CN109354627B - 一种提高海藻糖水解酶产量的方法 - Google Patents

一种提高海藻糖水解酶产量的方法 Download PDF

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CN109354627B
CN109354627B CN201811395710.3A CN201811395710A CN109354627B CN 109354627 B CN109354627 B CN 109354627B CN 201811395710 A CN201811395710 A CN 201811395710A CN 109354627 B CN109354627 B CN 109354627B
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谢艳萍
谭启程
崔楠
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Hunan Jindai Technology Development Co.,Ltd.
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Abstract

本发明公开了一种提高海藻糖水解酶产量的方法，属于基因工程技术领域以及酶工程技术领域。此方法通过在海藻糖水解酶亲本的N端通过氨基酸序列如SEQ ID NO.9所示的linker连接氨基酸序列如SEQ ID NO.2所示的短肽大大提高了海藻糖水解酶在宿主细胞中的表达量，对于推动海藻糖的大规模工业化、降低海藻糖的生产成本具有重大的意义。

Description

一种提高海藻糖水解酶产量的方法
技术领域
本发明涉及一种提高海藻糖水解酶产量的方法，属于基因工程技术领域以及酶工程技术领域。
背景技术
海藻糖(Trehalose)是由两个吡喃环葡萄糖以1,1-糖苷键连结而成，是一种稳定的非还原性二糖，它有3种光学异构体，即αα型、αβ型和ββ型。
海藻糖是一种安全的非还原性二糖，广泛存在于自然界中，具有保湿性，抗冻抗干燥性，热酸稳定性等特殊的生物学功能，对生物大分子有着非特异性的保护作用，因此在医学、农业、化妆品、食品等行业应用潜力巨大。自20世纪80年代后，各国相继开展了海藻糖生理生化和分子生物学的研究，该糖现已成为国际上最近开发的主要低聚糖之一。
酶法转化生产海藻糖是上世界90年代逐渐兴起的方法，主要有磷酸化酶法、海藻糖合成酶法和双酶法这三种方法。当前以淀粉为底物经过麦芽寡糖基海藻糖合成酶和麦芽寡糖基海藻糖水解酶的共同作用生成海藻糖，即双酶法生产海藻糖，受到广泛关注。以该方法生产海藻糖的转化率高达80％以上，在一定程度上降低了海藻糖的生产成本并极大的推动了海藻糖的工业化生产进程。
麦芽寡糖基海藻糖水解酶(maltooligosyl trehalose trehalohydrolase,EC3.2.1.141)由基因treZ编码，是双酶法生产海藻糖的其中一种酶。淀粉经过高温液化后加入支链淀粉酶作用生成麦芽糊精，麦芽寡糖基海藻糖合成酶作用于底物还原性末端的α,α-1,4-糖苷，产生α,α-1,4-糖苷键到α,α-1,1-糖苷键的分子内转糖苷作用，形成中间产物麦芽寡糖基海藻糖。麦芽寡糖基海藻糖水解酶则专一的内切该中间产物中麦芽寡糖基与海藻糖相连的α,α-1,4-糖苷键，使之分解产生海藻糖和减少两个葡萄糖单位的新麦芽寡糖，减少两个葡萄糖单位的新麦芽寡糖作为新底物进行下一轮反应，如此反复交替进行，两种酶反应就可以将麦芽寡糖转化成海藻糖为主，以及少量葡萄糖、麦芽糖、麦芽三糖的产物。
双酶法生产海藻糖以淀粉为底物，转化率高达80％以上，具有低成本的优点，但是麦芽寡糖基海藻糖水解酶基因在宿主菌中的可溶性表达很低，产生较多的包涵体，酶活力低，不利于其工业化应用。
发明内容
为解决上述问题，本发明提供了一种提高海藻糖水解酶产量的方法。此方法通过在海藻糖水解酶亲本的N端通过氨基酸序列如SEQ ID NO.9所示的linker连接氨基酸序列如SEQ ID NO.2所示的短肽大大提高了海藻糖水解酶在宿主细胞中的表达量，对于推动海藻糖的大规模工业化、降低海藻糖的生产成本具有重大的意义。
本发明的技术方案如下：
本发明提供了一种海藻糖水解酶突变体，在海藻糖水解酶亲本的N端通过氨基酸序列如SEQ ID NO.9所示的linker连接氨基酸序列如SEQ ID NO.2所示的短肽。
在本发明的一种实施方式中，所述海藻糖水解酶亲本的氨基酸序列如SEQ IDNO.10所示。
在本发明的一种实施方式中，所述海藻糖水解酶突变体的氨基酸序列如SEQ IDNO.11所示。
本发明提供了编码上述突变体的基因。
本发明提供了携带上述基因的重组质粒。
在本发明的一种实施方式中，所述质粒载体为pUC系列、pET系列、或pGEX中的任意一种。
本发明提供了携带上述基因或上述重组质粒的宿主细胞。
在本发明的一种实施方式中，所述宿主细胞为细菌或真菌细胞。
本发明提供了上述突变体或上述基因或上述重组质粒或上述宿主细胞在高效表达海藻糖水解酶以及生产海藻糖方面的应用。
本发明提供了一种高效表达海藻糖水解酶的方法，所述方法为将上述宿主细胞接种至发酵培养基中进行发酵，得到海藻糖水解酶。
有益效果：
本发明通过将来源于嗜酸热硫化叶菌(S.acidocaldarius)的海藻糖水解酶进行改造，在海藻糖水解酶亲本的N端通过氨基酸序列如SEQ ID NO.9所示的linker连接氨基酸序列如SEQ ID NO.2所示的短肽，大大提高了其在宿主细胞中的表达量(较野生型提高了2.6倍)，对于推动海藻糖的大规模工业化、降低海藻糖的生产成本具有重大的意义。
