Biodegradation Of Alkane, Cloning And Expression Of Alkane Hydroxylase Gene

A bacterium strain,2B5, was isolated from the sea mud of the crude oil-polluted Donghai area in China, which was capable of utilizing alkane as the sole carbon source for its growth. This bacterium was identified preliminarily as Alcanivorax sp. based on its physiological characteristics and analysis of its 16S rDNA sequence. The strain 2B5 was a round-shaped, gram-negative bacterium which produced biosurfactant. The strain 2B5 degraded 500 mg/L octadecane in 96h. Surgars could not be metabolized by the strain 2B5. The optimal temperature and pH for strain 2B5 growth and octadecane degradation were 30～37℃and pH 6.0～7.0, respectively. NaCl is required for strain 2B5 growth and octadecane degradation with the optimal concentrations of 20 to 90 g/L. Strain 2B5 was able to degrade C13～C3o n-alkanes and branched alkanes (pristane and phytane) in crude oil as the sole carbon source. The optimal temperature and inoculum for strain 2B5 alkanes in crude oil degradation were 30℃and 1%, respectively. The aeration has little effect on degrading ability of the strain 2B5. NH4NO3 (2 g/L) and yeast extract (0.25 g/L) could improve degrading ability largely. Based on these results, it is obvious that the strain 2B5 could potentially be useful for the remediation of hydrocarbon pollution in the marine environment.An extremely thermophilic alkane-degrading bacterium, strain MH-1 was isolated from the deep subterranean petroleum reservoir in Shengli oil field in China. The strain MH-1 was rod-shaped gram-positive strain. Based on its physiological characteristics and analysis of its 16S rRNA gene sequence, the strain MH-1 was identified as Geobacillus sp. The strain MH-1 was able to grow at temperatures ranging from 50 to 72℃and effectively degraded hexadecane as the sole carbon source at 70℃. The strain MH-1 degraded alkanes with different length chain (C12～C31) in crude oil, but it preferentially degraded middle chain-alkanes.A novel alkane hydroxylase gene (GenBank Accession No. FJ905614), obtained by self-formed adaptor PCR (SEFA-PCR), showed 41.9% deduced amino acid sequence identity with AlkB1 of Alcanivorax borkumensis SK2. Functional heterologous expression of the alkB gene was achieved in Pseudomona fluorescens KOB2△1. Recombinant Pseudomonas containing the alkB gene of Alcanivorax sp.2B5 recovered the ability to grow on C14 and C16 n-alkanes. The three conserved histidine motifs and a fourth motif found in the alkane hydroxylases were identified in the AlkB of the strain 2B5. RT-PCR analysis showed that expression of the alkB gene was induced by octadecane. The immediately upstream sequence of the alkB gene was found to encode a peptide with 58% sequence similarity with a putative AraC family transcriptional regulator protein. In the downstream region of the alkB gene, an open reading fame was 45% similar to the deduced amino acid sequence of the siderophore-interacting protein in Shewanella sp. W3-18-1.Cytochrome P450 gene and its cluster were also cloned by the same method. The cluster included transcriptional regulator, fdx, P450, alkJ and fdr which had a 73%,98%, 99%,99% and 99% sequence similarity respectively with transcriptional regulator of Limnobacter sp. MED 105 and putative ferrodoxin cytochrome (Fdx), P450 alkane hydroxylase, putative alcohol dehydrogenas, putative FAD-dependent oxidoreductase family protein (Fdr) of Alcanivorax dieselolei B-5T. The fdx, P450 and fdr genes were ligated into expression vector pET29a, then transformed into E.coli BL21 (DE3) which could not degrade alkane s.Alkane hydroxylase system of Geobacillus sp. MH-1 was characterized by self-formed adaptor PCR (SEFA-PCR). It contained at least three alkane monooxygenase gene homologs such as alkB-geol, alkB-geo4 and alkB-geo6, which had a high sequence similarity with alkB4, alkB3 and alkB2 in Rhodococcus sp. Q15, respectively. AlkB-geo6 cluster (GenBank Accession No. FJ977899) included putative transporter, alkB-geo6, rubA3, rubA4 and putative regulatory protein, which had a high sequence similarity (98%) with the alkB2 cluster of Rhodococcus sp. NRRL B-16531 and Q15. The conserved Hist-1, Hist-2, Hist-3 and HYG-motif were also found in the AlkB-geo6 of strain MH-1. AlkB-geo6 in strain MH-1 had a very high amino acid sequence similarity with AlkB2 of Rhodococcus sp. Q15 and Rhodococcus sp. NRRL-16531. There was only four amino acids variation among them.