CN114149981A - 一种比活提高的泛酸合成酶突变体及其应用 - Google Patents
一种比活提高的泛酸合成酶突变体及其应用 Download PDFInfo
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- CN114149981A CN114149981A CN202111494426.3A CN202111494426A CN114149981A CN 114149981 A CN114149981 A CN 114149981A CN 202111494426 A CN202111494426 A CN 202111494426A CN 114149981 A CN114149981 A CN 114149981A
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- pantothenate
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Abstract
本发明属于酶工程技术领域，公开了一种催化能力提高的泛酸合成酶突变体及其应用，该突变体的氨基酸序列如SEQ ID NO.2所示。与谷氨酸棒杆菌来源的野生型泛酸合成酶相比，本发明所得的泛酸合成酶突变体S31K酶活提高了70%。
Description
技术领域
本发明涉及一种比活提高的泛酸合成酶突变体，属于酶工程技术领域。
背景技术
泛酸又称维生素B5，是水溶性维生素B族的一种，具有旋光性，仅D型有生物活性。D-泛酸作为细胞中辅酶A合成的关键前体，参与人体和动物碳水化合物、脂肪和蛋白质等代谢反应，是维持正常生理机能不可缺少的微量物质。微生物和植物可以合成D-泛酸，但人类和其他动物不能合成，因此D-泛酸被广泛应用于医药、化妆品、食品和饲料行业等。临床上用于治疗维生素B缺乏症、周围神经炎、手术后肠梗阻﹑链霉素中毒及类风湿等疾病。D-泛酸在酸、碱、光和热条件下都不稳定，一般以钙盐形式（D-泛酸钙）进行储藏、运输和商品交易。
泛酸合成酶(pantothenate synthetase,PS)(EC 6.3.2.1)是panC基因的表达产物，是以同型二聚体形式存在的。它是泛酸生物合成途径的最后一个关键酶，催化泛解酸与β-丙氨酸在ATP依赖性反应中结合形成D-泛酸。SDS-PAGE凝胶电泳法测得亚基的分子量约为33 kDa。其酶反应动力学机制为“Bi Uni Uni Bi Ping Pong”，在该机制中，它首先在有Mg2+的环境下与ATP结合，然后与泛解酸结合，形成反应中间体泛酰腺苷酸，随后释放出焦磷酸盐，为β-丙氨酸的结合和反应腾出了空间，最终产生了两种产物泛酸和AMP。
目前，生产D-泛酸的方法有：①物理诱导结晶法，利用混旋泛酸钙的溶解度大于D-型或L-型这一特点进行诱导结晶，此方法工艺成熟，但只能生产泛酸钙，无法用于其他泛酸衍生物的生产。②化学拆分法，利用氯酶胺等手性拆分剂拆分，但拆分剂价格昂贵，分离困难，还存在毒性和环境污染问题。③微生物法，包含代谢工程法、发酵法和生物酶法，Zhang等利用代谢工程法生产D-泛酸，方法复杂，产量低，最高产量仅28 .45g/L；发酵法的产量较高，但存在发酵产物成分复杂，不利于下游提取等问题，发酵过程中产生的物质对食品品质也会产生一定影响；生物酶法是利用泛酸合成酶(PS)催化D-泛解酸和β-丙氨酸进行缩合反应生成D-泛酸，反应条件温和，没有副产物的生成，更有利于产品分离。
泛酸合成酶生产D-泛酸最重要的酶，但很少有研究集中在泛酸合成酶上。因此，有必要获得一种催化能力提高的泛酸合成酶突变体筛选泛酸合成酶，并将其在宿主细胞中进行优化表达，来提高泛酸的产量，促进D-泛酸产业的顺利发展，具有重大的经济和社会价值。
发明内容
为了解决上述问题，本发明的第一目的是提供了一种比活提高的泛酸合成酶突变体，所述泛酸合成酶突变体含SEQ ID NO.2所示的氨基酸序列。
在本发明的一种实施方式中，所述泛酸合成酶突变体是以氨基酸序列如SEQ IDNO.1所示的泛酸合成酶为亲本酶，将第31位的丝氨酸突变为赖氨酸。
在本发明的一种实施方式中，所述亲本酶来源于谷氨酸棒杆菌(Corynebacteriumglutamicum)，所述谷氨酸棒杆菌(Corynebacterium glutamicum)引用自文献：Tigu, F.,et al. (2018). "A highly active pantothenate synthetase from Corynebacteriumglutamicum enables the production of D-pantothenic acid with highproductivity." Appl Microbiol Biotechnol 102(14): 6039-6046.。
本发明的第二目的是提供一种编码上述比活提高的泛酸合成酶突变体的基因。
在本发明的一种实施方式中，所述基因的核苷酸序列如SEQ ID NO .4。
本发明的第三目的是提供含上述基因的质粒或载体。
本发明的第四目的是提供携带上述基因的细胞。
在本发明的一种实施方式中，所述细胞包括基因工程菌。
在本发明的一种实施方式中，所述基因工程菌以大肠杆菌为宿主。
在本发明的一种实施方式中，所述基因工程菌以pET-28a(+)为表达载体，以Escherichia coli BL21(DE3)为表达宿主。
