CN110079492B - Escherichia coli M4突变株及其制备方法和应用 - Google Patents
Escherichia coli M4突变株及其制备方法和应用 Download PDFInfo
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- CN110079492B CN110079492B CN201910407496.7A CN201910407496A CN110079492B CN 110079492 B CN110079492 B CN 110079492B CN 201910407496 A CN201910407496 A CN 201910407496A CN 110079492 B CN110079492 B CN 110079492B
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- mutant strain
- lipase
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- foldase
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- 241000617742 Escherichia coli M4 Species 0.000 title abstract description 19
- 238000002360 preparation method Methods 0.000 title abstract description 8
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- 239000004367 Lipase Substances 0.000 claims abstract description 35
- 235000019421 lipase Nutrition 0.000 claims abstract description 35
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims 1
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- 238000012216 screening Methods 0.000 abstract description 11
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Abstract
本发明属于生物工程领域,提供了Escherichia coli M4突变株及其制备方法和应用。将铜绿假单胞菌折叠酶基因进行易错PCR,然后将突变的折叠酶基因和铜绿假单胞菌脂肪酶基因克隆到pACYCDuet‑1重组质粒,转入E.coli BL21(DE3)进行共表达,经过初筛和复筛后,得到的一个突变株,其胞内脂肪酶的比活力是未突变株的2.1倍。与未突变株相比,突变株中重组折叠酶的一个氨基酸残基发生了变化,由甘氨酸突变为谷氨酸。该发明为提高脂肪酶活性提供了新方法。
Description
技术领域
本发明属于生物工程领域,具体涉及Escherichia coli M4(简写为E.coli M4)突变株及其制备方法和应用。
背景技术
脂肪酶不仅在工业、农业和畜牧业等传统产业中受到广泛的应用,在生物传感器、生物柴油合成、诊断工具酶等领域也越来越受到重视。脂肪酶来源非常广泛,它可以通过培养微生物提取,也可以从动植物体内获得。微生物性脂肪酶具有催化活性多样性、产量高、便于提取和微生物生长周期短、基因操作较容易等特点,因此相对于动物和植物来源的脂肪酶,微生物性脂肪酶运用更加广泛。来自铜绿假单胞菌的脂肪酶是研究历史较长的脂肪酶。它具有较高的活性、稳定性和选择性。但是铜绿假单胞菌中的脂肪酶依赖于特异性折叠酶,需在折叠酶的帮助下才能正确折叠为具有活性的构象。
脂肪酶和其特异性折叠酶具有较高的亲和力。研究证明它们之间主要是通过疏水相互作用、氢键和盐键这三种次级键来发生相互作用,形成中间复合物。实验证明,经过化学变性处理过的脂肪酶,在其折叠酶的协助下可以实现复性。在与脂肪酶结合的过程中,折叠酶的二级和三级结构都发生了明显的变化。有些研究表明:折叠酶协助相应的脂肪酶进行正确折叠后,不能再继续协助另一个脂肪酶的折叠,因而是单周转的催化剂。然而使用Northern印记实验却得出不同的结果。该实验表明虽然折叠酶基因和脂肪酶基因的转录是同步且等比例的,但是在它们转录完成后,编码折叠酶蛋白的mRNA有很大一部分被降解,因此产生的折叠酶的分子数会远远小于脂肪酶的分子数。这说明折叠酶蛋白协助对应脂肪酶正确折叠后,与该脂肪酶会发生分离,可以继续协助下一个对应脂肪酶分子的折叠,因而是多周转的催化剂(程蓝驿等,脂肪酶的特异性折叠酶,生物技术通报,2011,4:35-39)。
发明内容
本发明的目的在于提供一株Escherichia coli M4突变株及其制备方法和应用,至少在一定程度上解决现有技术中存在的问题。
本发明提供的一株Escherichia coli M4突变株,保藏于中国典型培养物保藏中心(武汉大学),保藏日期为2019年04月10日,保藏号为CCTCC NO:M 2019245。
上述的Escherichia coli M4突变株的制备方法,包括以下步骤:
(1)以携带Pseudomonas aeruginosa CS-2脂肪酶基因的pet28a-lip质粒为模板,进行PCR扩增,PCR扩增产物经BamH Ⅰ和Hind Ⅲ双酶切,将双酶切得到的基因片段与质粒pACYCDuet-1连接,得到重组质粒pACYCDuet-1-lip;
