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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (2): 30-37    DOI: 10.13523/j.cb.20180205
研究报告     
新型R-扁桃酸脱氢酶的基因挖掘及表达鉴定 *
唐存多1,2*,史红玲1*,马越1,丁朋举1,许建和2**(),阚云超1**(),姚伦广1**()
1 南阳师范学院昆虫生物反应器河南省工程实验室和河南省南水北调中线水源区生态安全重点实验室 南阳 473061
2华东理工大学生物反应器工程国家重点实验室 上海 200237
Gene Mining, Expression and Characterization of Novel R-mandelate Dehydrogenases
Cun-duo TANG1,2*,Hong-ling SHI1*,Yue MA1,Peng-ju DING1,Jian-he XU2**(),Yun-chao KAN1**(),Lun-guang YAO1**()
1 Henan Provincial Engineering Laboratory of Insect Bio-reactor and Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North, Nanyang Normal University, Nanyang 473061, China
2 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
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摘要：

R-扁桃酸脱氢酶在苯乙酮酸的生物合成中起着关键的作用,挖掘具有高催化活性及稳定性的新型R-扁桃酸脱氢酶具有重要的意义。为了获得理想的R-扁桃酸脱氢酶,采用了基因组挖矿技术从Lactobacillus harbinensis菌株中获得了一个新型的R-扁桃酸脱氢酶LhDMDH,重组LhDMDH的比酶活高达1264.3 U/mg,约为探针的4倍,在已报道的R-扁桃酸脱氢酶中处于领先水平。同时,考察了4个重组酶主要的酶学特性,它们的最适反应温度在25~30℃,最适反应pH在9.0~9.5。动力学参数的结果表明,LhDMDH对底物的Kcat值为30.28 S -1,明显高于其它重组酶。此外,底物谱分析的结果也表明LhDMDH在外消旋扁桃酸的手性拆分及苯乙酮酸的生物合成中更具优势。在R-扁桃酸脱氢酶基因挖掘方面取得了较为理想的结果,为进一步的改造及应用奠定了坚实的基础,也为其它酶的挖掘提供了可资借鉴的经验。

关键词： 扁桃酸脱氢酶;  基因组挖矿;  表达;  酶学特性;  生物催化    
Abstract:

R-mandelate dehydrogenase plays a key role in the biosynthesis of phenylglyoxylic acid, thus exploiting nove R-mandelate dehydrogenase with higher catalytic activity and stability has significant economic value. In order to obtain the perfect R-mandelate dehydrogenase, a novel R-mandelate dehydrogenase was obtained by genome mining, named as LhDMDH, which was from Lactobacillus harbinensis. The specific activity of LhDMDH was 1264.3 U/mg, which was near to four times that of the probe and leading in the reported enzyme. Meanwhile, the main enzymatic characterizations of the four recombinant enzymes were researched. Their temperature optima were 25 to 30 ℃, and their pH optima were 9.0 to 9.5. The Kcat of LhDMDH is 30.28 S -1, which is obviously higher than the others. In addtion, the results of substrate spectrum of R-mandelate dehydrogenases indicated that the LhDMDH could have advantages over other enzymes in chiral resolution of racemic mandelic acid and the biosynthesis of phenylglyoxylic acid. This gained ideal results in genome mining of R-mandelate dehydrogenases, established a solid foundation for further transformation and application, and provided a useful experience for the exploiting of other enzymes.

Key words: Mandelate dehydrogenase    Genome mining    Expression    Enzymatic characterization    Biocatalysis
收稿日期: 2017-10-24 出版日期: 2018-03-21
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(31372381);河南省科技攻关项目(162102210116);河南省科研服务平台专项(2016151);河南省南水北调中线水源区水生态安全创新型科技团队专项资助项目(17454)
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姚伦广

引用本文:

唐存多,史红玲,马越,丁朋举,许建和,阚云超,姚伦广. 新型R-扁桃酸脱氢酶的基因挖掘及表达鉴定 *[J]. 中国生物工程杂志, 2018, 38(2): 30-37.