本发明的第五目的是提供一种提高泛酸合成酶比活的方法，是将来源于谷氨酸棒杆菌(Corynebacterium glutamicum)的泛酸合成酶第31位的丝氨酸突变为赖氨酸，所述来源于谷氨酸棒杆菌(Corynebacterium glutamicum)的泛酸合成酶的氨基酸序列如SEQ IDNO.1所示，编码所示泛酸合成酶的基因的核苷酸序列如SEQ ID NO.3所示。
本发明的第六目的是提供泛酸合成酶突变体及表达所述泛酸合成酶突变体的细胞在合成泛酸中的应用。
本发明的有益效果：
本发明是通过将泛酸合成酶中的底物结合位点附近不带电的氨基酸残基突变为带正电荷的氨基酸残基，得到比活提高的泛酸合成酶突变体，与谷氨酸棒杆菌来源的野生型泛酸合成酶相比，本发明所得的泛酸合成酶突变体S31K酶活提高了70%。
具体实施方式
本发明的实施例仅作为本发明内容的进一步说明，不能作为本发明的限定内容或范围。
本发明涉及的检测方法如下：
（一）产物D-泛酸含量的测定方法：
将反应样品12000rpm离心1min后，上清液稀释一定倍数，使用 0.22μm水系滤膜过膜，用于高效液相色谱（HPLC）检测检测。
高效液相色谱操作条件如下：
A 流动相成分：95%超纯水、4.9%乙腈、0.1%磷酸，使用 0.20 μm 有机微孔滤膜过膜并超声除气泡；
B 色谱柱型号：ACQUITY UPLC BEH C18 column (100 mm×2.1 mm，1.7 μm，Waters，UK）；
C 参数设置：进样量：10 μL，柱温：30℃，流速：0.9 mL/min，检测波长： 200 nm。
D 保留时间：17min
（二）泛酸合成酶活力的测定方法：
混合物包括：50 mmol/L磷酸钠缓冲液(pH 7.0)，25 mmol/L D-泛解酸钠盐，25mmol/L β-丙氨酸，4.5 mmol/L ATP，10 mmol/L MgCl，15 mmol/L KCl，总体积1mL。加入100 uL泛酸合成酶开始反应，30℃培养30 min，随后添加l mol/L HCl和1 mol/L NaOH停止反应。
D-泛解酸钠的制备:为降低生产成本，使用l mol/L D-泛酰内酯与l mol/L NaOH在室温条件下完全反应3 h生成1 mol/L D-泛解酸钠。
酶活定义：一个单位的酶活为在30℃条件下每分钟催化形成1 μmol D-泛酸的酶量。
（三）培养基：
LB培养基：酵母粉5g/L，胰蛋白胨10g/L，NaCl 10g/L，pH 7.0。
实施例1：
泛酸合成酶突变体的制备
将来源于谷氨酸棒杆菌(Corynebacterium glutamicum)的泛酸合成酶的氨基酸序列用swissmodel 建模，再将建模结果用hotspotwizard预测，选择了12个点做丙氨酸筛选。其中只有31号位点为正突变。将31号位点做定点饱和突变，以表达载体pET-28a(+)/panC为模板，设计互补引物链(见表1)，S31K(氨基酸序列如SEQ ID NO.2所示，编码其的基因的核苷酸序列如SEQ ID NO.4所示)的酶活提高最多。
表1 泛酸合成酶突变位点的引物
1下划线碱基对应于相应突变氨基酸。
实施例2：
含有表达本发明泛酸合成酶突变体基因的基因工程菌的构建
在37℃下，用DpnI处理PCR产物30min，随后将处理好的PCR产物通过化学转化法转入表达宿主E.coli BL21(DE3)感受态中，将转化的E.coli BL21(DE3)涂布到含有50μg/mL卡那霉素的LB琼脂培养基中，在37℃恒温箱中过夜培养12h，从中挑选出单菌落接种到含有50μg/mL卡那霉素的LB液体培养基中，在37℃下、200r/min培养过夜并按照质粒提取试剂盒说明书所示方法提取质粒鉴定测序，最终得到基因工程菌E.coli BL21(DE3) (pET-28a(+)/panC)。
实施例3：
本发明泛酸合成酶突变体的表达
宿主菌活化培养：将E.coli BL21(DE3)(pET-28a(+)/panC)在LB固体培养基上进行划线分离，置于37℃恒温培养箱中过夜培养，挑取阳性单菌落接种于含有10mL LB液体培养基的试管中，180rpm 37℃下培养8-10h。
泛酸合成酶突变体的表达：将活化好的菌种培养液1mL接种于含有50mL LB液体培养基的250mL三角瓶中，180rpm 37℃下培养，使OD600达到0.6-0.8。加入终浓度为0.5mMIPTG，在180rpm 28℃条件下诱导培养16h。
各培养基在使用前添加终浓度为50μg/mL的卡那霉素。
实施例4：
本发明泛酸合成酶突变体的酶活检测
配制50 mmol/L磷酸钠缓冲液(pH 7.0)溶液，并按照上述所示方法配制反应液，加入一定量的泛酸合成酶野生型和突变体S31K分别在30℃下进行反应测定泛酸合成酶突变体的活力。
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。
序列表
<110> 浙江工业大学
<120> 一种比活提高的泛酸合成酶突变体及其应用
<141> 2021-12-09
<160> 6
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<213> Corynebacterium glutamicum
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His Leu Ser Val Gly Leu Val Pro Thr Met Gly Ala Leu His Ser Gly
20 25 30
His Ala Ser Leu Val Leu Ala Ala Ala Ala Gly Ala Ala Thr Val Val
35 40 45
