CN104004672A - 一种毕赤酵母整合高效表达胞外n-糖基化枯草芽孢杆菌亮氨酸氨肽酶的方法 - Google Patents
一种毕赤酵母整合高效表达胞外n-糖基化枯草芽孢杆菌亮氨酸氨肽酶的方法 Download PDFInfo
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- CN104004672A CN104004672A CN201410227057.5A CN201410227057A CN104004672A CN 104004672 A CN104004672 A CN 104004672A CN 201410227057 A CN201410227057 A CN 201410227057A CN 104004672 A CN104004672 A CN 104004672A
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- aminopeptidase
- pichia pastoris
- leucine aminopeptidase
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Abstract
本发明公开了一种毕赤酵母整合高效表达胞外N-糖基化枯草芽孢杆菌亮氨酸氨肽酶的方法，属于生物酶工程技术领域。本发明所构建的亮氨酸氨肽酶在重组毕赤酵母中的整合表达，不仅具有很好的遗传稳定性，且N-糖基化后的氨肽酶温度稳定性与底物亲和力都获得一定的提升，为氨肽酶的产业化和在食品领域中的应用奠定了优良的基础。
Description
技术领域
一种毕赤酵母整合高效表达胞外N-糖基化枯草芽孢杆菌亮氨酸氨肽酶的方法，属于生物酶工程技术领域。
背景技术
氨肽酶是一类从多肽链或蛋白质N端顺序水解氨基酸，使氨基酸逐个水解分离出来的酶，而亮氨酸氨肽酶则是其中一类主要水解N末端为亮氨酸的氨肽酶。氨肽酶来源广阔，包括动物、植物、微生物。氨肽酶依据作用底物的不同，催化中心所含金属离子的不同以及催化机理的不同可以分为好多类型。氨肽酶在食物中可用于去除蛋白水解物中的苦味，医药上可以用来合成一些药品，分子生物学上可用作一些分子序列测定的工具。
毕赤酵母，作为一种被广泛应用的宿主，已经成功的表达了上百种外源蛋白。它作为一种真核表达宿主与原核相比有许多的优点：高密度发酵，遗传稳定性高，不易染菌，诱导剂便宜，分泌表达等。毕赤酵母会对所表达的蛋白进行翻译后修饰，如糖基化。而糖基化往往会使所表达的蛋白酶的温度稳定性和酶催化效率有所提升。
因此，实现枯草芽孢杆菌氨肽酶在毕赤酵母中的表达将具有重要的意义。
发明内容
本发明的第一个目的是提供胞外分泌表达枯草芽孢杆菌Zj016亮氨酸氨肽酶的重组毕赤酵母的构建方法。
所述枯草芽孢杆菌Zj016，详见田亚平，须瑛敏，《一种枯草芽孢杆菌氨肽酶的纯化及酶学性质[J] 》，食品与发酵工业，2006，32（3）：7-10。
本发明的第二个目的是提供重组毕赤酵母分泌表达亮氨酸氨肽酶的方法。
本发明的第三个目的是通过对重组毕赤酵母N-糖基化的分析，探究其对重组表达亮氨酸氨肽酶温度稳定性及底物亲和力的影响。
本发明的第四个目的是酵母转化子遗传稳定性的验证。
本发明的技术方案：一种胞外分泌表达枯草芽孢杆菌Zj016亮氨酸氨肽酶的重组毕赤酵母的构建方法，将来源于枯草芽孢杆菌Zj016的氨肽酶基因导入到毕赤酵母中得到的基因工程菌，简称为重组毕赤酵母；步骤为：
（1）用枯草芽孢杆菌Zj016的亮氨酸氨肽酶基因序列设计一对引物P1、P2，以含有目的基因BSAP的质粒pUC19-BSAP为模板，P1和P2为引物扩增目的基因；
上游引物P1：5‘-CCGGAATTCA TGAAAAAGCT TTTGACTG- 3’，含有EcoR I酶切位点；，