(2)以携带Pseudomonas aeruginosa CS-2折叠酶基因的pet28a-fold质粒为模板,进行易错PCR扩增,易错PCR扩增产物经NdeI和XhoI双酶切,将双酶切得到的基因片段与重组质粒pACYCDuet-1-lip连接,得到重组质粒pACYCDuet-1-lip-fold;
(3)将重组质粒pACYCDuet-1-lip-fold转化至感受态细胞E.coli BL21(DE3),接种至筛选培养基培养,经初筛后和复筛后,得到Escherichia coli M4突变株。
在步骤(1)中,PCR扩增的正向引物序列为CCTTGGATCCGATGAAGAAGAAGTCTCTGCTCC,反向引物序列为CCTTAAGCTTCTACAGGCTGGCGTTCTTCAGG。
在步骤(2)中,易错PCR扩增的正向引物序列为GGAATTCCATATGATGAAGAAAATCCTCCTGC,反向引物序列为AATTCTCGAGGCGCTGCTCGGCCTG。
与未突变株相比,上述的Escherichia coli M4突变株中重组折叠酶的一个氨基酸残基发生了变化,由甘氨酸(未突变株)突变为谷氨酸(突变株)。因此,该Escherichiacoli M4突变株内脂肪酶的比活力得到了提高,脂肪酶活性测定结果显示,该Escherichiacoli M4突变株内脂肪酶的比活力是未突变株的2.1倍。基于该Escherichia coli M4突变株内脂肪酶的优异的比活力,该Escherichia coli M4突变株可以运用于生物催化领域。
本发明的有益效果:提供了一株Escherichia coli M4突变株,其胞内脂肪酶的比活力是未突变株的2.1倍,该Escherichia coli M4突变株可以应用于生物催化领域;提供了Escherichia coli M4突变株的制备方法,实现了通过折叠酶的分子改造来提高重组脂肪酶活性。
附图说明
图1是突变株和未突变株的脂肪酶的比活力图。
图2是脂肪酶和折叠酶的分子对接图。
图3是脂肪酶和折叠酶突变体的分子对接图。
图4是脂肪酶和折叠酶的分子对接PISA界面参数图。
图5是脂肪酶和折叠酶突变体的分子对接PISA界面参数图。
序列表中序列1是折叠酶突变体序列。
具体实施方式
下面将通过实施例来详细说明本发明所具有的有益效果,旨在帮助阅读者更好地理解本发明的实质,但不能对本发明的实施和保护范围构成任何限定。
本发明提供的Escherichia coli M4突变株是这样得到的:首先构建重组质粒pACYCDuet-1-lip;然后构建pACYCDuet-1-lip-fold突变体文库,经初筛和复筛得到Escherichia coli M4突变株。具体来说,包括以下步骤:
(1)以携带Pseudomonas aeruginosa CS-2脂肪酶基因的pet28a-lip质粒为模板扩增脂肪酶基因。其中正向引物序列为CCTTGGATCCGATGAAGAAGAAGTCTCTGCTCC,反向引物序列为CCTTAAGCTTCTACAGGCTGGCGTTCTTCAGG。PCR条件为:94℃2min;94℃20s,60℃20s,72℃50s,共20个循环;72℃3min。利用BamHⅠ和HindⅢ双酶切扩增得到的片段,然后连接至经BamHⅠ和HindⅢ双酶切的pACYCDuet-1后,转化至感受态细胞E.coli TOP10。挑选克隆,提取重组质粒pACYCDuet-1-lip进行酶切鉴定。
(2)以携带Pseudomonas aeruginosa CS-2折叠酶基因的pet28a-fold质粒为模板扩增折叠酶突变基因。其中正向引物序列为GGAATTCCATATGATGAAGAAAATCCTCCTGC,反向引物序列为AATTCTCGAGGCGCTGCTCGGCCTG。50μl易错PCR体系中含有3μl的Mn2+。易错PCR条件为:95℃10min;95℃1min,54℃50s,72℃1min,共30个循环;72℃3min。利用NdeI和XhoI双酶切扩增得到的片段,然后连接至经NdeI和XhoI双酶切的重组质粒pACYCDuet-1-lip后,转化至感受态细胞E.coli BL21(DE3),得到含有pACYCDuet-1-lip-fold突变体文库的重组菌。
(3)将含有pACYCDuet-1-lip-fold突变体文库的重组菌接种筛选培养基,挑选透明圈较大的菌株用于复筛。将初筛得到的菌株接种至含有氯霉素(34μg/ml)的LB培养基过夜培养,取2.5mL菌液加入50mL含有氯霉素(34μg/ml)的LB培养基中,37℃培养3h,加入终浓度为1.5mmol/L IPTG(异丙基硫代半乳糖苷),37℃诱导6h。离心收集菌体,用预冷的Tris-HCl(pH8.0)悬浮菌体,置于冰浴进行超声波破碎,离心后得到的上清液用于脂肪酶活性测定。经过初筛和复筛后,得到的一个突变株,其胞内脂肪酶的比活力是未突变株的2.1倍(图1)。与未突变株相比,突变株中重组折叠酶的一个氨基酸残基发生了变化,由甘氨酸(未突变株)突变为谷氨酸(突变株)。
利用I-TASSER构建脂肪酶和折叠酶以及折叠酶突变体的三维结构,然后用Z-Docking进行脂肪酶和折叠酶以及脂肪酶和折叠酶突变体之间的分子对接(图2和图3),PDBepisa分析结果表明脂肪酶和折叠酶复合物的△G(吉布斯自由能)为-24.3kcal/mol(图4),脂肪酶和折叠酶突变体复合物的△G为-38.9kcal/mol(图5)。这说明脂肪酶和折叠酶突变体复合物的亲和力大于脂肪酶和折叠酶复合物的亲和力。
突变的折叠酶序列:
