| Cytochrome P450s (P450s or CYPs), as a monooxygenases belong to the large family of hemoproteins, are widely existed and play an important role in the metabolism of endogenous substances and xenobiotics. As we know, P45Os are the most versatile biological catalyst, which can catalyze diverse reactions including hydroxylation, epoxidation, dealkylation, dehydrogenation, deamination and dehalogenation of the substrates, and can degrade recalcitrant compounds. Therefore, many researchers have focused on studying the potential applications of cytochrome 450 enzymes in environmental remediation in recent years.In the present study, a novel cytochrome P450 monooxygenase CYP102A16 from Bacillus sp. C3 was cloned and expressed in Escherichia coli BL21 (DE3); the expression of functional CYP102A16 was enhanced effectively by optimization process; CYP102A16 was purified using Ni-NTA 6×His-tag affinity chromatography; and the enzymatic properties of the purified product was characterized. The main results are as follows:1) Cloning and expression of CYP102A16The full-length P450 gene from Bacillus sp. C3 was obtained by using degenerate PCR, which was named as CYP102A16 by Cytochrome P450 Nomenclature Committee (GenBank number:FR775424). The 3198-bp CYP102A16 gene encodes a peptide consisting of 1065 amino acids with a molecular mass of approximately 119 kDa. CYP102A16 was cloned into pET28a(+) and expressed in E.coli BL21 (DE3). The enzyme was purified on a Ni-NTA 6×His-tag affinity resin, gaining a purity of 84% as estimated densitometrically from the SDS-PAGE.2) Optimal expression of CYP102A16The expression of CYP102A16 was greatly enhanced by applying one-factor-a-time experiments and statistical designs, for optimization of cultural condition and fermentation medium, respectively. The CYP102A16 production increased 3.65-fold and reached a maximum of 0.43μmol·L-1.3) Activities and stabilities of CYP102A16 The effects of temperature, pH, organic solvents, surfactants and metal ions on the activity of CYP102A16 were investigated. The optimum reaction temperature and pH for CYP102A16 were 35℃, pH 6.5-7.5, respectively, with stability up to 45℃, at pH 5.0-10.0. Purified CYP102A16 was completely tolerant to 20% DMSO,30% methanol,10% ethanol and 20% acetone, and could retain above 45% activity in 10% acetonitrile, n-propanol and isopropanol, but was almost inactivated in 10% n-butanol. Enzyme activity were enhanced to about 140% and 160% by low level of CPC (0.003-0.02 g·L-1) and Triton X-100 (0.1-0.2 g·L-1), respectively, but was affected insignificantly with low concentraion of SDS (0.004-0.008 g-L-1). Addition of 1-20 mM K+,20-50 mM Na+ and 0.05 mM Cd2+ could increase CYP102A16 activity slightly, while NH4+, Ca2+, Mg2+, Fe3+, Co2+, Mn2+, Zn2+ Cu2+ and EDTA exhibited negative effects on enzyme activity.4) Kinetics analysis of CYP102A16CYP102A16 showed preference for long-chain fatty acids (C12-C18), and could bind to unsaturated fatty acids more tightly than saturated fatty acids. The KD value towads saturated fatty acids would increase with the chain length getting longer, indicating a weaker binding. CYP102A16 exhibited a strongest affinity to palmitic acid (Km=10.1μM). The Km value towards saturated fatty acids was significantly higher than unsaturated fatty acids (palmitic acid excluded). The Kcat value of CYP102A16 towards these fatty acids was relatively low within 110～580 min-15) Substrate screening of CYP102A16Besides long chain fatty acids, CYP102A16 could oxide short long chain fatty acids (<C10), alkanes (n-octane, dodecane and n-hexadecane) and some kinds of aromatic compounds such as nitrobenzene, naphthalene, diphenylether and 2,4-D. The ability of CYP102A16 that they can degrade toxic compounds in the environment indicates that CYP102A16 is potential useful in remediation. |
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