CN109234220A - 一株枯草芽孢杆菌基因重组菌及其构建方法与应用 - Google Patents
一株枯草芽孢杆菌基因重组菌及其构建方法与应用 Download PDFInfo
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- CN109234220A CN109234220A CN201811300842.3A CN201811300842A CN109234220A CN 109234220 A CN109234220 A CN 109234220A CN 201811300842 A CN201811300842 A CN 201811300842A CN 109234220 A CN109234220 A CN 109234220A
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- ala
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- bacillus subtilis
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Abstract
本发明公开了一株枯草芽孢杆菌基因工程菌，该枯草芽孢杆菌中导入了耐热蔗糖磷酸化酶基因，所述耐热蔗糖磷酸化酶基因的核苷酸序列如SEQ ID NO.2所示。本发明还公开了利用上述耐热蔗糖磷酸化酶生物催化制备甘油葡萄糖苷的方法，得到的甘油葡萄糖苷纯度高、成本低，为甘油葡萄糖苷实现规模化生物制备奠定基础。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一株枯草芽孢杆菌基因重组菌及其构建方法与应用。
背景技术
Glycoin是一种新颖的皮肤保湿与修复因子，提取自名为卷柏的沙漠植物，其化学结构名为甘油葡萄糖苷(英文名2-O-(α-D-glucopyranosyl)-sn-glycerol，αGG，图1)。使用卷柏为原料提取Glycoin较难实现，受限于产量和较低的提取率等原因。开发生物技术制备Glycoin是迫切的需求，已报道的制备方法存在诸多问题，包括酶的稳定性差，酶活力低、副产物多，提取困难、使用大肠杆菌等不安全宿主作为生产菌株等。本发明希望解决以上问题，通过枯草芽孢杆菌系统与信号肽结合使用实现关键酶的高效分泌表达，通过微生物发酵去除杂质，一步法获取纯净的产品。
参考文献：
1.Goedl，C.,Sawangwan，T.，Mueller，M.,Schwarz，A.，&Nidetzky，B.(2008).AHigh‐Yielding Biocatalytic Process for the Production of 2‐O‐(α‐D‐glucopyranosyl)‐sn‐glycerol,a Natural Osmolyte and Useful MoisturizingIngredient.Angewandte Chemie International Edition,47(52),10086-10089.
2.Luley-Goedl,C.,Sawangwan,T.,Mueller,M.,Schwarz,A.,&Nidetzky,B.(2010).Biocatalytic process for production ofα-glucosylglycerol using sucrosephosphorylase.Food Technol Biotechnol,48,276-283.
发明内容
本发明要解决的技术问题是通过枯草芽孢杆菌系统与信号肽结合使用实现催化甘油葡萄糖苷关键酶的高效分泌表达，并获取纯净的发酵产品。
为解决上述技术问题，本发明采用如下技术方案：
一株枯草芽孢杆菌基因工程菌，该枯草芽孢杆菌中导入了耐热蔗糖磷酸化酶基因，所述耐热蔗糖磷酸化酶基因的核苷酸序列如SEQ ID NO.2所示。本发明中的耐热蔗糖磷酸化酶可以催化甘油、葡萄糖生成甘油葡萄糖苷，具有催化效率高的特点。
作为优选，所述枯草芽孢杆菌为枯草芽孢杆菌WB800。
作为优选，所述耐热蔗糖磷酸化酶基因前克隆有信号肽序列。
作为优选，所述信号肽的核苷酸序列如SEQ ID NO.3所示。
上述枯草芽孢杆菌基因工程菌的构建方法，包括如下步骤：
(1)将SEQ ID NO.3、SEQ ID NO.2所示的基因序列同时导入表达质粒中，得到重组质粒；
(2)将重组质粒转化枯草芽孢杆菌，既得到该枯草芽孢杆菌基因工程菌。
作为优选，所述表达质粒为pHT-01。
作为优选，所述枯草芽孢杆菌为枯草芽孢杆菌WB800。
上述草芽孢杆菌基因工程菌在制备甘油葡萄糖苷中的应用在本发明的保护范围之内。