具体实施方式
下面结合具体实施例和对比例对本发明进行进一步的阐述。
下述实施例中涉及的检测方法如下：
酶活检测方法：
预热：取0.45mL的2％麦芽糊精溶液(DE 9～13pH 6.0磷酸缓冲液)于试管内，置于60℃水浴锅中预热10min；
反应：加入0.05mL稀释后的发酵胞内粗酶液，振荡均匀，准确计时10min，加入0.1mL的NaOH溶液(4mol/L)终止反应，取0.3mL的反应液于具塞试管中，加入蒸馏水0.7mL和4mL DNS，振荡均匀，终止反应。煮沸7min，冷却；
测量：向上述反应体系中加入蒸馏水并定容至15mL，混匀；在540nm波长下测定吸光值并计算酶活力。
(酶活定义：每分钟相当于生成一微摩尔麦芽寡糖所需要的酶量)
实施例1：突变体的构建
(1)根据氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ IDNO.12、SEQ ID NO.13所示的短肽(分别命名为P1、P2、P3、P4、P5)，化学合成其基因并分别连接至氨基酸序列分别如SEQ ID NO.10所示的海藻糖水解酶基因序列的N端，并将其克隆至质粒pET24a(+)的Xho I和HindⅢ酶切位点之间，构建得到重组质粒pET24a(+)/P1-enzyme、pET24a(+)/P2-enzyme、pET24a(+)/P3-enzyme、pET24a(+)/P4-enzyme、pET24a(+)/P5-enzyme；
(2)根据氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ IDNO.7、SEQ ID NO.8、SEQ ID NO.9所示的linker(分别命名为L1、L2、L3、L4、L5、L6)，化学合成其基因并分别连接至pMD18-T载体上，得到重组质粒pMD18-T/L1、pMD18-T/L2、pMD18-T/L3、pMD18-T/L4、pMD18-T/L5、pMD18-T/L6；
(3)以上述重组质粒为模板，设计引物并通过PCR获得线性化的(1)中所述的重组质粒片段以及(2)中所述的linker片段，将得到的两种片段进行同源重组，获得混合质粒(或直接通过化学合成得到短肽、linker以及海藻糖水解酶基因连接后的片段并克隆至质粒pET24a(+)的Xho I和HindⅢ酶切位点之间，获得混合质粒)。
实施例2：突变体的验证
将混合质粒以及转化E.coli BL21(DE3)宿主菌，于LB液体培养基(含30μg/mL卡那霉素)生长8～10h，按5％接种量将种子发酵液接到TB培养基(含30μg/mL卡那霉素)中，在37℃摇床中培养48h后，将发酵液于4℃、8000rpm离心10min除菌体，收集离心上清液即为粗酶液。
将得到的粗酶液进行酶活检测，得到粗酶液酶活较含有重组质粒pET24a(+)/enzyme的重组菌更高的重组菌，这些重组菌分别含有重组质粒pET24a(+)/P2-L1-enzyme以及pET24a(+)/P2-L6-enzyme。
将含有重组质粒pET24a(+)/P2-L1-enzyme以及pET24a(+)/P2-L6-enzyme的重组菌发酵得到粗酶液中的海藻糖水解酶酶活与含有重组质粒pET24a(+)/enzyme的重组菌发酵得到粗酶液中的海藻糖水解酶进行比较。
结果如下：含有重组质粒pET24a(+)/P2-L6-enzyme的重组菌发酵得到粗酶液中的海藻糖水解酶酶活(79.3U/mL)较含有重组质粒pET24a(+)/enzyme的重组菌发酵得到粗酶液中的海藻糖水解酶酶活(30.5U/mL)有了明显的提高，为含有重组质粒pET24a(+)/enzyme的重组菌发酵得到粗酶液中的海藻糖水解酶酶活的2.5倍；而含有重组质粒pET24a(+)/P2-L1-enzyme的重组菌发酵得到粗酶液中的海藻糖水解酶酶活(38.2U/mL)较含有重组质粒pET24a(+)/enzyme的重组菌发酵得到粗酶液中的海藻糖水解酶没有明显改变，仅为含有重组质粒pET24a(+)/enzyme的重组菌发酵得到粗酶液中的海藻糖水解酶酶活的1.25倍。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
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Ile Glu Lys Asn Lys Gly Ile Phe Lys Leu Trp Ala Pro Tyr Val Asn
50 55 60
Ser Val Lys Leu Lys Leu Ser Lys Lys Leu Ile Pro Met Glu Lys Asn
65 70 75 80
Asp Glu Gly Phe Phe Glu Val Glu Ile Asp Asp Ile Glu Glu Asn Leu
85 90 95
Thr Tyr Ser Tyr Ile Ile Glu Asp Lys Arg Glu Ile Pro Asp Pro Ala
100 105 110
Ser Arg Tyr Gln Pro Leu Gly Val His Asp Lys Ser Gln Leu Ile Arg
115 120 125
Thr Asp Tyr Gln Ile Leu Asp Leu Gly Lys Val Lys Ile Glu Asp Leu
130 135 140
