CN114517191B - 热稳定性提升的酸性葡聚糖酶突变体及其应用 - Google Patents
热稳定性提升的酸性葡聚糖酶突变体及其应用 Download PDFInfo
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2448—Licheninase (3.2.1.73)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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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
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- 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)
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- C12Y302/01073—Licheninase (3.2.1.73)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
热稳定性提升的酸性葡聚糖酶突变体及其应用,对氨基酸序列如SEQ ID NO.1所示的葡聚糖酶BisGlu16B_∆C分别进行T40K、Q53L和S311Y突变,氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所示,获得所述热稳定性改良的葡聚糖酶突变体T40K、Q53L和S311Y。经过改造优化,获得的突变体在60℃下的热稳定性得到了明显提升,进一步满足了饲料用酶的要求,因此,本发明中热稳定性提升的葡聚糖酶突变体T40K、Q53L和S311Y在饲料工业中展现出巨大的应用潜力。
Description
技术领域
本发明涉及基因工程和酶工程技术领域,涉及一种热稳定性提升的酸性葡聚糖酶突变体及其应用。
背景技术
β-葡聚糖是植物细胞壁的重要多糖组分,由β-D-葡萄糖残基通过1,3-β-糖苷键和1,4-β-糖苷键连接而成。实现生物质多糖快速降解主要包括两种因素,一是加速纤维素和半纤维素大分子的彼此分离和释放,二是酶将多糖水解为寡糖。β-葡聚糖酶是降解β-葡聚糖的主要酶类,其在工业生产中具有重要应用价值,如在啤酒酿造中添加β-葡聚糖酶可增加啤酒澄清度从而改善啤酒口感,在饲料中添加β-葡聚糖酶可有效降低单胃动物肠道食糜黏度,消除葡聚糖的抗营养作用,提高饲料利用率。
根据切割糖苷键的类型可将β-葡聚糖酶分为以下4种类型:β-1,4-葡聚糖酶(EC3.2.1.4)、β-1,3-葡聚糖酶(EC 3.2.1.39)、β-1,3(4)-葡聚糖酶(EC 3.2.1.6)和β-1,3-1,4-葡聚糖酶(EC3.2.1.73)。其中β-1,3-1,4-葡聚糖酶(下称β-葡聚糖酶)的催化活性最高,可通过特异性切割1,3-键相邻的β-1,4-糖苷键将葡聚糖或地衣多糖降解为以纤维三糖和纤维四糖为主的低聚寡糖,因此具有最广泛的应用价值,然而目前报道的大部分β-葡聚糖酶的热稳定性依旧不能满足工业应用的要求。
提高酶的热稳定性可以有效拓宽酶在工业中的应用范围,同时有助于研究者了解酶结构与稳定性之间内在联系。在实际生产中,谷物内源性β-葡聚糖酶在制麦过程和麦汁糖化过程中会丧失大部分酶活。在饲料制粒过程中β-葡聚糖酶会快速丧失活性。为了满足工业生产的需求,需要通过新酶筛选和蛋白质工程手段对β-葡聚糖酶的稳定性进行改造研究。
发明内容
解决的技术问题:本发明的目的是采用理性设计方法通过对来源于丝状真菌Bispora sp.MEY-1的葡聚糖酶BisGlu16B_ΔC(WT)进行改造,获得耐高温的突变体,以更适应饲料添加,确保酶在饲料高温制粒过程中不会失活从而有效消除抗营养因子的负面作用。
技术方案:一种热稳定性提升的酸性葡聚糖酶突变体,对氨基酸序列如SEQ IDNO.1所示的葡聚糖酶BisGlu16B_ΔC分别进行T40K、Q53L和S311Y突变,氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所示,获得所述热稳定性改良的葡聚糖酶突变体T40K、Q53L和S311Y。
编码上述3个热稳定性改良的葡聚糖酶突变体的氨基酸序列,其核苷酸序列分别如SEQ ID NO.5、SEQ ID NO.6和SEQ ID NO.7所示。
包含任一上述热稳定性提升的酸性葡聚糖酶突变体基因的重组表达载体。
包含任一上述热稳定性提升的酸性葡聚糖酶突变体基因的重组载体pPIC9-T40K、pPIC9-Q53L和pPIC9-S311Y。
包含上述重组载体的重组菌株。
上述重组菌株为重组毕赤酵母菌GS115/T40K、GS115/Q53L和GS115/S311Y。
上述热稳定性提升的酸性葡聚糖酶突变体在饲料生产中的应用。
