pH和pH稳定性
参照国标方法测定原始AmyL及α-淀粉酶突变体BsaAmy6的最适反应pH。原始α-淀粉酶AmyL及α-淀粉酶突变体BsaAmy6的最适pH如图2所示。由图2可知，突变体BsaAmy6的最适pH并没有发生太大变化，几乎和原始α-淀粉酶一样。
将原始α-淀粉酶AmyL及α-淀粉酶突变体BsaAmy6分别在pH4-8条件下室温处理3小时，然后参照国标的方法测定酶活。原始α-淀粉酶AmyL及α-淀粉酶突变体BsaAmy6的pH稳定性如图3所示。由图3可知，相对于原始α-淀粉酶AmyL，突变体BsaAmy6在酸性条件下的稳定性更好。在pH4和5条件下突变体BsaAmy6的剩余酶活分别为95％和98％，原始α-淀粉酶AmyL则分别为81％和87％。
实施例8、突变体BsaAmy6和原始AmyL的最适反应温度和热稳定性
参照国标方法测定原始AmyL及α-淀粉酶突变体BsaAmy6的最适反应温度。原始α-淀粉酶AmyL及α-淀粉酶突变体BsaAmy6的最适反应温度如图4所示。由图4可知，突变体BsaAmy6的最适反应温度为65℃，原始AmyL的最适反应温度为60℃。
将原始α-淀粉酶AmyL及α-淀粉酶突变体BsaAmy6分别在50℃-90℃条件下水浴处理30分钟，然后参照国标的方法测定酶活。原始α-淀粉酶AmyL及α-淀粉酶突变体BsaAmy6的热稳定性如图5所示。由图5可知，突变体BsaAmy6的热稳定性要好于原始α-淀粉酶AmyL。在80℃和90℃条件下水浴处理30分钟后，突变体BsaAmy6的剩余酶活为80％和70％，而α-淀粉酶AmyL的剩余酶活为50％和40％。

Claims (7)

  1. 一种活性提高的α-淀粉酶突变体，其特征在于，其为氨基酸序列如SEQ ID NO.1所示的α-淀粉酶的突变体，氨基酸序列如SEQ ID NO.1所示的α-淀粉酶的+18N,+39S,+159Y,+220T,+281N,+363S,+474Y突变成+18D,+39N,+159D,+220K,+281D,+363C,+474K。
  2. 一种活性提高的α-淀粉酶突变体基因，其特征在于，编码权利要求1所述的活性提高的α-淀粉酶突变体。
  3. 如权利要求2所述的活性提高的α-淀粉酶突变体基因，其特征在于，其核苷酸序列如SEQ ID NO.3所示。
  4. 一种提高α-淀粉酶酶活的方法，其特征在于，将氨基酸序列如SEQ ID NO.1所示的α-淀粉酶的第18位用D替代N，第39位用N替代S，第159位用D替代Y，第220位用K替代T，第281位用D替代N，第363位用C替代S,第474位用K替代Y。
  5. 包含权利要求2或3所述的性提高的α-淀粉酶突变体基因的重组载体。
  6. 包含权利要求2或3所述的性提高的α-淀粉酶突变体基因的重组菌株。
  7. 一种制备权利要求1所述活性提高的α-淀粉酶突变体的方法，其特征在于，包括以下步骤：
    1)用权利要求6所述的重组载体转化宿主细胞，得到重组菌株；
    2)重组菌株进行发酵，诱导重组α-淀粉酶的表达；
    3)发酵结束后，回收并纯化所表达的α-淀粉酶突变体。
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