CN116656651A - 一种高比活生淀粉水解酶突变体、其编码基因、表达菌株及其应用 - Google Patents
一种高比活生淀粉水解酶突变体、其编码基因、表达菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一种高比活生淀粉水解酶突变体、其编码基因、表达菌株及其应用,本发明以稳定性欠佳的α‑淀粉酶Amy为出发酶,经过定点突变,获得了热稳定性提高的突变体Amy:ΔTG/W345A。以玉米生淀粉为底物时,突变体的比酶活为出发酶的85%,在30‑35℃条件下,热稳定性均有大幅度提升,在35℃、pH7.0条件下,半衰期可达到20h。在比酶活接近于出发酶的同时,热稳定性有大幅度提升。该突变体在以玉米生淀粉为底物的制糖工业领域具有潜在应用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及一种高比活生淀粉水解酶突变体、其编码基因、表达菌株及其应用。
背景技术
生淀粉水解酶指可以在淀粉的糊化温度以下直接降解生淀粉颗粒的酶。目前,在所有已发现的α-淀粉酶中,具有降解生淀粉能力的α-淀粉酶大约只有10%,分布于细菌、真菌和动物中。而这些α-淀粉酶可水解马铃薯、小麦、玉米等的生淀粉。但是,比活力普遍较低。在已报道的生淀粉水解酶中,比酶活在4.9-2370U/mg范围内。以玉米为底物时,来自Bacillus amyloliquefaciens的α-淀粉酶的比酶活为44.6U/mg,来自Streptomycesbadius DB-1的α-淀粉酶的比酶活为148.1U/mg,而来自Bacillus acidicola的α-淀粉酶最高比酶活达874.5U/mg。这些具生淀粉水解能力的α-淀粉酶因其偏低的酶活力限制了其在工业中的广泛应用。
在工业应用方面,淀粉加工工业普遍使用20-30%(W/V)的淀粉浆为原料,目前以生淀粉水解酶直接作用高浓度生淀粉水解的研究较少。热稳定性也是淀粉加工中使用淀粉分解酶的重要特性,许多淀粉酶由于其稳定性较差导致在水解过程中无法持续发挥水解作用。因此利用蛋白质工程技术,获得比酶活较高且热稳定性较好的生淀粉水解酶对于水解高浓度玉米生淀粉的应用具有重要作用。
发明内容
本发明提供了一种高比活生淀粉水解酶突变体、其编码基因、表达菌株及其应用。本发明以稳定性欠佳的生淀粉水解酶Amy为基础,通过定点突变,获得了热稳定性有大幅度提高、且保留出发酶Amy比活力的突变酶Amy:ΔTG/W345A。以玉米生淀粉为底物时,突变体的比酶活为出发酶的85%,动力学参数测定结果表明,突变酶与出发酶接近。但其热稳定性大大提高,在35℃,pH7.0的条件下,是出发酶的20倍。在水解高浓度玉米生淀粉实验中,反应4h后,水解率可达到43%。该突变酶在水解高浓度玉米生淀粉为基础的工业应用中具有潜在价值。
本发明高比活生淀粉水解酶突变体,其氨基酸序列是如SEQ ID No:1所示的生淀粉水解酶的氨基酸序列中第181位的苏氨酸和第182位的甘氨酸被去除,且第345位的色氨酸被突变为丙氨酸。
本发明生淀粉水解酶突变体编码基因的核苷酸序列如SEQ ID No:2所示。
本发明生淀粉水解酶突变体的表达菌株,分类命名为Bacillus subtilis WB600/pBHSs142-AmyZ1(ΔTG/W345A),已送至中国典型培养物保藏中心(CCTCC)保藏,保藏编号为CCTCC NO:CCTCC M 20221942,保藏时间为2022年12月12日,保藏地址:中国·武汉·武汉大学。
本发明生淀粉水解酶突变体表达菌株的构建方法,包括如下步骤:
首先以来自Bacillus licheniformis的α-淀粉酶BLA结构为模板,利用Swiss-Model对生淀粉水解酶Amy的结构进行同源建模。采用半理性设计策略,将Amy与BLA的序列与结构进行比对,分析确定突变的目标氨基酸。
根据生淀粉水解酶Amy的基因序列,设计并合成突变引物,以含有生淀粉水解酶Amy基因的重组质粒为模板,以上述的合成突变引物为引物,基于重叠延伸PCR的方法进行定点突变,获得了热稳定性有大幅度提高的生淀粉水解酶的突变基因。