Ala Ser Ile Pro Val Ala Pro Leu Gly Pro Gly Ala Leu Gly Ala Cys
50 55 60
Ala Ala Thr Ala Ala Thr Pro Ala Gly Leu Ala Ala Ala Leu Ala Leu
65 70 75 80
Leu Gly Gly Ala Gly Val Ala Ile Val Pro Ala Pro Ala Val Gly Gly
85 90 95
Met Thr Pro Gly Gly Leu Pro Leu Val Thr Ala Ala Thr Gly Ser Ile
100 105 110
Gly Thr Leu Leu Gly Gly Ala Ser Ala Pro Gly His Pro Ala Gly Val
115 120 125
Ala Thr Val Val Ala Leu Leu Pro Ala Leu Val Ala Pro Ala Ala Ala
130 135 140
Thr Pro Gly Gly Leu Ala Ala Gly Gly Val Ala Val Ile Ala Ala Leu
145 150 155 160
Val Ala Ala Leu Ala Ile Pro Val Gly Ile Ala Pro Val Pro Ile Ile
165 170 175
Ala Gly Ala Ala Gly Leu Ala Gly Ser Ser Ala Ala Gly Ala Leu Ser
180 185 190
Ala Ala Gly Ala Ala Gly Ala Leu Val Leu Pro Gly Val Leu Ser Gly
195 200 205
Leu Gly Ala Ala Leu Ala Ala Gly Gly Ala Leu Ala Ile Gly Gly Ala
210 215 220
Ala Ala Thr Leu Ala Ser Ala Ala Gly Val Ala Leu Ala His Leu Gly
225 230 235 240
Ile Val Ala Pro Ala Thr Leu Gly Pro Leu Gly Ile Ala Gly Leu Leu
245 250 255
Thr Gly Pro Ala Leu Val Val Gly Ala Ile Pro Val Gly Pro Val Ala
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Leu Ile Ala Ala Ile Gly Leu
275
<210> 2
<211> 279
<212> PRT
<213> Artificial Sequence
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Met Gly Val Ala Thr Thr Leu Gly Ala Leu Ile Ala Ala Leu Leu His
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His Leu Ser Val Gly Leu Val Pro Thr Met Gly Ala Leu His Leu Gly
20 25 30
His Ala Ser Leu Val Leu Ala Ala Ala Ala Gly Ala Ala Thr Val Val
35 40 45
Ala Ser Ile Pro Val Ala Pro Leu Gly Pro Gly Ala Leu Gly Ala Cys
50 55 60
Ala Ala Thr Ala Ala Thr Pro Ala Gly Leu Ala Ala Ala Leu Ala Leu
65 70 75 80
Leu Gly Gly Ala Gly Val Ala Ile Val Pro Ala Pro Ala Val Gly Gly
85 90 95
Met Thr Pro Gly Gly Leu Pro Leu Val Thr Ala Ala Thr Gly Ser Ile
100 105 110
Gly Thr Leu Leu Gly Gly Ala Ser Ala Pro Gly His Pro Ala Gly Val
115 120 125
Ala Thr Val Val Ala Leu Leu Pro Ala Leu Val Ala Pro Ala Ala Ala
130 135 140
Thr Pro Gly Gly Leu Ala Ala Gly Gly Val Ala Val Ile Ala Ala Leu
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Val Ala Ala Leu Ala Ile Pro Val Gly Ile Ala Pro Val Pro Ile Ile
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Ala Gly Ala Ala Gly Leu Ala Gly Ser Ser Ala Ala Gly Ala Leu Ser
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Ala Ala Gly Ala Ala Gly Ala Leu Val Leu Pro Gly Val Leu Ser Gly
