Heterologous Expression, Purification and Enzymatic Properties of a Novel Leucine 5-Hydroxylase

doi:10.13523/j.cb.20191204

China Biotechnology

2019, Vol. 39

Issue (12): 24-34 DOI: 10.13523/j.cb.20191204

Heterologous Expression, Purification and Enzymatic Properties of a Novel Leucine 5-Hydroxylase

ZHU Meng-lu^1,^2,³,WANG Xue-yu¹,LIU Xin¹,LU Fu-ping^1,^2,³,SUN Deng-yue^1,^2,^3,^**(

),QIN Hui-min^1,^2,^3,^**(

)

1 College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
2 Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, China
3 National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, China

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Abstract

Hydroxyl amino acids are a novel kind of amino acid derivatives, which are widely used as a chemically synthetic intermediate.A novel L-leucine-5-hydroxylase (NmLEH) from Nostoc minutum was cloned and inserted into recombinant plasmid, and its expression conditions were optimized. The results showed that when the enzyme was transferred into the BL21 (DE3) host, the induction temperature was 25℃ and the IPTG induction concentration was 0.5mmol/L, the expression levels of NmLEH was highest after induction of 10 (0.45 mg/ml). Using purification process of Ni-affinity chromatography and gel filtration, highly purified recombinant NmLEH was obtained. The enzymatic properties of NmLEH were characterized, and the optimum reaction temperature of the enzyme was 25℃, and the optimum pH was 7.5; the NmLEH enzyme was active at pH 7.0-9.0, and the optimum substrate for the NmLEH were the leucine and methionine. Sequence alignment and phylogenesis analysis implied that residues of H150, H236 and D152 constitute the catalytic triad of NmLEH, which is completely conserved in the Fe(II)/αKG-dependent dioxygenase [Fe(II)/αKG-Dos] superfamily. The formation mechanism of catalytic active site was analyzed based on NmLEH structural model analysis.

Key words： Leucine 5-hydroxylase Expression purification Enzymatic properties Substrate specificity Catalytic active site

Received: 24 April 2019 Published: 15 January 2020

ZTFLH:

Q814

Corresponding Authors: Deng-yue SUN,Hui-min QIN E-mail: dysun09@163.com;huiminqin@tust.edu.cn

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	Meng-lu ZHU
	Xue-yu WANG
	Xin LIU
	Fu-ping LU
	Deng-yue SUN
	Hui-min QIN

Cite this article:

ZHU Meng-lu,WANG Xue-yu,LIU Xin,LU Fu-ping,SUN Deng-yue,QIN Hui-min. Heterologous Expression, Purification and Enzymatic Properties of a Novel Leucine 5-Hydroxylase. China Biotechnology, 2019, 39(12): 24-34.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191204 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I12/24

Fig.1 Enzymatic reaction scheme for the NmLEH hydroxylation of L-leucine and L-methionine

Table 1 Gradient elution procedure

Fig.2 Enzyme digestion and Colony PCR of NmLEH-pQE-80L (a) 1-2: Colony PCR; M: DNA marker (b) 1: Double digestion; 2: BamH I single digestion; 3: NmLEH- pQE-80L; M: DNA marke

Fig.3 The concentration of soluble NmLEH in different induction conditions The induction conditions: 1-3.15℃, 0.1mmol/L, 0.5mmol/L, 0.75mmol/L IPTG; 4-6.25℃, 0.1mmol/L, 0.5mmol/L, 0.75mmol/L IPTG; 7-9. 37℃ 0.1mmol/L, 0.5mmol/L, 0.75mmol/L IPTG. The IPTG induction time was 10h

Fig.4 Purification of NmLEH (a) SDS-PAGE analysis of NmLEH expression in pQE-80L plasmidv Lanes 1: Supernatant; 2: Sediment; 3: Flow-through; 4: Wash buffer; 5: Resin before eluting; 6: Resin after eluting elution buffer; 7: Resin after eluting (b)Purification of NmLEH by gel filtration using a Superdex 200 HR 10/30 column The 47kDa protein mark was remarked by blue dotted line, and NmLEH protein was marked by red solid

Fig.5 Effect of temperature on NmLEH enzyme activity and thermal stability (a) The effect of temperature on activity of NmLEH (b) Thermostability of NmLEH

Fig.6 Effect of pH on the activity and stability of NmLEH (a) The effect of pH on activity of NmLEH (b) pH stability of NmLEH

Table 2 NmLEH enzyme kinetic parameters

Table 3 Substrate selectivity of NmLEH

Fig.7 Effect of enzyme cofactor on NmLEH activity a:Fe(Ⅱ)+αKG+ascorbate;b:αKG+Fe(Ⅱ)+ascorbate+EDTA;c:Fe(Ⅱ)+ascorbate;d:αKG+ascorbate; e:Fe(Ⅱ)+αKG

Fig.8 HPLC detection of leucine catalytic product

Fig.9 Sequence alignment of NmLEH

Fig.10 Phylogenetic analysis of NmLEH

Fig.11 Structural analysis of the NmLEH-substrate complex model (a) Ribbon representation of the NmLEH homology structure The α-KG and the coordinating side chains in stick mode and Fe as a sphere to illustrate the position of the metallocentre relative to the double-stranded β-helix fold (b) The predicted substrate binding model The residues, Fe, α-KG and L-leucine are colored as green, brown sphere, yellow and cyan stick, respectively


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