在E.coli BL21(DE3)中构建的未突变的生淀粉水解酶质粒为模板,采用重叠延伸PCR的方式构建突变基因;再通过POE-PCR的方法,将突变基因与载体pBHSs142进行连接,得到连接产物;连接产物转化宿主菌WB600,筛选阳性克隆,得到含有本发明突变基因的工程菌株。
上述构建方法中所述表达质粒载体包括pET22b、pBHSs142等。
上述构建方法中所述宿主菌包括E.coli BL21(DE3)和WB600等。
本发明生淀粉水解酶突变体可由所述表达菌株发酵获得。
本发明生淀粉水解酶突变体的应用,是将所述生淀粉水解酶突变体应用于水解高浓度玉米生淀粉(浓度20-30%,W/V)。以玉米生淀粉为底物时,在35℃,pH7.0条件下,突变酶的比酶活为野生型的85%。动力学参数测定结果表明突变体对玉米生淀粉的催化效率相较于出发酶差别不大。在保持比酶活的同时,突变酶的稳定性有大幅度的提升。在30℃和35℃条件下,热稳定性是出发酶的15倍和20倍。在水解高浓度玉米生淀粉实验中,反应4h后,水解率可达到43%。该突变体在水解高浓度玉米生淀粉为基础的工业应用中具有潜在价值。
本发明对突变体蛋白和原始野生型蛋白的比酶活、最适温度、最适pH、动力学参数、稳定性等进行了测定和比较。测定结果显示,以玉米生淀粉为底物时,本发明在接近比酶活的同时,在30℃和35℃条件下,稳定性较出发酶有大幅度提升。
附图说明
图1、2为本发明PCR扩增产物的电泳图谱:图1中各泳道分别为DNAmarker、PCR扩增的片段;图2中各泳道分别为DNAmarker、PCR扩增的载体。
图3为纯化的突变蛋白和出发酶Amy的SDS-PAGE图谱:1为出发酶Amy粗酶,2为出发酶Amy纯酶,3为Amy:ΔTG/W345A粗酶,4为Amy:ΔTG/W345A纯酶,M为蛋白marker。
图4中a为最适温度测定结果,b为最适pH测定结果。
图5中a为30℃、pH7.0条件下的稳定性,b为35℃、pH7.0条件下的稳定性。
具体实施方式
下列实施例中的实施方法,如无特别说明,均为常规方法。
(一)含有本发明生淀粉水解酶突变基因的表达菌株的构建
1、生淀粉水解酶基因突变位点的选择
基于序列比对,生淀粉水解酶Amy与来自Bacillus licheniformis的α-淀粉酶BLA氨基酸序列一致性为71%。以BLA的结构作为模板,利用Swiss-Model对生淀粉水解酶Amy的结构进行同源建模。
根据模拟结构及多序列比对,确定定点突变的位点为181位点的苏氨酸T和182位点的甘氨酸以及345位点的色氨酸W,突变方向为去除181位点的苏氨酸T和182位点的甘氨酸G,345位点的色氨酸W被丙氨酸A替代。
2、生淀粉水解酶突变基因工程菌株的构建
根据生淀粉水解酶Amy的基因序列:SEQ ID No:2(其氨基酸序列如SEQ ID No:1),以及选定的突变位点181T、182G和345A。
以包含AmyZ1基因的重组质粒为模板质粒,利用重叠延伸PCR扩增,得到目的片段(图1);再利用pBHS-F与pBHS-R来扩增载体pBHS(图2)。目的片段与载体互为模板和引物进行POE-PCR连接。将连接产物通过化转的方式转入枯草芽孢杆菌中,挑选序列正确的转化子得到本发明突变基因的工程菌株Bacillus subtilis WB600/pBHSs142-Amy(ΔTG/W345A)。
本发明的菌株Bacillus subtilis WB600/pBHSs142-AmyZ1(ΔTG/W345A)已送往中国典型培养物保藏中心(CCTCC)保藏,保藏编号为CCTCC NO:CCTCC M 20221942,保藏时间为2022年12月12日,保藏地址:中国·武汉·武汉大学。
(二)含本发明生淀粉水解酶突变基因工程菌的表达与蛋白纯化
将构建成功的突变体菌株接种到小体积含有Kana的5mL LB培养基中,37℃,200rpm的摇床中培养12h。将培养了12h的菌液作为种子液,接种5mL种子液到400mL含有30μg/mL Kana的TY液体培养基中,30℃,200rpm的摇床中发酵48-60h。将培养48h的发酵液于大型离心机中8000×g低温离心15min。得到的上清即为粗酶液。
粗酶液经过Ni-NTA柱层析进行纯化。洗脱液中咪唑浓度为200mM,洗脱3个柱体积。得到的蛋白经过检测达到SDS-PAGE纯度。
(三)含本发明生淀粉水解酶突变体比酶活的检测(DNS法)