上述枯草芽孢杆菌基因工程菌发酵得到的耐热蔗糖磷酸化酶在本发明的保护范围之内。
以枯草芽孢杆菌基因工程菌发酵得到的耐热蔗糖磷酸化酶为催化剂，蔗糖、甘油为底物催化制备甘油葡萄糖苷，催化结束后，再利用地衣芽孢杆菌ATCC14580消耗剩余的蔗糖、甘油，制备得到高纯度甘油葡萄糖苷。
有益效果：
本发明公开了利用耐热蔗糖磷酸化酶生物催化制备甘油葡萄糖苷的方法，得到的甘油葡萄糖苷纯度高、成本低，为甘油葡萄糖苷实现规模化生物制备奠定基础。
附图说明
图1蔗糖磷酸化酶编码基因的PCR产物，泳道1为DNA Marker，泳道2为蔗糖磷酸化酶基因。
图2pHT-01质粒图谱示意图。
图3蔗糖磷酸化酶的SDS-PAGE图谱，泳道1：本发明重组枯草芽孢杆菌WB800的发酵液、泳道2：80℃热水浴半小时离心去除沉淀蛋白的酶液、泳道3：蛋白质Marker，分子量25～166kDa。
图4本发明HPLC检测最终的Glycoin产品峰图，Glycoin的出峰时间为31.320分钟、未知杂质出峰时间为48.062分钟。
具体实施方式
根据下述实施例，可以更好地理解本发明。实施例所描述的具体物料比、工艺条件及其结果仅用于说明本发明，而不应该也不会限制本发明。
实施例1：克隆表达蔗糖磷酸化酶。
以序列SEQ ID No.1的氨基酸序列为模板，委托上海生工生物公司全合成编码基因DNA(序列SEQ ID No.2)，PCR扩增出目的片段(DNA序列N端接XbaI酶切位点，C端接SmaI，引物序列见附录)，胶回收纯化后备用(琼脂糖凝胶电泳图见附图1)。将pHT-01质粒(见附图2)用限制性内切酶XbaI和SmaI双酶切，胶回收纯化后备用。使用诺唯赞一步克隆酶连接DNA片段与载体，转化E.coli DH5α，涂布含100μg/mL的氨苄青霉素LB固体培养基(酵母粉5g/L、蛋白胨10g/L、氯化钠10g/L、琼脂20g/L)平板，获得单克隆。挑取单克隆于含100μg/mL氨苄青霉素的LB液体培养基(酵母粉5g/L、蛋白胨10g/L、氯化钠10g/L)过夜培养，用Axygen质粒提取试剂盒提取质粒。将质粒送南京金斯瑞生物科技有限公司测序，测序正确后即可用于下一步实验。通过PCR在编码基因5’端ATG前引入信号肽序列(序列见SEQ ID NO.3，氨基酸序列见SEQ ID NO.4)，方法与上述克隆方法相同，最终获得重组质粒。将1微升重组质粒转化枯草芽孢杆菌WB800，涂布含34μg/mL的氯霉素LB固体培养基平板，获得单克隆，挑取单克隆测序，测序正确的克隆即为阳性单克隆。将枯草芽孢杆菌单克隆于LB液体培养基培养(温度32℃、摇床转速220rpm、三角摇瓶带内置挡板、装液量20％)，当OD600达到1.0时加入终浓度1mM的IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-thiogalactoside)诱导剂，于25℃继续培养24小时。4000rpm离心30分钟去除菌体，保留发酵液。
实施例2：酶液的制备与催化方法。
将发酵液于80℃热水浴半小时，8000rpm离心20分钟去除变性蛋白质作为酶液4℃保存，待用(酶活力2.0U/mL酶液，酶纯度通过SDS-PAGE电泳鉴定，结果见附图3)。制备含700g/L蔗糖和200g/L甘油的水溶液，此溶液与酶液体积比1：1混匀，65℃水浴摇床反应24小时(200rpm摇速)，100℃热处理10分钟将酶灭活，8000rpm离心20分钟去除沉淀蛋白，反应液4℃保存，待用。
实施例3：发酵法去除杂质。
将地衣芽孢杆菌ATCC14580以10％体积接种量接种于反应液，并加入终浓度5g/L的安琪酵母粉，32℃发酵24小时，发酵结束后离心去除菌体。将终反应液通过平均分子量1000Da的纳滤膜，流出液即为高纯度的Glycoin，50℃抽真空旋蒸浓缩至200g/L终浓度-20℃冷冻保存。样品通过HPLC检测纯度＞95％、仅含有一种杂质(见附图4)，检测方法为如下：稀释样品至约10g/L浓度，使用液相系统为安捷伦1200LC型，液相柱Rezex RCM-Monosaccharide Ca2+column(美国菲罗门公司，尺寸300×7.8mm)，流动相为脱气后的纯水，温度80℃，流速0.5毫升/分钟；产物利用示差折光检测器SHODEX RI-101检测；Glycoin产物标准品购买自美国Sigma-Aldrich公司，使用浓度10g/L。
实施例4：利用7L全自动发酵罐培养重组枯草芽孢杆菌。
