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A 6.2-kb Apal fragment was cloned, which encoded a cytochrome P450, a ferrodoxin, and a ferrodixin reductase using N-terminal sequence information. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells.

Research has been conducted on Pseudomonas putida PAO1 alkane hydroxylases. The ability of the alkane hydroxylases to oxidize n-alkenes has been tested via the use of different recombination hosts and the results are reported.

Research has been conducted on rubredoxins. The sequence analysis and complementation studies indicate that these rubredoxins can be divided into AlkG1-type and AlkG2 rubredoxins and the mutagenesis results show which sequence elements determine whether rubredoxins can transfer electrons to alkane hydroxylase.

Research has been conducted on the N-benzylpyrrolidine. The hydroxylation of this compound has been carried out with the Sphingomonas sp. HXN-200 resting cells to produce N-benzyl-3-hydroxypyrrolidine and the results indicate that the change of the docking/protecting group in pyrrolidines improves the hydroxylation activity and enantioselectivity of the compound.

Results show that the alkane hydroxylase gene of Burkholderia cepacia RR10 exhibits catabolite repression in the presence of organic acids and sugars in complex media. Data indicate that transcription of the gene is induced by alkanes of 12 to 30 carbon chain length and by alkanols.

Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences have been analyzed with attention to regulation and evolution of the alk genes. Only the alkB and alkS promoter regions are conserved, other than the coding regions of the GPo1 and P1 alk genes.

The mechanisms that regulate the expression of Pseudomonas oleovorans alkB were analyzed in the Escherichia coli W3110 gene expression system. The levels of AlkB cytoplasmic membrane protein alkane hydroxylase were elevated by the expression of Pseudomonas oleovorans alkB gene in Escherichia coli W3110. The expression of alkB also increased phospholipid synthesis and induced the appearance of intracytoplasmic membrane structures. Furthermore, increase phospholipid synthesis was a consequence of high-level AlkB synthesis and a requirement for microbial growth.

A study was conducted to show that most of the strains that possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases, using PCR. Result provided evidence that the newly cloned P450s are indeed alkane hydroxylases.

Enzymes are being used in numerous new applications in the food, feed, agriculture, paper, leather, and textiles industries, resulting in significant cost reductions. At the same time, rapid technological developments are now stimulating the chemistry and pharma industries to embrace enzyme technology, a trend strengthened by concerns regarding health, energy, raw materials, and the environment. [Copyright &y& Elsevier]