CN114381448B - 一种葡聚糖酶突变体及其应用 - Google Patents
一种葡聚糖酶突变体及其应用 Download PDFInfo
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- CN114381448B CN114381448B CN202210022750.3A CN202210022750A CN114381448B CN 114381448 B CN114381448 B CN 114381448B CN 202210022750 A CN202210022750 A CN 202210022750A CN 114381448 B CN114381448 B CN 114381448B
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Classifications
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- C12N9/2405—Glucanases
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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
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Abstract
一种葡聚糖酶突变体及其应用,是以如SEQ ID NO:1所示的葡聚糖酶BisGlu16B_∆C为母本对氨基酸残基Ala299突变后得到的一种突变体A299W。热稳定性方面,突变体A299W在60℃下的半衰期较野生型延长7.7倍,长达130分钟;pH耐受性方面,突变体A299W在pH7‑pH12之间剩余酶活较野生型提高2.2‑58倍;在催化活力方面,以大麦葡聚糖为底物时,突变体的催化效率较野生型提高94%。这样一种耐热、耐酸碱且高催化的β‑1,3‑1,4‑葡聚糖酶突变体,在啤酒酿造、饲料添加以及生物能源等领域具有广阔的应用前景。
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(EC 3.2.1.73)。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、pH耐受性和催化活性依旧不能满足工业应用的要求。
提高酶的稳定性(包括热稳定性和pH耐受性)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:为了解决现有β-1,3-1,4-葡聚糖酶热稳定性差、pH耐受性差等问题,本发明提供一种真菌来源的pH耐受性及热稳定性均提高的葡聚糖酶BisGlu16B_ΔC的突变体A280W及其应用,该突变体具有耐酸碱、耐高温且高催化的特点。
技术方案:一种葡聚糖酶突变体,是以如SEQ ID NO:2所示的葡聚糖酶BisGlu16B_ΔC为母本对氨基酸残基Ala280突变后得到的一种突变体A280W。
氨基酸序列如SEQ ID NO:4所示。
编码所述葡聚糖酶突变体的核酸,核酸序列如SEQ ID NO:3所示。
含有上述基因的重组表达载体。
含有上述重组表达载体的菌株。
上述菌株在制备提升热稳定性和pH耐受性的葡聚糖酶突变体中的应用
有益效果:本发明通过以来源于丝状真菌Bispora sp.MEY-1的第16家族木聚糖酶BisGlu16B_ΔC为母本对Ala280位点突变后得到的突变体A280W。具体来讲,通过构建含有该突变体基因的重组菌株,诱导培养后筛选出pH耐受性和热稳定性提高的葡聚糖酶突变体A280W。
以地衣多糖为底物测定结果显示,突变体A280W与野生型的最适pH分别为3.5和4.0,相较于野生型,突变体A280W的pH范围明显提升,在pH1-7之间均能保持40%以上的活性,而野生型只能在pH2.5-4.5之间可维持40%以上的活性。在pH稳定性方面,突变体A280W明显优于野生型:在pH1-6之间野生型与突变体耐受性相近,均能维持在40%以上,然而在pH7-12之间,突变体A280W可维持60%以上的酶活,野生型的剩余酶活量低于40%。
在温度属性方面,突变体A280W与野生型的最适温度在55-60℃之间,相差不大,但在高温条件下(65-75℃)突变体A280W的相对酶活(38%-90%)明显高于野生型(0-10%);在热稳定性方面:突变体A280W在60℃下的半衰期较野生型延长7.7倍,长达130分钟。
在催化活力方面,突变体A280W的比活为57000U/mg,与野生型(62000U/mg)相比差距不大;催化效率为17200mL/s·mg,比野生型(11900mL/s·mg)提高45%。因此,本发明所提供的pH耐受性及热稳定性均提高的高催化β-1,3-1,4-葡聚糖酶在饲料、食品和生物能源领域具有巨大的应用潜力。
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、B分别为纯化的和脱糖基处理的野生酶BisGlu16B_ΔC;C和D分别为纯化的和脱糖基处理的突变酶A280W。
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(半衰期)。
具体实施方式
实施例所用的试验材料:
1.菌株及载体:质粒扩增大肠杆菌DMT感受态细胞购自全式金,表达宿主Pichiapastoris GS115为本实验室保存。
2.试剂盒及其它生化试剂:点突变试剂盒购自Fermentas公司,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:
1)LB培养基:1%蛋白胨,0.5%酵母提取物,1% NaCl,1%琼脂粉(固体);
2)MD培养基:1.5%琼脂糖,2%葡萄糖,0.00004% Biotin,1.34% YNB;
3)YPD培养基:2%葡萄糖,2%蛋白胨,1%酵母提取物;
4)BMGY培养基:2%蛋白胨,1%酵母提取物,1%甘油(V/V),0.00004% Biotin1.34%YNB;
5)BMMY培养基:2%蛋白胨,1%酵母提取物,1.34%YNB,0.5%甲醇(V/V),0.00004%Biotin。
实施例1葡聚糖酶突变体A280W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:1所示,氨基酸序列如SEQ ID NO:2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Ala280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_ΔN catalyticperformance through loop engineering for lignocellulosic biomass utilizationin feed and fuel industries;You,et al.,2021)。
表1引物合成清单
实施例2β-1,3-1,4-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/A280W。
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GS115/BisGlu16B_ΔC和突变体GS115/A280W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,野生型和突变体A280W的最适反应pH相近,分别为4.0和3.5,但突变体A280W的pH作用范围明显优于野生型,尤其是在pH1-2和pH5-6.5之间,突变体A280W的相对酶活均保持在40%以上,而野生型的相对酶活均低于10%。在pH稳定性方面:突变体A280W明显优于野生型,在pH1-6之间野生型与突变体耐受性相近,均能维持在40%以上,然而在pH7-12之间,突变体A280W可维持60%以上的酶活,野生型的剩余酶活量低于40%。
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为: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(37-95℃)下进行酶促反应。将野生型和突变体在60℃下分别处理一定时间(0-180分钟,间隔10分钟),处理时所有突变体和野生型的浓度为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A280W的最适温度分别为55℃和60℃,在高温条件下(65-75℃)突变体A280W的相对酶活(38%-90%)明显高于野生型(0-10%);在热稳定性方面:突变体A280W在60℃下的半衰期较野生型延长7.7倍,长达130分钟。
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 XYL10C_ΔN catalytic performancethrough loop engineering for lignocellulosic biomass utilization in feed andfuel industries;You,et al.,2021),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。
在各自最适条件下,突变体A280W的比活为57000U/mg,与野生型(62000U/mg)相比差距不大;催化效率为17200mL/s·mg,比野生型(11900mL/s·mg)提高45%。(见表2)。
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> 鑫缘茧丝绸集团股份有限公司
江苏科技大学
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Claims (5)
1.一种葡聚糖酶突变体,其特征在于,是以如SEQ ID NO:2所示的葡聚糖酶BisGlu16B_∆C为母本对氨基酸残基Ala280突变后得到的一种突变体A280W。
2.编码权利要求1所述葡聚糖酶突变体的核酸,其特征在于,核酸序列如SEQ ID NO:3所示。
3.含有权利要求2所述核酸的重组表达载体。
4.含有权利要求3所述重组表达载体的菌株。
5.权利要求4所述菌株在制备提升热稳定性和pH耐受性的葡聚糖酶突变体中的应用。
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