反应体系为900μL,玉米生淀粉用Na2HPO4-KH2PO4buffer(50mM,pH 7.0)进行溶液,使其终浓度为1%,35℃下进行孵育10min后,加入稀释好的酶液反应10min,加入300μLDNS置于冰上,终止反应。反应完成后,沸水浴煮沸15min显色。实验组做3个平行实验,对照组以缓冲液代替酶液。测定A540 nm下吸光值,在根据麦芽糖标准曲线公式计算还原糖量,1U即为每分钟生成1μM麦芽糖所需要的蛋白量。
测定结果显示,以玉米生淀粉为底物时,本发明获得的突变酶的比酶活为出发酶的85%。(四)含本发明生淀粉水解酶突变体最适pH、最适温度的检测
选择Na2HPO4-KH2PO4(50mM、pH7.0、1mM CaCl2)作为缓冲液,终浓度为1%的玉米生淀粉为底物,设置不同温度梯度。将突变酶提前稀释后,分装至2mL Ep管中,分别放置在不同温度的水浴锅中,测定不同温度梯度下突变酶的酶活。将最高酶活设置为100%,计算其他温度下突变酶的相对酶活。
在得到最适温度的结果后,在最适温度条件下,设置不同的pH梯度测定在不同pH条件下突变酶的酶活。将不同pH下测得的最高酶活设置为100%,计算剩余pH下突变酶的相对酶活。
以玉米生淀粉为底物时,突变酶最适温度为45℃,最适pH为6.0(图4),在pH5.5-7.5范围内均有80%以上的催化活性。
(五)含本发明生淀粉水解酶动力学参数的测定
使用玉米生淀粉作为底物测量突变酶的动力学常数,包括Km和Vmax。通过在不同浓度的玉米生淀粉(1.0-30mg/mL)存在下,在35℃下将酶在Na2HPO4-KH2PO4 buffer(50mM,pH7.0)中温育10min来进行反应。然后通过使用Origin 8.0将实验数据拟合到Michaelis-Menten模型的Lineweaver-Burt方程来计算动力学参数。
结果显示,突变酶对玉米生淀粉亲和力略有降低,但是其Kcat对比出发酶有所提高。
表3突变酶的动力学参数
(六)含本发明生淀粉水解酶稳定性的检测
在30-35℃、pH7.0条件下,对出发酶Amy与突变体进行热处理,每隔半小时或一小时取样,以初始酶活为100%,计算热处理一定时间后的酶活剩余率。
测定结果显示(图5),30℃条件下,突变酶的半衰期为28h,较出发酶提升15倍;35℃条件下,突变酶半衰期为20h,较出发酶提升了20倍。
(七)含本发明生淀粉水解酶突变体在水解高浓度玉米生淀粉的应用
水解体系为Na2HPO4-KH2PO4 buffer(50mM,pH 7.0)缓冲液中加入终浓度为1mM的CaCl2,随后加入30%(W/V)玉米生淀粉,以及所述的生淀粉水解酶突变体,35℃、200rpm摇床水浴下进行水解反应。间隔适当的时间取样,用DNS法测定水解体系中还原糖含量,同时加入相同酶量的商品酶BLA设置为对照组。
在水解30%的玉米生淀粉实验中,结果显示,突变酶在4h后水解基本达到平台期,其对于玉米的水解率可达到43%。相同条件下,可达到商业淀粉酶BLA对于高浓度玉米生淀粉的水解率,具有较大的应用潜能。
Claims (6)
1.一种高比活生淀粉水解酶突变体,其特征在于:
所述高比活生淀粉水解酶突变体的氨基酸序列是如SEQ ID No:1所示的生淀粉水解酶的氨基酸序列中第181位的苏氨酸和第182位的甘氨酸被去除,且第345位的色氨酸被突变为丙氨酸。
2.权利要求1所述生淀粉水解酶突变体的编码基因,其特征在于:
所述编码基因的核苷酸序列如SEQ ID No:2所示。
3.权利要求1所述生淀粉水解酶突变体的表达菌株,其特征在于:
所述表达菌株的分类命名为BacillussubtilisWB600/pBHSs142-AmyZ1(ΔTG/W345A),已送至中国典型培养物保藏中心(CCTCC)保藏,保藏编号为CCTCCNO:M20221942,保藏时间为2022年12月12日,保藏地址:中国·武汉·武汉大学。
4.权利要求1所述生淀粉水解酶突变体的应用,其特征在于:
以所述生淀粉水解酶突变体水解玉米生淀粉。
5.根据权利要求4所述的应用,其特征在于:
所述玉米生淀粉的浓度≤30%。
6.根据权利要求4所述的应用,其特征在于:
水解温度为30-45℃,pH5.5-7.5。
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