Expression optimization of recombinant cholesterol oxidase in Escherichia coli and its purification and characterization

Abstract

Cholesterol oxidase is a bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This enzyme has a great commercial value because of its wide applications in cholesterol analysis of clinical samples, synthesis of steroid-derived drugs, food industries, and potentially insecticidal activity. Accordingly, development of an efficient protocol for overexpression of cholesterol oxidase can be very valuable and beneficial. In this study, expression optimization of cholesterol oxidase from Streptomyces sp. SA-COO was investigated in Escherichia coli host strains. Various parameters that may influence the yield of a recombinant enzyme were evaluated individually. The optimal host strain, culture media, induction time, Isopropyl ß-d-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature were determined in a shaking flask mode. Applying the optimized protocol, the production of recombinant cholesterol oxidase was significantly enhanced from 3.2 to 158 U/L. Under the optimized condition, the enzyme was produced on a large-scale, and highly expressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver–Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were also determined. We report a straightforward and easy protocol for cholesterol oxidase production which can be performed in any laboratory.

Identification of cholesterol-assimilating actinomycetes strain and application of statistical modeling approaches for improvement of cholesterol oxidase production by Streptomyces anulatus strain NEAE-94

“…They found that the maximum cholesterol oxidase production by Streptomyces rimosus was achieved using the optimal levels of process variables which found to be inoculum size (3%, v/v), pH 7, incubation temperature (30°C), incubation time (48 h) and agitation speed (200 rpm). Fazaeli et al [46] achieved maximum cholesterol oxidase production by Escherichia coli when the induced culture production medium was incubated for 24 h at 15°C. Also, Niwas et al [30] studied the effect of process variables on cholesterol oxidase production by Streptomyces sp.…”

Identification of cholesterol-assimilating actinomycetes strain and application of statistical modeling approaches for improvement of cholesterol oxidase production by Streptomyces anulatus strain NEAE-94

“…[44] used a two-step statistical approach to optimize the production of cholesterol oxidase from [46] achieved maximum cholesterol oxidase production by Escherichia coli when the induced culture production medium was incubated for 24 hours at 15°C. Also, Niwas et al [30] studied the effect of process variables on cholesterol oxidase production by Streptomyces sp.…”

Cloning, expression, and in silico structural modeling of cholesterol oxidase of Acinetobacter sp. strain RAMD in E. coli

“…Typically, the cloning-expression of CHOXs using the heterologous host E. coli, a species Generally Regarded as Safe (GRAS), is considered the gold standard solution for avoiding these obstacles. In this regard, great attention has been paid in recent years toward the cloning and expression of CHOXs from a wide range of pathogenic and non-pathogenic bacteria [2,[26][27][28][29][30][31][32][33], especially after the publication of a plethora of microbial genome sequences by the NCBI.…”

“…In general, using high concentrations of sugar as additives in the growth media would lead to a drop in pH below 6.0. This, in turn, would lead to a reduction in cell density [29]. Interestingly, the cell density recombinant protein production profile is diverse with respect to various carbon sources with dissimilar cellular uptake and recombinant protein production in E. coli [43,44].…”