CN111621490A - 一种草酸青霉16的β-葡萄糖苷酶突变体SVS及应用 - Google Patents

一种草酸青霉16的β-葡萄糖苷酶突变体SVS及应用 Download PDF

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CN111621490A
CN111621490A CN202010456556.7A CN202010456556A CN111621490A CN 111621490 A CN111621490 A CN 111621490A CN 202010456556 A CN202010456556 A CN 202010456556A CN 111621490 A CN111621490 A CN 111621490A
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CN111621490B (zh
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赵喜华
李汉昕
黄秋霞
王可心
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Jiangxi Normal University
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Abstract

本发明涉及一种草酸青霉16的β‑葡萄糖苷酶突变体SVS及应用，所述的β‑葡萄糖苷酶突变体SVS的氨基酸序列为SEQ ID NO.3，其编码基因的核酸序列为SEQ ID NO.4。β‑葡萄糖苷酶突变体SVS最适反应温度为50℃，催化效率常数k cat / K m能达到1354.94mM‑1S‑1，产物抑制强度K appm/K I值为1.13（当20mmol/L葡萄糖时），分别是16BGL的1.68倍和0.89倍，葡萄糖耐受性能提高12%，体现了在较低温度下具有更高的产物耐受性和活性，可应用于饲料、医药、食品、化工、能源等众多领域。

Description

一种草酸青霉16的β-葡萄糖苷酶突变体SVS及应用
技术领域
本发明涉及一种草酸青霉16的β-葡萄糖苷酶突变体SVS及应用，具体属于酶工程技术领域。
背景技术
β-葡萄糖苷酶能将底物水解为β-D-葡萄糖和相应配基，但是由于原始β-葡萄糖苷酶具有较差的酶学特性，比如催化活性较低、葡萄糖耐受性较差以及催化温度较高，这很难应用于饲料、医药、食品、化工、能源等众多领域。
草酸青霉16的β-葡萄糖苷酶对呋喃衍生物和酚类化合物具有不受抑制的活性，并且对高浓度的KCl、NaCl和乙醇具有极好的耐受性，然而，野生型16BGL的最适温度约为70℃，其活性和葡萄糖耐受性较低。
本发明通过分子改造技术获得了一种突变体SVS，该突变体SVS在较低温度下具有较高催化效率和葡萄糖耐受性，可满足饲料、医药、食品、化工、能源等众多领域的要求，具有很好的应用前景。
发明内容
根据以上背景技术，本发明的目的是提供一种草酸青霉16的β-葡萄糖苷酶突变体SVS及其应用。本发明通过高通量筛选，最终获得了在较低温度下，具有较高葡萄糖耐受性和较高活性的突变体SVS，并重组构建了组成型表达的毕赤酵母GS115工程菌，为实现其应用打下了基础。
本发明一种草酸青霉16的β-葡萄糖苷酶突变体SVS的氨基酸序列为SEQ ID NO.3，其编码基因的核酸序列为SEQ ID NO.4；草酸青霉16的β-葡萄糖苷酶为SEQ ID NO.1所示的氨基酸序列的第414位甘氨酸突变为丝氨酸、第421位的天冬氨酸突变为缬氨酸以及第441位的苏氨酸突变为丝氨酸。
所述的编码基因通过插入到载体pGAPZaA，插入位点为NotI和EcoRI，获得重组载体pGAPZaA-SVS。
所述的重组载体pGAPZaA-SVS用BglII限制性内切酶线性化后，再电转到毕赤酵母GS115，获得重组毕赤酵母GS115工程菌。
所述的重组毕赤酵母GS115工程菌用于生产β-葡萄糖苷酶突变体SVS。
所述的β-葡萄糖苷酶突变体SVS应用的条件是反应温度为30-60℃，pH为3-6。
有益效果：本发明通过分子改造技术获得了一种突变体SVS，β-葡萄糖苷酶突变体SVS反应温度为50℃，催化效率常数kcat/Km能达到1354.94mM-1S-1，葡萄糖抑制强度Kappm/KI值为1.13(当20mmol/L葡萄糖时)，分别是16BGL的1.68倍和0.89倍，葡萄糖耐受性能提高了12％，体现了更高的产物耐受性和活性，表现出更高催化效率和高葡萄糖耐受性，可应用于饲料、医药、食品、化工、能源等领域，具有很好的应用前景。
