Heterologous Expression Of Cholesterol Oxidase And Study On Its Application

Cholesterol oxidase, which can catalyze the oxidation of cholesterol into cholest-4-en-3-one, is the key enzyme in cholesterol degradation. In recent years, cholesterol oxidase exhibits great potential value in food industry, medical care, clinical test and biological pesticide. Cholesterol oxidase has been found in many microorganisms including Arthrobacter, Brevibacterium sterolicum, Mycobacterium, Nocardia, Pseudomonas, Rhodococcus, et al.. Our lab has isolated a cholesterol oxidase-producing strain, Bordetella sp. B4. In this work, the cholesterol oxidase from Bordetella sp. B4 was heterologously expressed. The main results are as follows:1. Cloning of cholesterol oxidase gene and its expression in E. coliThe Bcod gene encoding cholesterol oxidase from Bordetella sp. B4 was cloned and sequenced. The sequencing result revealed that Bcod consists of 1656 base pairs and showed a sequence identity of 99% to cholesterol oxidase genes from Brevibacterium sp. DGCDC-82, Brevibacterium sterolicum, Arthrobacter sp. F2 and Rhodococcus equi. The Bcod was cloned in pET-22b (+) vector and transformed into E. coli Rosetta (DE3). The resulting recombinant strain was named as R/Bcod. R/Bcod could produce cholesterol oxidase (BCOD) when induced by IPTG, but almost all BCOD was expressed as insoluble, inactive inclusion bodies when induced at 37℃. Optimization experiments showed that when R/Bod was induced by 0.025 mM IPTG at 20℃for 8 h, the maximum active BCOD production could reach around 350 U/L.2. Chaperone coexpression increased active cholesterol oxidase production in recombinant E. coliThe constructed expression plasmid pET-Bcod and pACYC-mc, the expression vector of molecular chaperones of GroEL and GroES, were cotransformed into E. coli BL21 (DE3). The resulting recombinant strain was named as BL21/mc/Bcod. Optimization experiments showed that active BCOD production could reach 1000 U/L when BL21/mc/Bcod was induced by 1 mM IPTG at 20℃for 18 h. However, the control strain BL21/Bcod, which expressed BCOD alone, could only produce 450 U/L of BCOD when induced by 0.1 mM IPTG at 20℃for 10 h. SDS-PAGE analysis revealed that, production of insoluble BCOD in BL21/mc/Bcod was much decreased compared with that in BL21/Bcod. These results indicated that coexpression of GroEL, GroES and BCOD increased production of soluble, active BCOD and reduced insoluble, inactive BCOD production. BCOD expressed in BL21/mc/Bcod was purified and characterized. The purified enzyme presented a single band of 59 kDa on SDS-PAGE gel and its optimum pH and temperature were pH 7.0 and 40 C, respectively. The temperature stability and affinity of recombinant BCOD towards cholesterol were weaker than original BCOD. The inhibiting effect of Ag+ was much decreased. Cu2+ showed 1.7-fold enhancement on COD activity.3. Expression of cholesterol oxidase in Lactococcus lactisNICE system was first used for expression of cholesterol oxidase. The constructed recombinant strain, which produced BCOD by nisin induction, was named as LSc. The optimal induction conditions for BCOD production by single factor optimization experiment were as follows:When OD600 of LSc culture reached 0.4,4 ng/mL of nisin was added and cultivation was continued at 20℃for 8 h,340 U/L of BCOD production was obtained. Properties of the crude BCOD from induced LSc were characterized. The optimum pH and temperature were pH 7.0 and 37℃, respectively. Crude BCOD was very stable in a pH range of 5-10. Ag+ showed strong inhibiting effect on BCOD activity, while Cu2+ showed activity enhancement effect.4. Cholesterol degradation by recombinant Lactococcus lactis LScDegradation of cholesterol in culture medium by induced LSc and egg yolk cholesterol by crude BCOD from LSc, were determined. When the initial cholesterol concentration in the culture medium was 110μg/mL,20%,47%,67% of cholesterol were degraded after 20 h,48 h,72 h of transformation by LSc, respectively. When the initial egg yolk cholesterol was 1260μg/mL and in the presence of 0.5 M NaCl,40%, 70%,80% of cholesterol were degraded after 24 h,48 h,72 h of treatment by the crude enzyme from induced LSc, respectively.