下游引物P2：5‘-ATTTGCGGCC GCTTATTTGA TATCTTCAA- 3’，含有Not I酶切位点；
（2）将目的基因连接到表达载体pPIC9K上，获得重组载体pPIC9K-BSAP，转化E.coli JM109，涂布于含氨苄抗性的LB固体平板上，PCR扩增目的基因，双酶切进行验证阳性转化子，验证正确的重组载体pPIC9K-BSAP送去测序；
（3）测序正确的重组载体pPIC9K-BSAP用Bgl II进行单酶切，经酶切线性化之后，同毕赤酵母感受态细胞混匀，进行电击转化，参数设置1500 V、400Ω、25/50 μF、4-6 ms；将电转液涂布于MD平板，30℃培养4 d，待菌长出来以后分别点接于MM和MD平板，30℃培养2 d，其中在MD平板上正常生长、但在MM平板上几乎不长的，即为含有目的基因的重组菌；
（4）筛选获得重组毕赤酵母，进行表达验证。
用所述方法构建的重组毕赤酵母分泌表达亮氨酸氨肽酶的方法，所述分泌表达方法如下：
（1）挑取MD平板上的单菌落于25 mL BMGY培养基中 30℃ 230 rpm 培养2d；
（2）将培养液置于提前灭过菌的离心管中离心收集沉淀，将沉淀重溶于25 mL BMMY培养基中 23℃、230rpm培养96 h，每隔24 h向培养液中添加终浓度为1%的甲醇进行甲醇诱导；
（3）将获得的发酵液，10000 rpm 离心5 min，即得亮氨酸氨肽酶粗酶液。
所述亮氨酸氨肽酶基因BSAP来源于含有质粒pUC19-BSAP的大肠杆菌，亮氨酸氨肽酶基因的氨基酸序列如 SEQ ID NO.1 所示。
所述毕赤酵母为巴斯德毕赤酵母菌株GS115。
所获得的氨肽酶粗酶液，经过硫酸铵盐析、阴离子交换层析和凝胶过滤层析进行分离纯化，得到电泳纯的氨肽酶。
纯化得到的亮氨酸氨肽酶，特性如下：最适反应温度为60℃；在60℃下保温3 h后，相对酶活仍高于75%；在70℃下保温1 h后，相对酶活保留63.5%；酶的米氏常数Km及最大反应速度Vmax分别为：0.97 mmol/L及10.95 mmol/L/min。
通过对重组毕赤酵母N-糖基化的分析，探究其对重组表达亮氨酸氨肽酶温度稳定性及底物亲和力的影响。重组亮氨酸氨肽酶N-糖基化的分析方法如下：
1）将纯化得到的纯酶液适当稀释，加入去糖化酶Endo Hf，37℃恒温水浴4 h。
2）取少许酶解液跑SDS-PAGE蛋白胶。
N-糖基化对重组亮氨酸氨肽酶温度稳定性及底物亲和力的影响分析方法如下：
1）酶的温度稳定性：将酶液置于不同的温度（30、40、50、60、70、80℃）分别保温1 h及3 h后，测定氨肽酶酶活力，将最高的酶活力定义为100%。
2）酶的米氏常数Km及最大反应速度Vmax：以Leu-pNA作为底物，分别测定不同底物浓度(0.2、0.4、0.6、0.8、1.0、1.2、1.4 mmol/L)的酶活，并将测定结果按照双倒数法作1/v-1/s曲线。
酵母转化子遗传稳定性的验证。酵母转化子遗传稳定性的验证方法如下：
1）挑取酵母转化子单克隆菌落，接种于MD平板，30℃培养2 d直至长出单克隆菌落（第一代）；挑取第一代的转化子菌落于另一新鲜MD平板，30℃培养2 d直至长出新的单克隆菌落（第二代），按上述方法依次传代至第10代。
2）提取10代转化子的基因组，对提取的每代基因组同一条件下做PCR，取PCR产物6μL做1%的琼脂糖凝胶电泳。与此同时，对各代菌株进行发酵培养，并测定各代培养上清液中氨肽酶的酶活力。
氨肽酶酶活测定方法：
以L-亮氨酸-对硝基苯胺为底物，加入2 mL pH 8.5的Tris-HCl缓冲液，1 mL稀释适当倍数的酶液和1 mL底物（2 mM），50℃反应10 min，在405 nm处测定吸光值，计算酶活力。
氨肽酶酶活定义：
