构建重组枯草芽孢杆菌催化制备D-对羟基苯甘氨酸

doi:10.13523/j.cb.20190310

中国生物工程杂志

2019, Vol. 39

Issue (3): 75-86 DOI: 10.13523/j.cb.20190310

技术与方法

构建重组枯草芽孢杆菌催化制备D-对羟基苯甘氨酸

李法彬,刘露,杜燕,班睿(

)

天津大学化工学院天津大学系统生物工程教育部重点实验室天津 300350

Construction of Recombinant Bacillus subtilis as Catalyst for Preparing D- p-Hydroxyphenylglycine

Fa-bin LI,Lu LIU,Yan DU,Rui Ban(

)

1 School of Chemical Engineering and Technology, Key Laboratory of Systems Biotechnology of Ministry of Education,Tianjin University, Tianjin 300350, China

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摘要：

目的: 在Bacillus subtilis中表达异源D-海因酶基因(hyd)和D-氨甲酰水解酶基因(adc),构建重组细胞作为催化剂,用于生产D-对羟基苯甘氨酸(D-HPG)。方法: 构建hyd表达质粒,考察培养基中二价金属离子对D-海因酶活性的影响。过表达acoR基因,考察AcoR蛋白胞内水平与P_acoA-hyd基因拷贝数的关系。筛选表达adc基因的启动子,构建hyd和adc基因共表达质粒,考察双酶活性菌株的催化特性。结果: 成功构建了海因酶表达质粒pHPS和pUBS,培养基中添加0.8mmol/L的MnCl₂·4H₂O,使168N/pUBS菌株的D-海因酶活性达到956U/gDCW。整合表达P_cdd-acoR基因,使LSL02/pUBS菌株的D-海因酶活性达到1 470U/gDCW。单拷贝P_AE-adc基因的表达水平相对最高。双酶共表达质粒pUBSC被成功构建,菌株LSL02/pUBSC的最适催化温度为40℃45℃,催化活性能够持续12h,当底物起始浓度为20g/L时,反应12h生成的D-HPG达到14.32g/L,转化率达到95%,收率超过80%。结论: 构建具有D-海因酶和D-氨甲酰水解酶双酶活性的重组Bacillus subtilis作为全细胞催化剂,用于海因酶法生产D-HPG,具有技术上的可行性和优势。

关键词： 枯草芽孢杆菌; D-海因酶; D-氨甲酰水解酶; 表达质粒; D-对羟基苯甘氨酸

Abstract:

Objective: To construct the recombinant Bacillus subtilis with D-hydantoinase(DHase, hyd ene) and D-carbamoylase(DCase, adc gene) activity as catalyst to produce D-p-hydroxyphenylglycine(D-HPG) by hydantoinase process. Methods: The hyd gene expression plasmids were constructed. The effects of divalent metal ions in medium on the DHase activity was investigated. The acoR gene was over-expressed to investigate the correlation between the activator protein AcoR and P_acoA-hyd gene expression. The optimal promoter used to express adc gene was screened from the P_AE, P_spoVG, P_cdd and P_lytR. The hyd and adc gene co-expression plasmid was constructed and its catalytic properties was characterized. Results: The hyd gene expression plasmid pHPS and pUBS were successfully constructed. Mn ²⁺ has a strong activation effect on DHase and the activity of the 168N/pUBS reached 956U/gDCW when 0.8mmol/L MnCl₂·4H₂O was added to the medium. Adding a copy of the P_cdd-acoR gene, DHase activity of the LSL02/pUBS reached 1 470 U/gDCW. The LN04 strain integrated with the P_AE-adc had the highest DCase activity. The co-expression plasmid pUBSC was constructed, and under the optimum conditions of pH 8.0 and 40℃, with initial substrate concentration of 20g/L, the catalytic activity of LSL02/pUBSC could last for 12h to generate 14.32g/L of D-HPG with the conversion rate of 95%, yield of 82.4%. Conclusion: The recombinant strain with higher dual enzyme activity can be obtained when heterologous hyd and adc were expressed in Bacillus subtilis and it is technically feasible and has the application prospect for preparing D-HPG by hydantoinase process.

Key words: Bacillus subtilis Heterologous expression D-hydantoinase D-carbamoylase D-p-Hydroxyphenylglycine

收稿日期: 2018-09-20 出版日期: 2019-04-12

ZTFLH:

Q78

通讯作者: 班睿 E-mail: rbprofessor@163.com

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引用本文:

李法彬,刘露,杜燕,班睿. 构建重组枯草芽孢杆菌催化制备D-对羟基苯甘氨酸[J]. 中国生物工程杂志, 2019, 39(3): 75-86.