Ile Ile Tyr Glu Leu His Val Gly Thr Phe Ser Gln Glu Gly Asn Phe
145 150 155 160
Lys Gly Val Ile Glu Lys Leu Asp Tyr Leu Lys Asp Leu Gly Ile Thr
165 170 175
Gly Ile Glu Leu Met Pro Val Ala Gln Phe Pro Gly Asn Arg Asp Trp
180 185 190
Gly Tyr Asp Gly Val Phe Leu Tyr Ala Val Gln Asn Thr Tyr Gly Gly
195 200 205
Pro Trp Glu Leu Ala Lys Leu Val Asn Glu Ala His Lys Arg Gly Ile
210 215 220
Ala Val Ile Leu Asp Val Val Tyr Asn His Ile Gly Pro Glu Gly Asn
225 230 235 240
Tyr Leu Leu Gly Leu Gly Pro Tyr Phe Ser Asp Arg Tyr Lys Thr Pro
245 250 255
Trp Gly Leu Thr Phe Asn Phe Asp Asp Arg Gly Cys Asp Gln Val Arg
260 265 270
Lys Phe Ile Leu Glu Asn Val Glu Tyr Trp Phe Lys Thr Phe Lys Ile
275 280 285
Asp Gly Leu Arg Leu Asp Ala Val His Ala Ile Phe Asp Asn Ser Pro
290 295 300
Lys His Ile Leu Gln Glu Ile Ala Glu Lys Ala His Gln Leu Gly Lys
305 310 315 320
Phe Val Ile Ala Glu Ser Asp Leu Asn Asp Pro Lys Ile Val Lys Asp
325 330 335
Asp Cys Gly Tyr Lys Ile Asp Ala Gln Trp Val Asp Asp Phe His His
340 345 350
Ala Val His Ala Phe Ile Thr Lys Glu Lys Asp Tyr Tyr Tyr Gln Asp
355 360 365
Phe Gly Arg Ile Glu Asp Ile Glu Lys Thr Phe Lys Asp Val Phe Val
370 375 380
Tyr Asp Gly Lys Tyr Ser Arg Tyr Arg Gly Arg Thr His Gly Ala Pro
385 390 395 400
Val Gly Asp Leu Pro Pro Arg Lys Phe Val Val Phe Ile Gln Asn His
405 410 415
Asp Gln Val Gly Asn Arg Gly Asn Gly Glu Arg Leu Ser Ile Leu Thr
420 425 430
Asp Lys Thr Thr Tyr Leu Met Ala Ala Thr Leu Tyr Ile Leu Ser Pro
435 440 445
Tyr Ile Pro Leu Ile Phe Met Gly Glu Glu Tyr Tyr Glu Thr Asn Pro
450 455 460
Phe Phe Phe Phe Ser Asp Phe Ser Asp Pro Val Leu Ile Lys Gly Val
465 470 475 480
Arg Glu Gly Arg Leu Lys Glu Asn Asn Gln Met Ile Asp Pro Gln Ser
485 490 495
Glu Glu Ala Phe Leu Lys Ser Lys Leu Ser Trp Lys Ile Asp Glu Glu
500 505 510
Val Leu Asp Tyr Tyr Lys Gln Leu Ile Asn Ile Arg Lys Arg Tyr Asn
515 520 525
Asn Cys Lys Arg Val Lys Glu Val Arg Arg Glu Gly Asn Cys Ile Thr
530 535 540
Leu Ile Met Glu Lys Ile Gly Ile Ile Ala Ser Phe Asp Asp Ile Val
545 550 555 560
Ile Asn Ser Lys Ile Thr Gly Asn Leu Leu Ile Gly Ile Gly Phe Pro
565 570 575
Lys Lys Leu Lys Lys Asp Glu Leu Ile Lys Val Asn Arg Gly Val Gly
580 585 590
Val Tyr Gln Leu Glu
595
<210> 12
<211> 8
<212> PRT
<213> 人工序列
<400> 12
Leu Glu Leu Glu Leu Lys Leu Lys
1 5
<210> 13
<211> 16
<212> PRT
<213> 人工序列
<400> 13
Leu Glu Leu Glu Leu Lys Leu Lys Leu Glu Leu Glu Leu Lys Leu Lys
1 5 10 15