附图说明
图1：本发明实施例4原始酶和突变体SVS在不同温度下的相对活性；
图2：本发明实施例5原始酶、对照和突变体SVS的纤维素乙醇产量；
其中：对照为没有加入任何β-葡萄糖苷酶。
具体实施方式
下面结合说明书附图和实施例对本发明作出更进一步的说明，以便本领域技术人员了解本发明，但本发明的保护内容不局限于以下实施案例。
实施例1
获得β-葡萄糖苷酶
本发明以草酸青霉16为材料，对其进行接种、诱导培养、收集菌丝体。采用Trizol法提取总RNA，然后再进行反转录获得cDNA，所用到的都是常规技术，本发明使用了Tarkara公司的Trizol试剂以及反转录试剂盒得到了cDNA。
采用常规PCR方法，通过特异性引用物SP5’-GAATTCAAGGATCTTGCCTACTCTCCCCCCT-3’(直划线为EcoRI限制性酶酶切位点)和XM5’GGCGGCCGCCTCAATGGTGATGGTGATGATGCTGCACCTTGGGCAGATCGGCTGAAAG-3’(直划线为NotI限制性酶酶切位点，波浪线为6个组氨酸标签)，以cDNA为模板扩增β-葡萄糖苷酶的基因，连接到组成型表达载体pGAPZaA，得到重组载体，对其进行测序得到其核苷酸序列如SEQ ID NO.2所示，其编码的氨基酸序列如SEQ ID NO.1所示。
实施例2
发现β-葡萄糖苷酶突变体基因
采用定向进化技术对上述核苷酸序列为SEQ ID NO.2进行分子改造，获得突变体基因，将其与组成型表达载体pGAPZaA连接得到重组载体，通过平板颜色筛选和96孔板高通量筛选，获得了在低温下有较高活性和葡萄糖耐受性的突变体SVS，对其进行测序得到其核苷酸序列如SEQ ID NO.4所示，其编码的氨基酸序列如SEQ ID NO.3所示。
实验例3
制备β-葡萄糖苷酶突变体SVS
定向进化条件：3μL的10×PCRbuffer，3μL的dNTP，0.5mmol/L的MnCl2，0.3μM的上游引物SP，0.3μM的下游引物XM，5U的TaqDNA聚合酶，5ng的β-葡萄糖苷酶DNA模板，加灭菌去离子水至30μL。
将来自定向进化的突变基因连接到载体pGAPZαA中，然后转化到大肠杆菌Top10中，并涂布于含有博莱霉素的LB平板上，将所有来自LB平板的阳性突变子回收到装有25mLLB(pH7.5)的250mL锥形瓶中，含有50μg/mL的博莱霉素，于37℃条件下120rpm培养4-6h。提取大肠杆菌Top10的重组载体并进行酶切，将酶切后的重组载体电转到毕赤酵母GS115中，然后将转化子涂布于含有5g葡萄糖，0.15％七叶苷，1.5％柠檬酸铁铵和100ug/mL博莱霉素的YPG(pH6.5)平板中，于30℃培养48-96h。
从第一轮筛选中筛选出所有周围有大黑圈的阳性突变体，用于第二轮筛选。将选择的阳性突变体接种到含有100μLYPG液体培养基和100μg/mL博莱霉素的96孔板中，在30℃下培养48h。将菌液10000rpm离心5分钟，并分别保存其上清液和沉淀物。取25μL稀释上清液与25μL1％水杨苷(溶于50mM柠檬酸盐缓冲液，pH5)在50℃下于96孔板中混合15分钟。然后，添加50μLDNS到混合物中以终止反应。再将反应混合物煮沸10分钟，然后在室温下冷却。最后，使用酶标仪测定混合物在540nm的OD值。
从第二轮筛选中选择具有较高OD值的阳性突变体进行进一步筛选，并接种到装有25mLYPG(pH6.5)的液体培养基中，于30℃培养2天，其中含有100μg/mL的博莱霉素。然后，将菌液在10000g下离心15分钟，以获得上清液。根据DNS法，筛选在50℃下具有较高活性和葡萄糖耐受性的突变体SVS。
实施例4
表征β-葡萄糖苷酶及其突变体SVS的酶学性质
酶活的测定：取50μL适当稀释后酶液，加入450μL1％水杨苷(水杨苷事先溶于pH5.0，50mM柠檬酸盐缓冲液中)，混匀后，于50℃反应一段时间，最后加入500μLDNS试剂终止反应，并立即将反应物于沸水中煮沸10-15min，待冷却至室温后，采用分光光度计于540nm处检测OD值。
最适温度的测定：将β-葡萄糖苷酶及其突变体SVS分别与含有水杨苷的50mM醋酸盐缓冲液，pH5在不同温度下(35-80℃下持续30分钟)反应，并通过DNS方法测定其活性。