Cun-duo TANG,Hong-ling SHI,Yue MA,Peng-ju DING,Jian-he XU,Yun-chao KAN,Lun-guang YAO. Gene Mining, Expression and Characterization of Novel R-mandelate Dehydrogenases. China Biotechnology, 2018, 38(2): 30-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180205    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I2/30

Primer name Product description Primer sequence (5'-3') Accession number
SeDMDH-F DMDH from Salmonella enterica CGCGGATCCATGAAAATTGCAATCGCAGG WP_058112521.1
SeDMDH-R CCGCTCGAGTTGCGCTTCTTTAGCGTGAA
LlDMDH1-F DMDH1 from Lactococcus lactis CGCGGATCCATGAAAATCGCAATAGCAGG WP_075525716.1
LlDMDH1-R CCGCTCGAGATGCGCCTCTAAAATTTCTTC
LlDMDH2-F DMDH2 from Lactococcus lactis CGCGGATCCATGAGAATCACAATTGCCGGT WP_011676318.1
LlDMDH2-R CCGCTCGAGTTTCGCCTTTAATAACTCTT
SaDMDH-F DMDH from Staphylococcus aureus CGCGGATCCATGTACAAAATAGCCATAG WP_031797626.1
SaDMDH-R CCGCTCGAGTAATTTTGATTCTAATTGGT
Primer name Product description Primer sequence (5'-3') Accession number
KoDMDH-F DMDH from Klebsiella oxytoca CGCGGATCCATGAAAATTGCAATCGCAG WP_004104561.1
KoDMDH-R CCGCTCGAGTTGCGCTTCTTTTGCGTGAAT
LcDMDH-F DMDH from Leuconostoc citreum CGCGGATCCATGAAAATAGCTATTGCAGGA WP_036058586.1
LcDMDH-R CCGCTCGAGAATTTCAAAGTTTTCTTGCT
表1  新型R-扁桃酸脱氢酶基因克隆及表达所需的引物
图1  4个R-扁桃酸脱氢酶基因的PCR扩增
图2  重组大肠杆菌的菌落PCR鉴定
图3  代表性重组R-扁桃酸脱氢酶的SDS-PAGE分析
图4  重组R-扁桃酸脱氢酶的最适温度
图5  重组R-扁桃酸脱氢酶的温度稳定性
图6  重组R-扁桃酸脱氢酶的最适pH
图7  重组R-扁桃酸脱氢酶的pH稳定性
Enzyme Kinetic parameter of substrate
Km (mmol/L) Kcat (S-1) Ki (mmol/L) Kcat/Km (S-1 mmol/L-1)
LbDMDH 1.45 8.82 24.37 6.08
LhDMDH 1.05 30.28 26.40 28.80
LcDMDH 1.75 1.56 24.86 0.89
LlDMDH-2 1.00 2.29 16.25 2.29
表2  R-扁桃酸脱氢酶对R-扁桃酸的动力学参数
Enzyme Kinetic parameter of NAD+
Km (mmol/L) Kcat (S-1) Ki (mmol/L) Kcat/Km (S-1 mmol/L-1)
LbDMDH 0.78 9.23 N 11.83
LhDMDH 0.40 29.05 N 72.00
LcDMDH 0.85 1.86 N 2.19
LlDMDH-2 0.42 1.85 N 4.40
表3  R-扁桃酸脱氢酶对NAD+的动力学参数
Substrate Relative activity (%)
LbDMDH LhDMDH LcDMDH LlDMDH-2
R-mandelate acid 100 100 100 100
R-2-chloromandelic acid 0.59 0.28 2.51 0.05
S-mandelate acid 0.04 0.02 0 0
S-2-chloromandelic acid 0 0 0 0
L-lactic acid 0 0 0 0
表4  R-扁桃酸脱氢酶的底物谱
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