MMKKILLLIPLAFAASLAWFVWLEPSPAPETAPPASPQAGADRAPPAASAGEAVPAPQVMPAKVAPLPTSFRGTSVDGSFSVDASGNLLITRDIRNLFDYFLSAVGEEPLQQSLDRLRAYIAAELQEPARGQALALMQQYIDYKKELVLLERDLPRLADLDALRQREAAVKALRARIFSNEAHVTFFADEETYNQFTLERLAIRQDGKLSTEEKAAAIDRLRASLPEDQQESVLPQLQSELQQQTAALQAAGAGPEAIRQMRQQLVGAEATTRLEQLDRQRSAWKGRLDDYFAEKSRIEGNTGLSEADRRAAVERLAEERFSEQERLRLGALEQMRQAEQRLELESGKETAAAKFERQHMDSSTSAA
注:下划线为表达载体中核甘酸翻译的氨基酸残基,和未突变株相比,突变株中重组折叠酶由甘氨酸突变为谷氨酸(黑色的方框)。
序列表
<110> 江西师范大学
<120> Escherichia coli M4突变株及其制备方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 367
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Met Lys Lys Ile Leu Leu Leu Ile Pro Leu Ala Phe Ala Ala Ser
1 5 10 15
Leu Ala Trp Phe Val Trp Leu Glu Pro Ser Pro Ala Pro Glu Thr Ala
20 25 30
Pro Pro Ala Ser Pro Gln Ala Gly Ala Asp Arg Ala Pro Pro Ala Ala
35 40 45
Ser Ala Gly Glu Ala Val Pro Ala Pro Gln Val Met Pro Ala Lys Val
50 55 60
Ala Pro Leu Pro Thr Ser Phe Arg Gly Thr Ser Val Asp Gly Ser Phe
65 70 75 80
Ser Val Asp Ala Ser Gly Asn Leu Leu Ile Thr Arg Asp Ile Arg Asn
85 90 95
Leu Phe Asp Tyr Phe Leu Ser Ala Val Gly Glu Glu Pro Leu Gln Gln
100 105 110
Ser Leu Asp Arg Leu Arg Ala Tyr Ile Ala Ala Glu Leu Gln Glu Pro
115 120 125
Ala Arg Gly Gln Ala Leu Ala Leu Met Gln Gln Tyr Ile Asp Tyr Lys
130 135 140
Lys Glu Leu Val Leu Leu Glu Arg Asp Leu Pro Arg Leu Ala Asp Leu
145 150 155 160
Asp Ala Leu Arg Gln Arg Glu Ala Ala Val Lys Ala Leu Arg Ala Arg
165 170 175
Ile Phe Ser Asn Glu Ala His Val Thr Phe Phe Ala Asp Glu Glu Thr
180 185 190
Tyr Asn Gln Phe Thr Leu Glu Arg Leu Ala Ile Arg Gln Asp Gly Lys
195 200 205
Leu Ser Thr Glu Glu Lys Ala Ala Ala Ile Asp Arg Leu Arg Ala Ser
210 215 220
Leu Pro Glu Asp Gln Gln Glu Ser Val Leu Pro Gln Leu Gln Ser Glu
225 230 235 240
Leu Gln Gln Gln Thr Ala Ala Leu Gln Ala Ala Gly Ala Gly Pro Glu
245 250 255
Ala Ile Arg Gln Met Arg Gln Gln Leu Val Gly Ala Glu Ala Thr Thr
260 265 270
Arg Leu Glu Gln Leu Asp Arg Gln Arg Ser Ala Trp Lys Gly Arg Leu
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Asp Asp Tyr Phe Ala Glu Lys Ser Arg Ile Glu Gly Asn Thr Gly Leu
290 295 300
Ser Glu Ala Asp Arg Arg Ala Ala Val Glu Arg Leu Ala Glu Glu Arg
305 310 315 320
Phe Ser Glu Gln Glu Arg Leu Arg Leu Gly Ala Leu Glu Gln Met Arg
325 330 335
Gln Ala Glu Gln Arg Leu Glu Leu Glu Ser Gly Lys Glu Thr Ala Ala
340 345 350
Ala Lys Phe Glu Arg Gln His Met Asp Ser Ser Thr Ser Ala Ala
355 360 365
Claims (1)
1.Escherichia coli M4突变株生产脂肪酶的用途,其特征在于:所述Escherichia coli M4突变株保藏于中国典型培养物保藏中心,保藏日期为2019年4月10日,保藏编号为CCTCC NO: M 2019245。
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