葡萄糖耐受性的测定：取25μL稀释的16BGL或其突变体以及不同浓度的4-硝基苯基-β-D-葡萄糖苷(pNPG)和葡萄糖混合，加入pH5.0柠檬酸缓冲液至总体积为500μL。在50℃下反应15分钟后，向混合物中加入等体积10％(w/v)碳酸钠以终止反应，并在420nm处获得混合物的OD值，其中，pNPG的终浓度分别为0.1、0.2、0.4、0.6、0.8和1mmol/L，并且控制葡萄糖终浓度分别为0、20和40mmol/L。根据Lineweaver-Burk倒数图，使用Michaelis-Menten方程(1)和竞争抑制方程(2)计算Vmax，Km和KI(葡萄糖抑制常数)。
Figure BDA0002509432150000041
Figure BDA0002509432150000042
由表1及图1可知，突变体SVS的催化效率常数kcat/Km达到了1354.94mM-1S-1，葡萄糖抑制强度Kappm/KI值为1.13(当20mmol/L葡萄糖时)，分别是原始酶的1.68倍和0.89倍(这个突变体的葡萄糖耐受性提高了12％)，体现了更高的产物耐受性和活性。
表1分子动力学常数和葡萄糖抑制分子动力学常数
Figure BDA0002509432150000043
实施例5
β-葡萄糖苷酶突变体SVS在纤维素乙醇生产上的应用
取250mL的锥形瓶摇瓶，加入10g微晶纤维素为底物，20IU的滤纸酶液，0.06mg的β-葡萄糖苷酶或其突变体SVS，以及等量的经发酵48h的酿酒酵母UV-20，再定容至100mL。对照则不加入任何β-葡萄糖苷酶，其余均不变。
于40℃培养箱静置培养，每隔12h从中取1mL菌液进行离心，取上清使用重铬酸钾方法检测其中酒精浓度。
如图2所示，发酵120小时后，突变体SVS产生6.85g/L的纤维素乙醇，分别比草酸青霉16的β-葡萄糖苷酶和对照组高出10％和36％。因此，突变体SVS在纤维素乙醇生产上具有潜在的应用价值。
序列表
<110> 江西师范大学
<120> 1、一种草酸青霉16的β-葡萄糖苷酶突变体SVS及应用
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Leu Tyr Leu Trp Pro Phe Val Asp Ser Val Arg Ala Gly Val Gly Ser
210 215 220
Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Ser Asn
225 230 235 240
Ser Tyr Thr Leu Asn Lys Leu Leu Lys Gly Glu Leu Gly Phe Gln Gly
245 250 255
Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly Val Gly Asp Ala
260 265 270
Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Ile Leu Gly Ser
275 280 285
Pro Tyr Ser Phe Trp Gly Thr Asn Leu Thr Val Ser Val Leu Asn Ser
290 295 300
Thr Ile Pro Glu Trp Arg Leu Asp Asp Met Ala Val Arg Ile Met Ala
305 310 315 320
Ala Tyr Tyr Lys Val Gly Arg Asp Arg His Arg Thr Pro Pro Asn Phe
325 330 335
Ser Ser Trp Thr Arg Asp Lys Tyr Gly Tyr Glu His Phe Ile Val Gln
340 345 350
Glu Asn Tyr Val Lys Leu Asn Glu Arg Val Asn Val Gln Arg Asp His
355 360 365
Ala Asn Val Ile Arg Lys Ile Gly Ser Asp Ser Ile Val Met Leu Lys
370 375 380
Asn Asn Gly Gly Leu Pro Leu Thr His Gln Glu Arg Leu Val Ala Ile
385 390 395 400
Leu Gly Glu Asp Ala Gly Ser Asn Ala Tyr Gly Ala Asn Ser Cys Ser
405 410 415
Asp Arg Gly Cys Val Asn Gly Thr Leu Ala Met Gly Trp Gly Ser Gly