有益效果:本发明利用酶工程手段对来源于丝状真菌Bispora sp.MEY-1的葡聚糖酶BisGlu16B_ΔC进行热稳定性改良,以解决该葡聚糖酶在饲料制粒过程中易失活的缺点,经过改造优化,获得的突变体在60℃下的热稳定性得到了明显提升,进一步满足了饲料用酶的要求,因此,本发明中热稳定性提升的葡聚糖酶突变体T40K、Q53L和S311Y在饲料工业中展现出巨大的应用潜力。
附图说明
图1葡聚糖酶野生型及其突变体的SDS-PAGE分析,其中,M为低分子量蛋白质Marker;A、C、E、G分别为纯化的野生酶BisGlu16B_ΔC及其3个突变体T40K、Q53L和S311Y;B、D、F、H分别为纯化后脱N-糖基处理的野生酶BisGlu16B_ΔC及其3个突变体T40K、Q53L和S311Y。
图2为葡聚糖酶突变体与野生型的最适pH;
图3为葡聚糖酶突变体与野生型的pH稳定性;
图4为葡聚糖酶突变体与野生型的最适温度;
图5为葡聚糖酶突变体与野生型的热稳定性(60℃下的半衰期)。
具体实施方式
实验条件:
1.载体和菌株:表达载体pPIC9质粒和毕赤酵母GS115购自Invitrogen公司,大肠杆菌DMT感受态细胞购自全式金。
2.酶类、试剂盒及其它生化试剂:taq酶购自TaKaRa公司,内切酶和点突变试剂盒购自全式金公司,底物地衣多糖购自Magzyme公司。其它都为国产分析纯试剂均购自国药试剂公司。
3.培养基:
大肠杆菌LB培养基:1%蛋白胨,0.5%酵母提取物,1%NaCl,1%琼脂粉(固体);酵母YPD培养基:2%葡萄糖,2%蛋白胨,1%酵母提取物;酵母MD培养基:1.5%琼脂糖,2%葡萄糖,生物素4×10-4g/L,YNB 13.4g/L;酵母BMGY培养基:2%蛋白胨,1%酵母提取物,1%甘油(V/V),生物素4×10-4g/L,YNB 13.4g/L;酵母BMMY诱导培养基:2%蛋白胨,1%酵母提取物,YNB 13.4g/L,0.5%甲醇(V/V),生物素4×10-4g/L。
实施例1葡聚糖酶突变体编码基因的获得
以来源于丝状真菌Bispora sp.MEY-1的葡聚糖酶基因Bisglu16b_ΔC(核苷酸序列如SEQ ID NO:8所示,氨基酸序列如SEQ ID NO:1所示)的重组表达载体pic9r-Bisglu16b_ΔC为模板,采用定点突变的方法对Thr40、Gln53和Ser311位点进行定点突变,引物设计如表1所示,突变方法以及克隆方法参考文献(Improvement of XYL10C_ΔNcatalytic performance through loop engineering for ligNOcellulosic biomassutilization in feed and fuel industries;You,et al.,2021)。
表1 引物合成清单
实施例2葡聚糖酶突变体的制备
将经实施例1PCR获得的线性重组表达载体经DMT酶消化后转化大肠杆菌DMT感受态细胞,菌落PCR验证,获得目标位点突变的重组表达质粒(核苷酸序列如SEQ ID NO:3所示,氨基酸序列如SEQ ID NO:4所示),将质粒用内切酶BglⅡ线性化后电转化毕赤酵母P.pastoris GS115,获得重组酵母菌株GS115/T40K、GS115/Q53L和GS115/S311Y。
将含有重组质粒的酵母菌株,接种于含有2mL BMGY培养基的10mL试管中,30℃,220rpm摇床培养48h后将菌液3000g离心5min,弃上清,沉淀用2mL含有0.5%甲醇的BMMY培养基重悬,并再次置于30℃,220rpm条件下诱导培养48h。取上清用于酶活性检测,筛选到酶活最高的酵母单克隆。
将野生型和三个突变体酵母菌株放大培养诱导产酶,首先接种于30mL YPD培养基中30℃,220rpm摇床培养48h后获得种子培养液,按1%接种量接种于含有300mL BMGY培养基的1L三角瓶中,30℃,220rpm摇床培养48h;后将培养液3000g离心5min,弃上清,沉淀用200mL含有0.5%甲醇的BMMY培养基重悬,并再次置于30℃,220rpm条件下诱导培养48小时。每隔12h补加1mL甲醇,同时取上清用于酶活性检测。最后将上清液浓缩至30mL,磷酸氢二钠-柠檬酸缓冲液(pH6.0,10mM)脱盐后采用阴离子交换法纯化蛋白用于酶学性质测定和比较。所表达的酶经过纯化之后,其蛋白质纯度达到90%以上(如图1所示)。实施例3突变体和野生型的酶学性质比较分析
一、DNS法测定
具体方法如下:在各自最适pH、最适温度条件下,1mL的反应体系包括100μL稀释酶液,900μL底物(0.5%的地衣多糖),反应10min后加入1.5mL DNS终止反应,沸水煮5min。冷却后测定OD540值。在给定的条件下,每分钟水解葡聚糖生成1μmoL还原糖所需的酶量为1个酶活单位(U)。
二、突变体和野生型葡聚糖酶性质测定
1、最适pH和pH稳定性测定方法