在一定条件下，每分钟分解L-亮氨酸-对硝基苯胺产生1μM的对硝基苯胺所需要的酶量，即为一个酶活单位。
本发明的有益效果：本发明所构建的亮氨酸氨肽酶在重组毕赤酵母中的整合表达，不仅具有很好的遗传稳定性，且N-糖基化后的氨肽酶温度稳定性与底物亲和力都获得一定的提升，为氨肽酶的产业化和在食品领域中的应用奠定了优良的基础。
附图说明
图 1 亮氨酸氨肽酶（BSAP）的扩增结果（M：Marker，1、2：目的基因）。
图 2 重组质粒pPIC9K-BSAP的双酶切验证（M：Marker，1、2：上边条带为pPIC9K，下边为目的基因）。
图 3 重组质粒pPIC9K-BSAP示意图。
图 4 重组毕赤酵母的筛选（MD和MM平板）。
图 5 重组亮氨酸氨肽酶的去糖基化分析（SDS-PAGE）（M：Marker，1、2：纯化后的重组氨肽酶，3、4：Endo Hf去糖基化后的氨肽酶）。
图 6 重组亮氨酸氨肽酶的热稳定性。
图 7 重组亮氨酸氨肽酶的米氏常数Km及最大反应速度Vmax的测定。
图 8 重组毕赤酵母的基因遗传稳定性（M：Marker，1-10：连续10代的基因组PCR产物）。
具体实施方式
所用培养基：
LB（g/L）：胰蛋白胨 10，酵母膏 5，NaCl 10，pH 7.0
YPD（g/L）：蛋白胨 20，葡萄糖20，琼脂 20
MD（g/L）：葡萄糖20，琼脂20，YNB 13.4，生物素 0.4
MM (g/L)：甲醇 5 mL/L，琼脂20，YNB 13.4，生物素 0.4
BMGY (g/L): 蛋白胨 20，甘油 10，酵母膏10，YNB 13.4，生物素 0.4，100 mM 磷酸盐缓冲液 pH 6.0
BMMY (g/L): 蛋白胨 20，甲醇 10，酵母膏10，YNB 13.4，生物素 0.4，100 mM 磷酸盐缓冲液 pH 7.0
实施例1 胞外分泌表达亮氨酸氨肽酶的重组毕赤酵母的构建方法。
为实现亮氨酸氨肽酶基因在毕赤酵母中的表达，从亮氨酸氨肽酶基因的信号肽处设计两对基因引物。
上游引物P1：5‘-CCGGAATTCA TGAAAAAGCT TTTGACTG- 3’（含有EcoR I酶切位点），
下游引物P2：5‘-ATTTGCGGCC GCTTATTTGA TATCTTCAA- 3’（含有Not I酶切位点）。
以含目的基因的质粒pUC19-BSAP为模板，P1及P2为引物扩增目的基因。PCR体系为（50μL）：rTaq酶 0.25μL；PCR Buffer 5μL；dNTP 4μL；Template 1μL；P1 1μL；P2 1μL；ddH2O 37.75μL。PCR条件为：95℃预变性5 min；95℃变性30 s，55 ℃退火30 s，72 ℃延伸90 s；72 ℃后延伸5 min；35个循环。
将扩增获得的目的基因同质粒pPIC9K用EcoR I和Not I进行双酶切，双酶切产物胶回收纯化后，16℃连接过夜，涂布于含氨苄抗性（100 μg/mL）的LB培养基中培养12-16 h。挑取5株进行PCR扩增及双酶切验证，验证正确的进行序列测定，测序正确的即为重组载体pPIC9K-BSAP。
毕赤酵母感受态细胞的制作：
（1）挑取毕赤酵母GS115单菌落接种到5 mL YPD试管培养过夜（10 h）；
（2）以1 %接种量接到50 mL YPD三角摇瓶中，30℃培养过夜，至OD600达到1.5左右（12 h）；
（3）取50 mL灭菌的离心管，每个离心管中装入25 mL菌液，置于冰上半小时；
（4）4 ℃、4000 rpm离心6 min，用50 mL预冷的无菌水悬浮；
（5）4 ℃、4000 rpm离心6 min，用25 mL预冷的无菌水悬浮；
（6）按步骤（5），离心10 min，用2 mL预冷的山梨醇洗涤；
（7）将沉淀重悬于80 μL 山梨醇以得到感受态细胞。