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Leu Gly Ala Ala Leu Ala Ala Gly Gly Ala Leu Ala Ile Gly Gly Ala
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Ile Val Ala Pro Ala Thr Leu Gly Pro Leu Gly Ile Ala Gly Leu Leu
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atgcaggttg ccaccaccaa acaggccctg attgacgccc tgttacacca caaaagcgtt 60
ggcctggtgc cgaccatggg ggccctgcat agcggccacg caagcttagt taaagcagcc 120
cgtgcagaaa atgacaccgt tgttgccagc atttttgtta atcccctgca gttcgaggca 180
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ttagaggaag caggcgttga tattgttttt gccccggatg ttgaagagat gtatccgggt 300
ggtctgccgc tggtttgggc acgtaccggc tctattggta ccaaactgga gggtgccagc 360
cggccgggtc attttgatgg tgttgccacc gttgttgcta agctgtttaa tctggttcgt 420
ccggatcgtg cgtattttgg tcagaaagat gcccagcagg tggctgttat tcgtcgttta 480
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cgtgcagaaa atgacaccgt tgttgccagc atttttgtta atcccctgca gttcgaggca 180
ttaggtgatt gcgatgatta tcgtaattat ccgcgccagc tggatgcaga tttagcatta 240
ttagaggaag caggcgttga tattgttttt gccccggatg ttgaagagat gtatccgggt 300
ggtctgccgc tggtttgggc acgtaccggc tctattggta ccaaactgga gggtgccagc 360
cggccgggtc attttgatgg tgttgccacc gttgttgcta agctgtttaa tctggttcgt 420
ccggatcgtg cgtattttgg tcagaaagat gcccagcagg tggctgttat tcgtcgttta 480
gttgcggatc tggatattcc ggttgaaatt cggccggtgc ctattatacg tggtgcagat 540
ggtttagcag agagcagtcg taaccagaga ctttccgcag atcagcgggc ccaggccctg 600
gttttacctc aggttttaag tggtctgcag cgtcgtaaag cagcaggtga ggcactggat 660
attcagggtg cacgggatac cttagcatca gccgatgggg ttcgtctgga tcatttagaa 720
attgttgatc cggccaccct ggaaccatta gaaatagatg ggctgttaac ccaaccggca 780
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tgcgtggcct ttatgcaggg cccccatggt cg 32
Claims (10)
1.一种比活提高的泛酸合成酶突变体，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
2.如权利要求1所述的一种比活提高的泛酸合成酶突变体，其特征在于，所述泛酸合成酶突变体是以氨基酸序列如SEQ ID NO.1所示的泛酸合成酶为亲本酶，将第31位的丝氨酸突变为赖氨酸。
3.编码权利要求1所述的泛酸合成酶突变体的基因。
4.含权利要求3所述基因的载体。
5.表达权利要求1所述泛酸合成酶突变体的细胞。
6.如权利要求5所述的细胞，其特征在于，所述细胞以大肠杆菌为宿主。
7.如权利要求5所述的细胞，其特征在于，所述细胞为基因工程菌，所述基因工程菌以pET 28a(+)为表达载体，以Escherichia coli BL21(DE3)为表达宿主。
8.一种提高泛酸合成酶比活的方法，其特征在于，将氨基酸序列如SEQ ID NO.1所示的泛酸合成酶的第31位的丝氨酸突变为赖氨酸。
9.权利要求1-2任一项所述泛酸合成酶突变体在合成泛酸中的应用。
10.权利要求5-7任一所述细胞在合成泛酸中的应用。
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| CN106676051A (zh) * | 2016-10-31 | 2017-05-17 | 中国科学院微生物研究所 | 一种制备高效合成泛酸基因工程菌的方法及其应用 |
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