420 425 430
Thr Ala Asn Phe Pro Tyr Leu Ile Ser Pro Glu Gln Ala Ile Gln Asn
435 440 445
Glu Val Leu Asn Tyr Gly Asn Gly Asp Thr Asn Val Phe Ala Val Thr
450 455 460
Asp Asn Gly Ala Leu Ser Gln Met Ala Ala Leu Ala Ser Thr Ala Ser
465 470 475 480
Val Ala Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile Ser
485 490 495
Val Asp Gly Asn Glu Gly Asp Arg Lys Asn Met Thr Leu Trp Lys Asn
500 505 510
Gly Glu Glu Leu Ile Lys Thr Ala Thr Ala Asn Cys Asn Asn Thr Ile
515 520 525
Val Ile Met His Thr Pro Asn Ala Val Leu Val Asp Ser Trp Tyr Asp
530 535 540
Asn Glu Asn Ile Thr Ala Ile Leu Trp Ala Gly Met Pro Gly Gln Glu
545 550 555 560
Ser Gly Arg Ser Leu Val Asp Val Leu Tyr Gly Arg Thr Asn Pro Gly
565 570 575
Gly Lys Thr Pro Phe Thr Trp Gly Lys Glu Arg Lys Asp Trp Gly Ser
580 585 590
Pro Leu Leu Thr Lys Pro Asn Asn Gly His Gly Ala Pro Gln Asp Asp
595 600 605
Phe Thr Asp Val Leu Ile Asp Tyr Arg Arg Phe Asp Lys Asp Asn Val
610 615 620
Glu Pro Ile Phe Glu Phe Gly Phe Gly Leu Ser Tyr Thr Lys Phe Glu
625 630 635 640
Phe Ser Asp Ile Gln Val Lys Ala Leu Asn His Gly Glu Tyr Asn Ala
645 650 655
Thr Val Gly Lys Thr Lys Pro Ala Pro Ser Leu Gly Lys Pro Gly Asn
660 665 670
Ala Ser Asp His Leu Phe Pro Ser Asn Ile Asn Arg Val Arg Gln Tyr
675 680 685
Leu Tyr Pro Tyr Leu Asn Ser Thr Asp Leu Lys Ala Ser Ala Asn Asp
690 695 700
Pro Asp Tyr Gly Met Asn Ala Ser Ala Tyr Ile Pro Pro His Ala Thr
705 710 715 720
Asp Ser Asp Pro Gln Asp Leu Leu Pro Ala Ser Gly Pro Ser Gly Gly
725 730 735
Asn Pro Gly Leu Phe Glu Asp Leu Ile Glu Val Thr Ala Thr Val Thr
740 745 750
Asn Thr Gly Ser Val Thr Gly Asp Glu Val Pro Gln Leu Tyr Val Ser
755 760 765
Leu Gly Gly Ala Asp Asp Pro Val Lys Val Leu Arg Ala Phe Asp Arg
770 775 780
Val Thr Ile Ala Pro Gly Gln Lys Leu Arg Trp Thr Ala Thr Leu Asn
785 790 795 800
Arg Arg Asp Leu Ser Asn Trp Asp Val Pro Ser Gln Asn Trp Ile Ile
805 810 815
Ser Asp Ala Pro Lys Lys Val Trp Val Gly Asn Ser Ser Arg Lys Leu