在三角摇瓶培养重组枯草芽孢杆菌的基础上，利用7L全自动发酵罐培养重组菌可以获得更高的单位体积酶活力。方法如下：在7L发酵罐中配置4L发酵培养基，成分为：15g/L蔗糖,15g/L安琪酵母粉,5g/L奥博星蛋白胨,5g/L氯化钠,0.5g/L无水硫酸镁,6g/L磷酸氢二钾·三水和2g/L磷酸二氢钾。121℃高温湿热灭菌20分钟后待用(蔗糖、无水硫酸镁与其他组分分开灭菌)，按照前述三角摇瓶培养菌种的方法制备种子液，过夜培养，以体积比5％接种到发酵罐中，培养温度37℃，搅拌桨转速400rpm，通气量3L/分钟，以50％体积比的氨水作为碱液实时调节pH。3小时后降低温度到32℃，加入终浓度1mM的IPTG，继续发酵16小时，发酵过程中采取恒速补料，补料流速25mL/小时，补料液为600g/L葡萄糖、10g/L硫酸镁的水溶液(121℃湿热灭菌20分钟)，当菌体OD600数值超过30时调整转速为600rpm，通气量6L/分钟。发酵完成后OD600在80左右，离心去除菌体，按照前述方法制备为酶液，酶活力一般在20U/mL，是三角摇瓶法的10倍。
实施例5：酶活力测定方法。
1个单位(U)的酶活力定义为单位时间内在65℃催化蔗糖和甘油产生1μmolGlycoin所需的酶量。一般方法如下：取蔗糖和甘油配置成终浓度50g/L蔗糖和20g/L甘油的水溶液，取0.5毫升此水溶液与0.5毫升酶液在EP管中混匀，65℃水浴反应30分钟。100℃反应10分钟灭活，12000rpm离心10分钟去除沉淀蛋白。将反应液用纯水稀释5倍，用注射器过0.2μm水系滤膜，通过HPLC检测Glycoin产物峰，计算产物浓度。
序列表
<110> 南京工业大学
<120> 一株枯草芽孢杆菌基因重组菌及其构建方法与应用
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Ser Tyr Leu Leu Ala Arg Ala Ile Gln Cys Phe Ala Pro Gly Ile Pro
355 360 365
Gln Ile Tyr Tyr Val Gly Leu Leu Ala Gly Glu Asn Asp Ile Glu Leu
370 375 380
Leu Glu Arg Thr Lys Glu Gly Arg Asn Ile Asn Arg His Tyr Tyr Thr
385 390 395 400
Leu Glu Glu Ile Glu Arg Glu Val Glu Arg Pro Val Val Lys Gln Leu
405 410 415
Phe Arg Leu Leu Lys Phe Arg Asn Thr Cys Pro Ala Phe Asp Gly Thr
420 425 430
Val Glu Ala Glu Gln Ala Asp Ile Asn Asn Leu Arg Ile Ile Trp Lys
435 440 445
Asn Gly Ala Ser Glu Ala Lys Leu Glu Ala Asn Leu Ala Thr Lys Glu
450 455 460
Phe Ser Ile Arg Tyr Lys Asp Ala Gly Thr Asn Trp Thr Leu Leu Met
465 470 475 480
<210> 2
<211> 1443
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ala Thr Gly Ala Ala Ala Ala Thr Cys Ala Ala Ala Ala Ala Cys Gly
1 5 10 15
Ala Ala Gly Cys Thr Ala Thr Gly Cys Thr Thr Ala Thr Cys Ala Cys
20 25 30
Ala Thr Ala Cys Gly Cys Thr Gly Ala Thr Thr Cys Thr Cys Thr Thr
35 40 45
Gly Gly Cys Ala Cys Ala Ala Ala Cys Cys Thr Thr Ala Ala Ala Gly
50 55 60
Ala Ala Cys Thr Thr Ala Ala Cys Cys Ala Ala Gly Thr Thr Cys Thr
65 70 75 80
Thr Gly Ala Thr Ala Ala Ala Cys Ala Thr Cys Thr Thr Cys Ala Ala
85 90 95
Gly Gly Cys Gly Thr Thr Gly Thr Thr Gly Gly Cys Gly Gly Cys Gly
100 105 110
Thr Thr Cys Ala Thr Cys Thr Thr Cys Thr Thr Cys Cys Thr Thr Thr
115 120 125
Cys Thr Ala Cys Cys Cys Thr Thr Cys Thr Thr Cys Thr Gly Gly Cys
130 135 140
Gly Ala Thr Cys Gly Thr Gly Gly Cys Thr Thr Cys Gly Cys Thr Cys
