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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (2): 30-37    DOI: 10.13523/j.cb.20180205
Orginal Article     
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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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     
Received: 24 October 2017      Published: 21 March 2018
ZTFLH:  Q819  
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Cun-duo TANG
Hong-ling SHI
Yue MA
Peng-ju DING
Jian-he XU
Yun-chao KAN
Lun-guang YAO
Cite this article:

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.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180205     OR     https://manu60.magtech.com.cn/biotech/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
Table 1 Sequences of the primers used for cloning and expression of novel R-mandelate dehydrogenases
Fig.1 The PCR amplification for the four R-mandelate dehydrogenase geneM: DNA marker; 1: LlDMDH1; 2: LlDMDH2; 3: KoDMDH; 4: SeDMDH; 5: SaDMDH; 6: LcDMDH
Fig.2 The bacterial colonies PCR identification for the recombinant E.coliM: DNA marker; 1: BL21/pET28a-LlDMDH1; 2: BL21/pET28a-LlDMDH2; 3: BL21/pET28a-KoDMDH; 4: BL21/pET28a-LcDMDH; 5: BL21/pET28a-LpDMDH; 6: BL21/pET28a-SsDMDH; 7: BL21/pET28a-CbDMDH; 8: BL21/pET28a-LhDMDH
Fig.3 The SDS-PAGE analysis for the representative recombinant R-mandelate dehydrogenasesM: PageRuler Prestained Protein Ladder; 1: Expressed products of BL21/pET28a; 2: Crude LbDMDH; 3: Purified LbDMDH; 4: Crude LhDMDH; 5: Purified LhDMDH; 6: Crude LcDMDH; 7: Purified LcDMDH; 8: Crude LlDMDH-2; 9: Purified LlDMDH-2
Fig.4 The temperature optima of recombinant R-mandelate dehydrogenases
Fig.5 The temperature stability of recombinant R-mandelate dehydrogenases
Fig.6 The pH optima of recombinant R-mandelate dehydrogenases
Fig.7 The pH stability of recombinant R-mandelate dehydrogenases
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
Table 2 The kinetic parameter of R-mandelate dehydrogenases for R-mandelate acid
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
Table 3 The kinetic parameter of R-mandelate dehydrogenases for NAD+
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
Table 4 The substrate spectrum of R-mandelate dehydrogenases
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