Heterologous Expression, Purification and Enzymatic Characterization of Cholesterol Oxidase PsCO<sub>4</sub>

doi:10.13523/j.cb.20180605

China Biotechnology

2018, Vol. 38

Issue (6): 34-42 DOI: 10.13523/j.cb.20180605

Heterologous Expression, Purification and Enzymatic Characterization of Cholesterol Oxidase PsCO₄

Qian-qian GUO,Deng-ke GAO,Xiao-tao CHENG,Fu-ping LU,Tanokura Masaru(

),Hui-min QIN(

)

State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology Ministryof Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China

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Abstract

Cholesterol oxidase, which catalyzes the reaction of cholesterol to cholest-4-en-3-one, is widely used in clinical and food processing industry. Cholesterol oxidase from Pimelobacter simplex (PsCO₄) was transformed into E.coli BL21(DE3), Rosetta(DE3), and C41(DE3). The bacteria were induced at different temperatures (15℃, 25℃, 37℃), and different IPTG concentrations (0.01mmol/L, 0.1mmol/L, 0.5mmol/L). The results showed that PsCO₄ was expressed greatly in supernatant of Rosetta(DE3) (0.63mg/ml), at the conditions of 0.1mmol/L IPTG and 15℃. The optimum temperature and pH of heterologous expressed PsCO₄ was 30℃ and 7.5. The product of cholesterol catalyzed by PsCO₄ was identified by TLC and GC-MS. The substrate specificity of PsCO₄ towards cholesterol, β-sitosterol, stigmasterol and pregnenolone was determined. The K_m/k_cat value of cholesterol (0.08s ^-1·μM ^-1) is higher than β-sitosterol (0.04s ^-1·μM ^-1), stigmasterol (0.005s ^-1·μM ^-1) and pregnenolone (0.02s ^-1·μM ^-1).

Key words： Cholesterol oxidase Catalytic activity Enzymatic property Substrate specificity

Received: 08 January 2018 Published: 06 July 2018

ZTFLH:

Q786

Corresponding Authors: Tanokura Masaru,Hui-min QIN E-mail: amtanok@mail.ecc.u-tokyo.ac.jp;huiminqin@tust.edu.cn

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	Qian-qian GUO
	Deng-ke GAO
	Xiao-tao CHENG
	Fu-ping LU
	Tanokura Masaru
	Hui-min QIN

Cite this article:

Qian-qian GUO,Deng-ke GAO,Xiao-tao CHENG,Fu-ping LU,Tanokura Masaru,Hui-min QIN. Heterologous Expression, Purification and Enzymatic Characterization of Cholesterol Oxidase PsCO₄. China Biotechnology, 2018, 38(6): 34-42.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180605 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I6/34

Fig.1 Three-step catalytic reaction of cholesterol oxidase

Fig.2 Enzyme digestion and colony PCR of PsCO₄-pET28a olony PCR;2:Double enzyme digestion;3:EcoR I digestion; 4:PsCO₄-pET28a;5:DNA marker KB ladder

Fig.3 The concentration of soluble PsCO₄ in different induction conditions Induction conditions: 1~3: 15℃ 0.05, 0.1, 0.5mmol/L IPTG; 4~6: 25 ℃ 0.05, 0.1, 0.5mmol/L IPTG; 7~9: 37℃ 0.05, 0.1, 0.5mmol/L IPTG.

Fig.4 SDS-PAGE of affinity chromatography and ion exchange chromatography a)1:Supernatant; 2:Precipitant; 3:Flowthrough; 4: Washing buffer; 5:Elution buffer (b)Ion exchange chromatography

Fig.5 Gel filtration chromatography

Fig.6 Product analysis with (a)TLC and(b)(c)GC-MS (a)1:Vector pET-28a; 2: PsCO₄-pET28a; 3:Cholesterol; 4: Standard of 4-Cholesten-3-one

Fig.7 Effect of temperature (a) and pH (b) on the PsCO₄

Table 1 Cholesterol oxidase enzymatic properties

Fig.8 PsCO₄ active center substrate binding key amino acid comparison

Fig.9 Cholesterol oxidase substrate binding domains from different sources (a)PsCO₄ mimic substrate binding channel (b)PsChO substrate binding channel (c)Cholesterol oxidase 1coy substrate-bound channel from Brevibacterium sterolicum

Fig.10 Cholesterol oxidase catalytic mechanism (a) ^[16] and cholesterol oxidase sequence alignment (b) F. sp.: Frankiasp.EAN1pec, WP_020461426; S. arenicola: Sarenicola arenicola CNS-205 WP_012182946; S. sp.: Streptomyces sp., AAA26719


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