145 150 155 160
Cys Thr Ala Thr Gly Gly Ala Thr Thr Ala Cys Ala Cys Ala Ala Ala
165 170 175
Ala Gly Thr Thr Gly Ala Thr Cys Cys Thr Gly Cys Thr Thr Thr Cys
180 185 190
Gly Gly Cys Gly Ala Thr Thr Gly Gly Thr Cys Thr Gly Ala Thr Gly
195 200 205
Thr Thr Gly Ala Ala Cys Ala Ala Ala Thr Gly Thr Cys Thr Cys Ala
210 215 220
Ala Ala Ala Ala Thr Ala Cys Thr Ala Cys Ala Thr Gly Ala Thr Gly
225 230 235 240
Thr Ala Cys Gly Ala Thr Thr Thr Cys Ala Thr Gly Ala Thr Cys Ala
245 250 255
Ala Cys Cys Ala Thr Ala Thr Cys Thr Cys Thr Cys Gly Thr Cys Ala
260 265 270
Ala Thr Cys Thr Cys Cys Thr Thr Ala Cys Thr Thr Cys Cys Ala Ala
275 280 285
Gly Ala Thr Thr Thr Cys Cys Thr Thr Gly Ala Ala Ala Ala Cys Ala
290 295 300
Ala Ala Gly Ala Thr Gly Ala Ala Thr Cys Thr Gly Cys Thr Thr Ala
305 310 315 320
Cys Ala Ala Ala Gly Ala Thr Cys Thr Thr Thr Thr Cys Ala Thr Cys
325 330 335
Cys Gly Thr Thr Ala Cys Ala Ala Ala Ala Ala Cys Thr Thr Cys Thr
340 345 350
Gly Gly Cys Cys Thr Gly Gly Cys Gly Gly Cys Gly Ala Ala Cys Cys
355 360 365
Thr Ala Cys Ala Cys Cys Thr Gly Ala Ala Gly Ala Thr Gly Thr Thr
370 375 380
Gly Ala Thr Cys Thr Thr Ala Thr Cys Thr Ala Cys Ala Ala Ala Cys
385 390 395 400
Gly Thr Ala Ala Ala Cys Cys Thr Cys Gly Thr Gly Cys Thr Cys Cys
405 410 415
Thr Thr Ala Cys Gly Thr Thr Gly Ala Ala Gly Thr Thr Ala Cys Ala
420 425 430
Thr Thr Cys Ala Ala Ala Gly Ala Thr Gly Gly Cys Thr Cys Thr Ala
435 440 445
Cys Ala Gly Ala Ala Ala Ala Ala Gly Thr Thr Thr Gly Gly Thr Gly
450 455 460
Cys Ala Cys Ala Thr Thr Cys Gly Ala Thr Gly Ala Ala Cys Ala Ala
465 470 475 480
Cys Ala Ala Ala Thr Cys Gly Ala Thr Cys Thr Thr Gly Ala Thr Gly
485 490 495
Thr Thr Ala Cys Ala Ala Cys Ala Gly Ala Thr Ala Cys Ala Ala Cys
500 505 510
Ala Ala Ala Ala Ala Ala Ala Thr Thr Cys Ala Thr Cys Cys Gly Thr
515 520 525
Gly Ala Thr Ala Ala Cys Cys Thr Thr Ala Cys Ala Thr Thr Cys Cys
530 535 540
Thr Thr Gly Cys Thr Cys Ala Ala Ala Ala Ala Gly Gly Cys Gly Cys
545 550 555 560
Thr Thr Cys Thr Ala Thr Cys Ala Thr Cys Cys Gly Thr Cys Thr Thr
565 570 575