Fa-bin LI,Lu LIU,Yan DU,Rui Ban. Construction of Recombinant Bacillus subtilis as Catalyst for Preparing D- p-Hydroxyphenylglycine. China Biotechnology, 2019, 39(3): 75-86.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190310 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I3/75

图1 D, L-HPH生成D-HPG的双酶催化反应过程

表1 本研究所用的菌株和质粒

Primer name	Primer sequence (5'-3')	Size(bp)
ASU1	ATCTCGTTTCGGGAAATTAC	20
ASU2	TCAGTCTTCTCATCTTCACT	20
Sd1	AGTGAAGATGAGAAGACTGA	20
Sd2	GCGACATCCTCACCGATCAACAATCACAATGGAGGACAAT	40
ASD1	TTGATCGGTGAGGATGTCGC	20
ASD2	ATGGGTGCTTTAGTTGAAGATTATCTGGTGTCGGCAAAT	39
CR1/ CR2	TCTTCAACTAAAGCACCCAT/TTATTCATTCAGTTTTCGTG	20/20
ASG1	CACGAAAACTGAATGAATAACACATTGCGGATCTTGATAA	40
ASG2	CCATCTCTTACATTCCTCCTT	21
PHSD1	CCCAAGCTTAACGGCACGATAGACTGTATG	30
PHSD2	CGGGATCCCAATCACAATGGAGGACAATATG	31
PURS1	AAAACTGCAGCAATCACAATGGAGGACAATATG	33
PURS2	AACGGCACGATAGACTGTATG	21
PUB1	CATACAGTCTATCGTGCCGTTAGTGCCGACCAAAACCATAAAAC	44
PUB2	AAAACTGCAGCAGCACAATTCCAAGAAAAACA	32
sacBU1	TCCTCATAGCCAAGAATCC	19
sacBU2	GCAATGTTTCCGATAAAGTCTGGTAGCCGTGATAGTT	37
PacoR1/ PacoR2	GACTTTATCGGAAACATTGC/AATAGGAACGCCGTTATATG	20/20
AP1/AP2	GACAAACATCACGCTCTTG/GTGTAAATTCCTCCCTTACCT	19/21
sacBUAE	GATCAGTATATCACAGCGTTCTTGGTAGCCGTGATAGTT	39
sacBPAE1	AGAACGCTGTGATATACTGATC	22
sacBpAE2	CGTTTGGGACCGAGTTCATTCTTTACCCTCTCCTTTT	37
acoR1	ATGAACTCGGTCCCAAACG	19
sacBD1	CATATAACGGCGTTCCTATTTTGATCCTAACGATGTAACC	40
sacBG2	GGAGTCAGTGAACAGGTAC	19
pelU1/ pelU2	CGTTGTTATTCTGGCTTGAT/TGTTCCGCTATCCTATTGC	40
AP1s	GCAATAGGATAGCGGAACAGACAAACATCACGCTCTTG	38
AP2s	AAGTATCATCTGACGTGTCATGTGTAAATTCCTCCCTTACCT
spoVG1	GCAATAGGATAGCGGAACATGCGGAAGTAAACGAAGTG	38
spoVG2	GTGTACATTTCACCTCCTTTCTATATAAAAGCATTAGTGT	40
peladcA /peladcB	ATGACACGTCAGATGATACTT/GCATCGTTTGACTGAATAGC	42
pelD1	TACCAAGGAGGAGTTATAGCGGATCAAGTGACAGCAA	37
pelG2	AGTTAGCACCGTTGGAAG	18
lytRU1/ lytRU2	CTACACTATCACTGACGCTAA/AAATTACTTTCATTATGAG	21/19
AEC1/ AEC2	AGAACGCTGTGATATACTGATC/GCATCGTTTGACTGAATAGC	22/20
PUBR1	CGGGATCCCAGCACAATTCCAAGAAAAACA	30
PUBC2	GATCAGTATATCACAGCGTTCTAGTGCCGACCAAAACCATAAAAC	45
adc1	AGAACGCTGTGATATACTGATC	22
cdh2	CATACAGTCTATCGTGCCGTTCGCTATAACTCCTCCTTGGTA	42
acoA1	AACGGCACGATAGACTGTATG	21
hyd2	CGGGATCCCAATCACAATGGAGGACAATATG	31

表2 本研究所用引物

图2 D-海因酶表达质粒pHPS (a)和pUBS (b)的结构示意图

图3 反应前后反应液的HPLC分析图谱(a)和重组菌株胞内蛋白的SDS-PAGE分析(b)

图4 二价金属离子对D-海因酶活性的影响

表3 重组菌株acoR基因的qRT-PCR 转录分析

图5 胞内AcoR蛋白水平与不同拷贝数PacoA-hyd基因表达水平的关系

表4 不同启动子表达adc基因的qRT-PCR 转录分析

图6 不同重组菌株D-氨甲酰水解酶活性比较

图7 双酶共表达质粒pUBSC的结构示意图

图8 培养基组分对全细胞催化活性的影响

图9 D-HPG浓度随反应时间变化

图10 40℃不同反应时间反应液液相色谱图

图11 不同D, L-HPH浓度下LSL02/pUBSC全细胞催化反应过程

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