Claims (9)

1.一种海藻糖水解酶突变体，其特征在于，在海藻糖水解酶亲本的N端通过氨基酸序列如SEQ ID NO.9所示的linker连接氨基酸序列如SEQ ID NO.2所示的短肽；所述海藻糖水解酶亲本的氨基酸序列如SEQ ID NO.10所示。
2.如权利要求1所述的海藻糖水解酶突变体，其特征在于，所述海藻糖水解酶突变体的氨基酸序列如SEQ ID NO.11所示。
3.编码权利要求1或2所述突变体的基因。
4.携带权利要求3所述基因的重组质粒。
5.如权利要求4所述的重组质粒，其特征在于，所述质粒载体为pUC系列、pET系列、或pGEX中的任意一种。
6.携带权利要求3所述基因或权利要求4或5所述重组质粒的宿主细胞。
7.如权利要求6所述的宿主细胞，其特征在于，所述宿主细胞为细菌或真菌细胞。
8.权利要求1或2所述的突变体或权利要求3所述的基因或权利要求4或5所述的重组质粒或权利要求6或7所述的宿主细胞在高效表达海藻糖水解酶以及生产海藻糖方面的应用。
9.一种提高海藻糖水解酶产量的方法，其特征在于，所述方法为将权利要求6或7所述的宿主细胞接种至发酵培养基中进行发酵，得到海藻糖水解酶。
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