Gly Ala Thr Gly Cys Thr Thr Thr Cys Gly Cys Thr Thr Ala Cys Gly
580 585 590
Cys Thr Ala Ala Cys Ala Ala Ala Ala Ala Ala Ala Thr Cys Gly Gly
595 600 605
Cys Ala Cys Ala Ala Ala Cys Thr Gly Cys Thr Thr Cys Thr Thr Cys
610 615 620
Gly Thr Thr Gly Ala Ala Cys Cys Thr Gly Ala Thr Ala Thr Cys Thr
625 630 635 640
Gly Gly Gly Ala Thr Ala Thr Gly Cys Thr Thr Cys Gly Thr Thr Ala
645 650 655
Cys Thr Cys Thr Ala Ala Ala Gly Ala Thr Ala Thr Cys Ala Thr Cys
660 665 670
Thr Cys Thr Cys Cys Thr Thr Cys Thr Gly Gly Cys Ala Thr Cys Ala
675 680 685
Cys Ala Gly Thr Thr Cys Thr Thr Cys Cys Thr Gly Ala Ala Ala Thr
690 695 700
Cys Cys Ala Thr Gly Ala Ala Cys Ala Thr Thr Ala Cys Thr Cys Thr
705 710 715 720
Ala Thr Cys Cys Ala Ala Cys Thr Thr Ala Ala Ala Ala Thr Cys Gly
725 730 735
Cys Thr Gly Ala Ala Cys Ala Ala Gly Ala Thr Thr Ala Cys Thr Ala
740 745 750
Cys Gly Thr Thr Thr Ala Cys Gly Ala Thr Thr Thr Cys Gly Cys Thr
755 760 765
Cys Thr Thr Cys Cys Thr Ala Thr Gly Cys Thr Thr Gly Thr Thr Cys
770 775 780
Thr Thr Cys Ala Thr Gly Cys Thr Cys Thr Thr Thr Ala Cys Thr Cys
785 790 795 800
Thr Gly Gly Cys Cys Ala Ala Gly Thr Thr Cys Ala Thr Cys Gly Thr
805 810 815
Cys Thr Thr Gly Thr Thr Cys Ala Thr Thr Gly Gly Cys Thr Thr Ala
820 825 830
Ala Cys Ala Thr Cys Thr Gly Cys Cys Cys Thr Cys Gly Thr Ala Ala
835 840 845
Ala Cys Ala Ala Thr Thr Cys Ala Cys Ala Ala Cys Ala Cys Thr Thr
850 855 860
Gly Ala Thr Ala Cys Ala Cys Ala Thr Gly Ala Thr Gly Gly Cys Ala
865 870 875 880
Thr Cys Gly Gly Cys Gly Thr Thr Gly Thr Thr Gly Ala Thr Gly Thr
885 890 895
Thr Ala Ala Ala Gly Ala Thr Cys Thr Thr Cys Thr Thr Thr Cys Thr
900 905 910
Gly Ala Thr Gly Ala Ala Gly Ala Ala Thr Gly Cys Gly Ala Ala Ala
915 920 925
Thr Gly Ala Cys Ala Cys Gly Thr Gly Ala Ala Thr Cys Thr Cys Thr
930 935 940
Thr Thr Ala Cys Thr Cys Thr Cys Ala Ala Gly Gly Cys Gly Cys Thr
945 950 955 960
Ala Ala Cys Gly Thr Thr Ala Ala Ala Ala Ala Ala Ala Thr Cys Thr
965 970 975
Ala Cys Thr Cys Thr Ala Cys Ala Gly Ala Ala Gly Cys Thr Thr Ala
980 985 990
Cys Ala Ala Cys Ala Ala Cys Cys Thr Thr Gly Ala Thr Ala Thr Cys
995 1000 1005