将纯化好的突变体和野生型葡聚糖酶在不同的pH(1.0-6.5)下进行酶促反应,以测定其最适pH。底物地衣多糖用不同pH(1.0、1.5、2.0、2.5、3、3.5、4、4.5、5、5.5、6、6.5)的0.1mol/L磷酸氢二钠-柠檬酸缓冲液中55℃下进行酶活力测定;将酶液在不同pH(1-12)的缓冲液中37℃孵育1小时,再测定剩余酶活以显示酶的pH稳定性。
结果如图2所示,野生型WT和三个突变体的最适反应pH和pH作用范围相近,最适pH均为为4.0,且在pH2.5-5.0之间均能维持40%以上的相对酶活。在pH稳定性方面:碱性环境(pH7.0-9.0)下三个突变体的pH稳定性明显优于野生型,如在pH8.0条件下,三个突变体的相对酶活菌在75%以上,而野生型只有20%。
2、最适温度和热稳定性测定方法
葡聚糖酶最适温度的测定方法:在pH 4.0,0.1mol/L磷酸氢二钠-柠檬酸缓冲液缓冲液及不同温度(25-75℃)下进行酶促反应。
热稳定性测定方法:将野生型和突变体在60℃下分别处理一定时间(0-240分钟),处理时所有突变体和野生型的蛋白浓度为100μg/mL,体积为100μL,在不同时间点取样后迅速置于冰上,并在55℃、pH 4.0的条件下测定剩余酶活。
结果如图3所示,野生型和突变体T40K、S311Y的最适温度均为60℃,但突变体Q53L的最适温度为70℃,比野生型提高10℃,且在75℃下突变体Q53L的相对酶活(26%)明显高于野生型(1%);在热稳定性方面:三个突变体T40K、Q53L和S311Y在60℃下的半衰期分别为51min、240min和66min,比野生型(16min)分别延长35min、224min和50min。三个突变提具有较好的热稳定性,在饲料添加中表现出巨大的应用潜力。
3、葡聚糖酶动力学参数和比活测定方法
检测方法参照文献(Improvement of enzyme activity ofβ-1,3-1,4-glucanasefrom Paenibacillus sp.X4 by error-prone PCR and structural insights ofmutated residues.DOI 10.1007/s00253-017-8145-4,Baek et al.,2017),测定反应的一级反应时间。确定测定Km值及Vmax的反应时间为5min。用不同浓度的地衣多糖(0.625,0.5,0.4,0.2,0.1,0.075和0.05mg/mL)为底物,在最适条件(温度、pH)下测定酶活性,计算出相应的反应速度,利用GraFit7软件计算Km值及Vmax。
尤其指出的是,在各自最适条件下,突变体Q53L的比活和催化效率(kcat/Km)较野生型分别提高65%和22%。(见表2),另外两个突变体T40K和S311Y在催化活力方面较野生型无明显降低。
表2 以地衣多糖为底物野生酶和突变体的比活力和催化效率比较
序列表
<110> 江苏科技大学
鑫缘茧丝绸集团股份有限公司
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<213> 人工序列(Artificial Sequence)
<400> 5
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgagac ttattgcaaa 120
gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgac gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc agctggggtc catctgcttc tgcaaatgtg 960
gcagtgtga 969
<210> 6
<211> 969
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgagac ttattgcacc 120
gacaacgatc tcatcagcag ttccagcacg aacgtgctta ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgac gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc agctggggtc catctgcttc tgcaaatgtg 960
gcagtgtga 969
<210> 7
<211> 969
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgagac ttattgcacc 120
gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgac gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc tattggggtc catctgcttc tgcaaatgtg 960
gcagtgtga 969
<210> 8
<211> 969