820 825 830
Pro Leu Ser Ala Asp Leu Pro Lys Val Gln
835 840
<210> 4
<211> 2526
<212> DNA
<213> Mutant gene
<400> 4

Claims (5)

1.一种草酸青霉16的β-葡萄糖苷酶突变体SVS，其特征在于：所述的β-葡萄糖苷酶突变体SVS的氨基酸序列为SEQ ID NO. 3，其编码基因的核酸序列为SEQ ID NO. 4；草酸青霉16的β-葡萄糖苷酶为SEQ ID NO. 1所示的氨基酸序列的第414位甘氨酸突变为丝氨酸、第421位的天冬氨酸突变为缬氨酸以及第441位的苏氨酸突变为丝氨酸。
2.根据权利要求1所述的一种草酸青霉16的β-葡萄糖苷酶突变体SVS，其特征在于：所述的编码基因通过插入到载体pGAPZaA，插入位点为Not I和EcoR I，获得重组载体pGAPZaA-SVS。
3.根据权利要求2所述的一种草酸青霉16的β-葡萄糖苷酶突变体SVS，其特征在于：所述的重组载体pGAPZaA- SVS用Bgl II限制性内切酶线性化后，再电转到毕赤酵母GS115，获得重组毕赤酵母GS115工程菌。
4.根据权利要求3所述的一种草酸青霉16的β-葡萄糖苷酶突变体SVS，其特征在于：所述的重组毕赤酵母GS115工程菌用于生产β-葡萄糖苷酶突变体SVS。
5.一种草酸青霉16的β-葡萄糖苷酶突变体SVS应用，其特征在于：所述的β-葡萄糖苷酶突变体SVS应用的条件是反应温度为30-60℃，pH为3-6。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114703165A (zh) * 2022-04-02 2022-07-05 天津科技大学 一种β-葡萄糖苷酶突变体及其应用
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106854237A (zh) * 2016-12-29 2017-06-16 广西大学 功能蛋白pox08415及其编码基因与应用
CN109134630A (zh) * 2018-09-10 2019-01-04 广西大学 功能蛋白pox03016及其编码基因与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106854237A (zh) * 2016-12-29 2017-06-16 广西大学 功能蛋白pox08415及其编码基因与应用
CN109134630A (zh) * 2018-09-10 2019-01-04 广西大学 功能蛋白pox03016及其编码基因与应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
QIUXIA HUANG等: "Enhancing cellulosic ethanol production through coevolution of multiple enzymatic characteristics of β-glucosidase from Penicillium oxalicum 16", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY VOLUME》 *
ZHAO,X.等: "beta-glucosidase, partial [Penicillium oxalicum]", 《GENBNAK》 *
易拾: "木质纤维素酶的生产优化、定向进化和生物乙醇应用研究", 《中国优秀硕士学位论文全文数据库》 *
赵喜华等: "β-葡萄糖苷酶最适温度及耐酸性的定向进化", 《 中国生物工程学会第十二届学术年会暨2018年全国生物技术大会》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114703165A (zh) * 2022-04-02 2022-07-05 天津科技大学 一种β-葡萄糖苷酶突变体及其应用
CN114703165B (zh) * 2022-04-02 2023-06-27 天津科技大学 一种β-葡萄糖苷酶突变体及其应用
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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