Thr Ala Cys Cys Ala Ala Ala Thr Cys Ala Ala Cys Thr Gly Cys Ala
1010 1015 1020
Cys Ala Thr Ala Cys Thr Ala Cys Thr Cys Thr Gly Cys Thr Cys Thr
1025 1030 1035 1040
Thr Gly Gly Cys Ala Ala Cys Ala Ala Cys Gly Ala Thr Cys Ala Ala
1045 1050 1055
Thr Cys Thr Thr Ala Cys Cys Thr Thr Cys Thr Thr Gly Cys Thr Cys
1060 1065 1070
Gly Thr Gly Cys Thr Ala Thr Cys Cys Ala Ala Thr Gly Cys Thr Thr
1075 1080 1085
Cys Gly Cys Thr Cys Cys Thr Gly Gly Cys Ala Thr Cys Cys Cys Thr
1090 1095 1100
Cys Ala Ala Ala Thr Cys Thr Ala Cys Thr Ala Cys Gly Thr Thr Gly
1105 1110 1115 1120
Gly Cys Cys Thr Thr Cys Thr Thr Gly Cys Thr Gly Gly Cys Gly Ala
1125 1130 1135
Ala Ala Ala Cys Gly Ala Thr Ala Thr Cys Gly Ala Ala Cys Thr Thr
1140 1145 1150
Cys Thr Thr Gly Ala Ala Cys Gly Thr Ala Cys Ala Ala Ala Ala Gly
1155 1160 1165
Ala Ala Gly Gly Cys Cys Gly Thr Ala Ala Cys Ala Thr Cys Ala Ala
1170 1175 1180
Cys Cys Gly Thr Cys Ala Thr Thr Ala Cys Thr Ala Cys Ala Cys Ala
1185 1190 1195 1200
Cys Thr Thr Gly Ala Ala Gly Ala Ala Ala Thr Cys Gly Ala Ala Cys
1205 1210 1215
Gly Thr Gly Ala Ala Gly Thr Thr Gly Ala Ala Cys Gly Thr Cys Cys
1220 1225 1230
Thr Gly Thr Thr Gly Thr Thr Ala Ala Ala Cys Ala Ala Cys Thr Thr
1235 1240 1245
Thr Thr Cys Cys Gly Thr Cys Thr Thr Cys Thr Thr Ala Ala Ala Thr
1250 1255 1260
Thr Cys Cys Gly Thr Ala Ala Cys Ala Cys Ala Thr Gly Cys Cys Cys
1265 1270 1275 1280
Thr Gly Cys Thr Thr Thr Cys Gly Ala Thr Gly Gly Cys Ala Cys Ala
1285 1290 1295
Gly Thr Thr Gly Ala Ala Gly Cys Thr Gly Ala Ala Cys Ala Ala Gly
1300 1305 1310
Cys Thr Gly Ala Thr Ala Thr Cys Ala Ala Cys Ala Ala Cys Cys Thr
1315 1320 1325
Thr Cys Gly Thr Ala Thr Cys Ala Thr Cys Thr Gly Gly Ala Ala Ala
1330 1335 1340
Ala Ala Cys Gly Gly Cys Gly Cys Thr Thr Cys Thr Gly Ala Ala Gly
1345 1350 1355 1360
Cys Thr Ala Ala Ala Cys Thr Thr Gly Ala Ala Gly Cys Thr Ala Ala
1365 1370 1375
Cys Cys Thr Thr Gly Cys Thr Ala Cys Ala Ala Ala Ala Gly Ala Ala
1380 1385 1390
Thr Thr Cys Thr Cys Thr Ala Thr Cys Cys Gly Thr Thr Ala Cys Ala
1395 1400 1405
Ala Ala Gly Ala Thr Gly Cys Thr Gly Gly Cys Ala Cys Ala Ala Ala