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caatataccc ttcagcagga ttacatggca gacggcaact tttttagcca attttcattt 60
tgggataccg ccgaccctac agatggcttt gtggcttata aaaatgagac ttattgcacc 120
gacaacgatc tcatcagcag ttccagcacg aacgtgcaga ttcgggtgga cagctccaat 180
gttacaccga atggacggcc tagtgttcgc attaccagca accagtcgta caatccaggc 240
acacttgtaa tcctggacct tgaacacatg ccaggtggca tctgcggtac ctggccagca 300
ttttggatgg ttgggccgaa ttggcccgac gatggggaaa tcgacatcat tgagggtgtc 360
aaccagcaaa ctaccaatga catgaccctc cacactagtg aaggctgcac aatatccagc 420
agtggcgatt tctcgggctc gatagttagc accgactgct gggtcgatga ccccaaccaa 480
tccgacaatg aaggctgtca gatcactacg agcaataccg aaacttacgg ttccggtttt 540
aatgctaaca atggcggcgt ctatgcgacg gacttccaag acgccgctat cagcatctat 600
ttcttccccc gtggttccat accttcggac attacagacg gctctccaga cccgtccggc 660
tggggtacgc caattgcgca gttcacggat agcagctgtg acattcaaag ctatttcacc 720
gatttacaga tcgttttcga tacgacgttc tgtggacaat gggctggcaa cgtctggtca 780
agtggctctt gtgcctctgt ggcaagtacc tgcgacgact acgtggaaaa caacccggct 840
gccttcgtcg atgcatactg gtcgatcaac agtcttcagg tttattcggg aacctccaat 900
ggtcccatgc agaatgatac ttcgagcagc agctggggtc catctgcttc tgcaaatgtg 960
gcagtgtga 969
<210> 9
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tgagacttat tgcaaagaca acgatctcat c 31
<210> 10
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tttgcaataa gtctcatttt tataagccac a 31
<210> 11
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cagcacgaac gtgcttattc gggtggacag c 31
<210> 12
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aagcacgttc gtgctggaac tgctgatgag a 31
<210> 13
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tacttcgagc agctattggg gtccatctgc t 31
<210> 14
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atagctgctc gaagtatcat tctgcatggg a 31
Claims (7)
1.一种热稳定性提升的酸性葡聚糖酶突变体,其特征在于,氨基酸序列如SEQ ID NO.2所示。
2.热稳定性提升的酸性葡聚糖酶突变体基因,其特征在于,编码权利要求1所述的热稳定性提升的酸性葡聚糖酶突变体的核苷酸序列如SEQ ID NO.5所示。
3.包含权利要求2所述热稳定性提升的酸性葡聚糖酶突变体基因的重组表达载体。
4.包含权利要求3所述热稳定性提升的酸性葡聚糖酶突变体基因的重组载体 pPIC9- T40K。
5.包含权利要求4所述重组载体的重组菌株。
6.根据权利要求5所述的重组菌株,其特征在于,所述重组菌株为重组毕赤酵母菌GS115/T40K。
7.权利要求1所述热稳定性提升的酸性葡聚糖酶突变体在饲料生产中的应用。
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