取上述感受态细胞与Bgl II线性化处理的pPIC9K-BSAP混合，于1500 V、400Ω、25/50 μF、4-6 ms条件下进行电转，将电转液涂布于MD平板上，30℃培养4 d，待转化子长出后，挑取50株分别点样到MM和MD平板上，30℃培养2 d，其中在MD平板上正常生长但是在MM平板上几乎不长的即为含有目的基因的重组毕赤酵母。
实施例2 重组毕赤酵母分泌表达亮氨酸氨肽酶的方法
挑取上述获得的重组毕赤酵母单菌落于25/50 mL的BMGY 培养基中，30℃230 rpm培养2d。而后将培养液3000 rpm离心5 min收集菌体，用BMMY重悬菌体并置于25/50 mL的三角瓶中，23℃ 230 rpm培养96 h，中间每隔24 h添加终浓度为1 %的甲醇进行诱导表达。将获得的发酵液，10000 rpm 离心5 min，即得氨肽酶粗酶液，测得的发酵液酶活为28.4 U/mL。
实施例3 重组毕赤酵母N-糖基化的分析，及其对重组亮氨酸氨肽酶温度稳定性及底物亲和力的影响。
对亮氨酸氨肽酶的氨基酸序列通过ExPASy在线软件进行糖基化位点的预测，结果如图5，潜在的糖基化位点分别为，188-191：NLSG；224-227：NQTS；263-266：NGSG。并且都是N-糖基化修饰，不存在O-糖基化修饰位点。
通过硫酸铵盐析，阴离子交换层析及凝胶过滤层析，得到了纯的氨肽酶，取纯酶液上样跑SDS-PAGE，得到相邻很近的两条条带（分别命名为up和down）。
取9μL适当稀释的纯酶液与1 μL 10×糖蛋白变性缓冲液混匀，100℃加热煮沸10 min；再添加2 μL 10×G5缓冲液、5 μL糖苷内切酶 Endo Hf、3 μL ddH2O，混匀后于37℃恒温水浴4 h。取少许酶解液跑SDS-PAGE蛋白胶。结果显示，糖化酶酶解后两条蛋白条带（up和down）全部消失，而有一条新的条带出现，它的蛋白分子量正好跟野生枯草芽孢杆菌所产氨肽酶分子量相一致，这正好说明了毕赤酵母对所表达的亮氨酸氨肽酶进行了N-糖基化修饰。
按上述分离方法得到纯的氨肽酶，该N-糖基化酶具有以下性质：最适反应温度为60℃；在60℃下保温3 h后，相对酶活仍高于75%；在70℃下保温1 h后，相对酶活保留约63.5%；酶的米氏常数Km及最大反应速度Vmax分别为：0.97 mmol/L及10.95 mmol/L/min。与野生菌产亮氨酸氨肽酶(60℃保温3 h后，相对酶活仅为60%；Km及Vmax值分别为2.8 mmol/L和5 mmol/L/min)相比，本发明所产N-糖基化氨肽酶的温度稳定性与底物亲和力分别提升约1.25和2.89倍，这将为氨肽酶的产业化和在食品领域中的应用奠定优良的基础。
实施例4 酵母转化子遗传稳定性的验证。
对传代获得的10代菌株，提取基因组进行PCR鉴定，发现转化子的插入基因没有发生任何改变（如图8所示）；与此同时，对发酵培养上清液进行氨肽酶酶活力测定，发现表达水平差异很小，说明获得的重组毕赤酵母具有很好的遗传稳定性。
<160> 1
<210> SEQ ID NO: 1
<211> 455
<212> PRT
<213> 枯草芽孢杆菌（Bacillus Subtilis）Zj016的亮氨酸氨肽酶基因BSAP
Met Lys Lys Leu Leu Thr Val Met Thr Met Ala Val Leu Thr Ala
5 10 15
Gly Thr Leu Leu Leu Pro Ala Gln Ser Val Thr Pro Ala Ala His
20 25 30
Ala Val Gln Ile Ser Asn Ser Glu Arg Glu Leu Leu Phe Lys Ala
35 40 45
Lys His Ala Tyr Ser Thr Ile Ser Gln Leu Ser Glu Ala Ile Gly
