Effect of Prolines in the Loop of CPC Acylase Substrate Binding Region on Its Catalytic Properties

doi:10.13523/j.cb.20171205

China Biotechnology

2017, Vol. 37

Issue (12): 34-39 DOI: 10.13523/j.cb.20171205

Orginal Article

Effect of Prolines in the Loop of CPC Acylase Substrate Binding Region on Its Catalytic Properties

Cun-duo TANG^1,^2*,Hong-ling SHI^1*,Zhu-jin JIAO¹,Fei LIU¹,Jian-he XU^2**(

),Yun-chao KAN^1**(

),Lun-guang YAO^1**(

)

1 Henan Provincial Engineering Laboratory of Insect Bio-reactor, 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

Cold-active CPC acylase plays an important role and has significant advantage in the biosynthesis of 7-ACA, thus developing cold-active CPC acylase has significant economic value. In order to obtain the CPC acylase with higher catalytic activity at low temperature, on the basis of previous research, the previously obtained CA III^M was taken as a parent, confirmed its substrate binding region by the method of molecular docking, and found the key proline residues in the loop of CPC acylase substrate binding region by pyMOL. After analyzing, the selected prolines were mutated into glycines, respectively. The study of soluble expression was carried out in E. coli BL21(DE3) with pET32a plasmid, and soluble expression was achieved in the other mutants except P272G. Catalytic activity of P238G, P582G and P679G to CPC was 1.25, 1.04 and 1.38 U/mg respectively at 13℃, and there was a significant improvement compared with parental 0.85 U/mg. Moreover, the stability of parent and mutants was investigated, and there was no obvious difference between them. Afterwards, the low temperature biosynthesis of 7-ACA was carried out at 13℃, and the results showed that the conversion rate of CPC can reach 80% and above after 24h . The ideal results in cold adaptation improvement of CPC acylase were gained. So a solid foundation for further transformation and application was established, it provided a useful experience for the creating of other low-temperature enzymes.

Key words： CPC acylase Cold adaptation Molecular modification Rational design Activity assay

Received: 25 May 2017 Published: 16 December 2017

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	Cun-duo TANG
	Hong-ling SHI
	Zhu-jin JIAO
	Fei LIU
	Jian-he XU
	Yun-chao KAN
	Lun-guang YAO

Cite this article:

Cun-duo TANG,Hong-ling SHI,Zhu-jin JIAO,Fei LIU,Jian-he XU,Yun-chao KAN,Lun-guang YAO. Effect of Prolines in the Loop of CPC Acylase Substrate Binding Region on Its Catalytic Properties. China Biotechnology, 2017, 37(12): 34-39.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20171205 OR https://manu60.magtech.com.cn/biotech/Y2017/V37/I12/34

Fig.1 The molecule-docked conformation between 3-D structures of a CPC and CAⅢ^M

Table 1 Sequences of the primers used for site-directed mutagenesis of CA III^M-encoding gene

Fig.2 The SDS-PAGE analysis for the expressed products of recombinant E. coli
M: PageRuler Prestained Protein Ladder; 1: Expressed products of BL21/pET32a; 2: Expressed products of BL21/pET32a-caIII^P238G; 3: Expressed products of BL21/pET32a-caIII^P272G; 4: Expressed products of BL21/pET32a-caIII^P582G; 5: Expressed products of BL21/pET32a-caIII^P679G

Fig.3 The SDS-PAGE analysis for the purified recombinant cephalosporin C acylases
M: PageRuler Prestained Protein Ladder; 1: Purified reCAIII^P238G; 2: Purified reCAIII^P582G; 3: Purified reCAIII^P679G

Table 2 Temperature stability of the parent and mutants

Fig.4 The conversion rate of CPC at low temperature


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