1410 1415 1420
Cys Thr Gly Gly Ala Cys Ala Cys Thr Thr Cys Thr Thr Ala Thr Gly
1425 1430 1435 1440
Thr Ala Ala
<210> 3
<211> 39
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ala Thr Gly Cys Gly Thr Ala Ala Ala Ala Ala Ala Ala Cys Gly Ala
1 5 10 15
Ala Ala Ala Ala Cys Cys Gly Thr Cys Thr Gly Ala Thr Thr Thr Cys
20 25 30
Gly Thr Cys Gly Gly Thr Thr
35
<210> 4
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Arg Lys Lys Thr Lys Asn Arg Leu Ile Ser Ser Val Leu Ser Thr
1 5 10 15
<210> 5
<211> 46
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Ala Gly Gly Ala Gly Gly Ala Ala Gly Gly Ala Thr Cys Cys Thr
1 5 10 15
Cys Thr Ala Gly Ala Ala Thr Gly Ala Ala Ala Ala Thr Cys Ala Ala
20 25 30
Ala Ala Ala Cys Gly Ala Ala Gly Cys Thr Ala Thr Gly Cys
35 40 45
<210> 6
<211> 45
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Cys Ala Thr Thr Ala Gly Gly Cys Gly Gly Gly Cys Thr Gly Cys Cys
1 5 10 15
Cys Cys Gly Gly Gly Cys Ala Thr Ala Ala Gly Ala Ala Gly Thr Gly
20 25 30
Thr Cys Cys Ala Gly Thr Thr Thr Gly Thr Gly Cys Cys
35 40 45
Claims (10)
1.一株枯草芽孢杆菌基因工程菌，其特征在于，该枯草芽孢杆菌中导入了耐热蔗糖磷酸化酶基因，所述耐热蔗糖磷酸化酶基因的核苷酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的枯草芽孢杆菌基因工程菌，其特征在于，所述枯草芽孢杆菌为枯草芽孢杆菌WB800。
3.根据权利要求1所述的枯草芽孢杆菌基因工程菌，其特征在于，所述耐热蔗糖磷酸化酶基因前克隆有信号肽序列。
4.根据权利要求3所述的枯草芽孢杆菌基因工程菌，其特征在于，所述信号肽的核苷酸序列如SEQ ID NO.3所示。
5.权利要求1所述枯草芽孢杆菌基因工程菌的构建方法，其特征在于，包括如下步骤：
(1)将SEQ ID NO.3、SEQ ID NO.2所示的基因序列同时导入表达质粒中，得到重组质粒；
(2)将重组质粒转化枯草芽孢杆菌，既得到该枯草芽孢杆菌基因工程菌。
6.根据权利要求6所述枯草芽孢杆菌基因工程菌的构建方法，其特征在于，所述表达质粒为pHT-01。
7.根据权利要求6所述枯草芽孢杆菌基因工程菌的构建方法，其特征在于，所述枯草芽孢杆菌为枯草芽孢杆菌WB800。
8.权利要求1～4任一所述枯草芽孢杆菌基因工程菌在制备甘油葡萄糖苷中的应用。
9.权利要求1～4任一所述枯草芽孢杆菌基因工程菌发酵得到的耐热蔗糖磷酸化酶。
10.根据权利要求8所述的应用，其特征在于，以枯草芽孢杆菌基因工程菌发酵得到的耐热蔗糖磷酸化酶为催化剂，蔗糖、甘油为底物催化制备甘油葡萄糖苷，催化结束后，再利用地衣芽孢杆菌ATCC14580消耗剩余的蔗糖、甘油，制备得到高纯度甘油葡萄糖苷。
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| CN111172127A (zh) * | 2020-01-17 | 2020-05-19 | 浙江工业大学 | 一种蔗糖磷酸化酶在制备甘油葡萄糖苷中的应用 |
| WO2021109467A1 (zh) * | 2019-12-02 | 2021-06-10 | 天津科技大学 | 生产l-精氨酸的基因工程菌及其构建方法与应用 |
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