In vivo and in vitro studies on the carotenoid cleavage oxygenases from Sphingopyxis alaskensis RB2256 and Plesiocystis pacifica SIR-1 revealed their substrate specificities and non-retinal-forming cleavage activities.

Carotenoid cleavage oxygenases are nonheme iron enzymes that specifically cleave carbon-carbon double bonds of carotenoids. Their apocarotenoid cleavage products serve as important signaling molecules that are involved in various biological processes. A database search revealed the presence of putative carotenoid cleavage oxygenase genes in the genomes of Sphingopyxis alaskensis RB2256 and Plesiocystis pacifica SIR-1. The four genes sala_1698, sala_1008, ppsir1_15490 and ppsir1_17230 were cloned and heterologously expressed in carotenoid-producing Escherichia coli JM109 strains. Two of the four encoded proteins exhibited carotenoid cleavage activity. S. alaskensis RB2256 carotenoid cleavage oxygenase (SaCCO), which is encoded by sala_1698, was shown to cleave acyclic and monocyclic substrates. Coexpression of sala_1698 in carotenoid-producing E. coli JM109 strains revealed cleavage activity for lycopene, hydroxylycopene, and dihydroxylycopene. The monocyclic substrate apo-8'-carotenal was cleaved in vitro by purified SaCCO at the 9'/10' and 11'/12' double bonds. The second enzyme, P. pacifica SIR-1 carotenoid cleavage oxygenase (PpCCO), is encoded by ppsir1_15490. PpCCO-mediated carotenoid cleavage requires the presence of either hydroxy or keto groups. PpCCO cleaved zeaxanthin, hydroxylycopene, and dihydroxylycopene, and also the C(50) carotenoids decaprenoxanthin, sarprenoxanthin and sarcinaxanthin, in carotenoid-producing E. coli JM109 strains. Whole cells of E. coli JM109 overexpressing ppsir1_15490mut, a mutant of ppsir1_15490 with enhanced gene expression, were applied for the conversion of carotenoids. Analysis of the carotenoid cleavage products revealed a single cleavage site at the 13'/14' double bond for astaxanthin, and two cleavage sites at the 11'/12' or 13'/14' double bond for zeaxanthin, nostoxanthin, and canthaxanthin.

Thiol-functionalization of acrylic ester monomers catalyzed by immobilized Humicola insolens cutinase.

Immobilized cutinase HiC from the ascomycete Humicola insolens was applied as a novel biocatalyst for the synthesis of functionalized acryclic esters by transesterification. As a model reaction, transesterification of methyl acrylate with 6-mercapto-1-hexanol at a high molar ratio in a solvent free system was chosen. Besides two minor Michael-addition by-products, 6-mercaptohexyl acrylic ester was identified as the main product with the thiol as the functional end group. Reaction conditions were optimized regarding the influence of water (0-1.72 M), temperature (22-50 °C), product inhibition and addition of the radical inhibitor butylated hydroxytoluol (BHT; 0.14-0.71 M) on conversion and by-product formation. Highest conversion of 6-mercapto-1-hexanol to 6-mercaptohexyl acrylic ester (95.4 ± 0.3%) was achieved after 6h at 40 °C in the presence of 0.025% (w/w) water without formation of by-products in a solvent free system. Applying methyl methacrylate, transesterification with 6-mercapto-1-hexanol was significantly lower (43.6 ± 0.1%) compared to transesterification of methyl acrylate with 6-mercapto-1-hexanol.

Biosynthesis of zeaxanthin in recombinant Pseudomonas putida.

Pseudomonas putida KT2440 strain was investigated for biosynthesis of the valuable xanthophyll zeaxanthin. A new plasmid was constructed harboring five carotenogenic genes from Pantoea ananatis and three genes from Escherichia coli under control of an L: -rhamnose-inducible promoter. Pseudomonas putida KT2440 wild type hardly tolerated the plasmids for carotenoid production. Mating experiments with E. coli S17-1 strains revealed that the carotenoid products are toxic to the Pseudomonas putida cells. Several carotenoid-tolerant transposon mutants could be isolated, and different gene targets for relief of carotenoid toxicity were identified. After optimization of cultivation conditions and product processing, 51 mg/l zeaxanthin could be produced, corresponding to a product yield of 7 mg zeaxanthin per gram cell dry weight. The effect of various additives on production of hydrophobic zeaxanthin was investigated as well. Particularly, the addition of lecithin during cell cultivation increased volumetric productivity of Pseudomonas putida by a factor of 4.7 (51 mg/l vs. 239 mg/l).

Identification of the GDP-N-acetyl-d-perosamine producing enzymes from Escherichia coli O157:H7.

GDP-N-acetyl-d-perosamine is a precursor of the LPS-O-antigen biosynthesis in Escherichia coli O157:H7. Like other GDP-6-deoxyhexoses, GDP-N-acetyl-d-perosamine is supposed to be synthesized via GDP-4-keto-6-deoxy-d-mannose, followed by a transamination- and an acetylation-reaction catalyzed by PerA and PerB. In this study, we have overproduced and purified PerA and PerB from E. coli O157:H7 in E. coli BL21. The recombinant proteins were partly characterized and the final product of the reaction catalyzed by PerB was shown to be GDP-N-acetyl-d-perosamine by chromatography, mass spectrometry, and 1H-NMR. The functional expression of PerB provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which is needed to further study the corresponding glycosyltransferases in vitro.