50 55 60
Pro Arg Ile Ala Gly Thr Ala Ala Glu Lys Lys Ser Ala Leu Leu
65 70 75
Ile Ala Ser Ser Met Arg Lys Leu Lys Leu Asp Val Lys Val Gln
80 85 90
Arg Phe Asn Ile Pro Asp Arg Leu Glu Gly Thr Leu Ser Ser Ala
95 100 105
Gly Arg Asp Ile Leu Leu Gln Ala Ala Ser Gly Ser Ala Pro Thr
110 115 120
Glu Glu Gln Gly Leu Thr Ala Pro Leu Tyr Asn Ala Gly Leu Gly
125 130 135
Asn Gln Lys Gly Phe Thr Ala Asp Ala Lys Gly Lys Ile Ala Leu
140 145 150
Ile Ser Arg Gly Asp Leu Thr Tyr Tyr Glu Lys Ala Lys Asn Ala
155 160 165
Glu Ala Ala Gly Ala Lys Ala Val Ile Ile Tyr Asn Asn Lys Glu
170 175 180
Ser Leu Val Pro Met Thr ProAsnLeu Ser GlyAsn Lys Val Gly
185 190 195
Ile Pro Val Val Gly Ile Lys Lys Glu Asp Gly Glu Ala Leu Thr
200 205 210
Gln Gln Lys Glu Ala Thr Leu Lys Leu Lys Ala Phe ThrAsnGln
215 220 225
ThrSerGln Asn Ile Ile Gly Ile Lys Lys Pro Lys Asn Ile Lys
230 235 240
His Pro Asp Ile Val Tyr Val Thr Ala His Tyr Asp Ser Val Pro
245 250 255
Phe Ser Pro Gly Ala Asn AspAsnGly Ser GlyThr Ser Val Met
260 265 270
Leu Glu Met Ala Arg Val Leu Lys Ser Val Pro Ser Asp Lys Glu
275 280 285
Ile Arg Phe Ile Ala Phe Gly Ala Glu Glu Leu Gly Leu Leu Gly
290 295 300
Ser Ser His Tyr Val Asp His Leu Ser Glu Lys Glu Leu Lys Arg
305 310 315
Ser Glu Val Asn Phe Asn Leu Asp Met Val Gly Thr Ser Trp Glu
320 325 330
Lys Ala Ser Glu Leu Tyr Val Asn Thr Leu Asp Gly Gln Ser Asn
335 340 345
Tyr Val Trp Glu Ser Ser Arg Thr Ala Ala Glu Lys Ile Gly Phe
350 355 360
Asp Ser Leu Ser Leu Thr Gln Gly Gly Leu Ser Asp His Val Pro
365 370 375
Phe His Glu Ala Gly Ile Asp Ser Ala Asn Phe Ile Trp Gly Asp
380 385 390
Pro Glu Thr Glu Glu Val Glu Pro Trp Tyr His Thr Pro Glu Asp
395 400 405
Ser Ile Glu His Ile Ser Lys Glu Arg Leu Gln Gln Ala Gly Asp
410 415 420
Leu Val Thr Ala Ala Val Tyr Glu Ala Val Lys Lys Glu Lys Lys
425 430 435
Pro Lys Thr Ile Lys Lys Gln Met Lys Ala Lys Ala Ser Asp Ile
440 445 450
Phe Glu Asp Ile Lys
455
Claims (6)
1.一种胞外分泌表达枯草芽孢杆菌Zj016亮氨酸氨肽酶的重组毕赤酵母的构建方法，其特征在于将来源于枯草芽孢杆菌Zj016的亮氨酸氨肽酶基因导入到毕赤酵母中得到的基因工程菌，简称为重组毕赤酵母；步骤为：
（1）用枯草芽孢杆菌Zj016的亮氨酸氨肽酶基因序列设计一对引物P1、P2，以含有目的基因BSAP的质粒pUC19-BSAP为模板，P1和P2为引物扩增目的基因；
上游引物P1：5‘-CCGGAATTCA TGAAAAAGCT TTTGACTG- 3’，含有EcoR I酶切位点；，
下游引物P2：5‘-ATTTGCGGCC GCTTATTTGA TATCTTCAA- 3’，含有Not I酶切位点；
（2）将目的基因连接到表达载体pPIC9K上，获得重组载体pPIC9K-BSAP，转化E.coli JM109，涂布于含氨苄抗性的LB固体平板上，PCR扩增目的基因，双酶切进行验证阳性转化子，验证正确的重组载体pPIC9K-BSAP送去测序；
（3）测序正确的重组载体pPIC9K-BSAP用Bgl II进行单酶切，经酶切线性化之后，同毕赤酵母感受态细胞混匀，进行电击转化，参数设置1500 V、400Ω、25/50 μF、4-6 ms；将电转液涂布于MD平板，待菌长出来以后分别点接于MM和MD平板，获取重组菌；
（4）筛选获得重组毕赤酵母，进行表达验证。
2.用权利要求1所述的方法构建的重组毕赤酵母分泌表达亮氨酸氨肽酶的方法，其特征在于所述分泌表达方法如下：
（1）挑取MD平板上的单菌落于25 mL BMGY培养基中 30℃ 230 rpm 培养2d；
（2）将培养液置于提前灭过菌的离心管中离心收集沉淀，将沉淀重溶于25 mL BMMY培养基中 23℃ 230 rpm培养96 h，每隔24 h向培养液中添加终浓度为1%的甲醇进行甲醇诱导；
（3）将获得的发酵液，10000 rpm 离心5 min，即得亮氨酸氨肽酶粗酶液。
3.根据权利要求1所述胞外分泌表达枯草芽孢杆菌Zj016亮氨酸氨肽酶的重组毕赤酵母的构建方法，其特征在于所述亮氨酸氨肽酶基因BSAP来源于含有质粒pUC19-BSAP的大肠杆菌，亮氨酸氨肽酶基因的氨基酸序列如 SEQ ID NO.1 所示。
4.根据权利要求1所述胞外分泌表达枯草芽孢杆菌Zj016亮氨酸氨肽酶的重组毕赤酵母的构建方法，其特征在于所述毕赤酵母为巴斯德毕赤酵母菌株GS115。
5.根据权利要求2所述的重组毕赤酵母分泌表达亮氨酸氨肽酶的方法，其特征在于所获得的氨肽酶粗酶液，经过硫酸铵盐析、阴离子交换层析和凝胶过滤层析进行分离纯化，得到电泳纯的氨肽酶。
6.根据权利要求5所述的重组毕赤酵母分泌表达亮氨酸氨肽酶的方法，其特征在于纯化得到的氨肽酶，特性如下：最适反应温度为60℃；在60℃下保温3 h后，相对酶活仍高于75%；在70℃下保温1 h后，相对酶活保留63.5%；酶的米氏常数Km及最大反应速度Vmax分别为：0.97 mmol/L及10.95 mmol/L/min。
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