Monooxygenases as biocatalysts: Classification, mechanistic aspects and biotechnological applications.

Monooxygenases are enzymes that catalyze the insertion of a single oxygen atom from O(2) into an organic substrate. In order to carry out this type of reaction, these enzymes need to activate molecular oxygen to overcome its spin-forbidden reaction with the organic substrate. In most cases, monooxygenases utilize (in)organic cofactors to transfer electrons to molecular oxygen for its activation. Monooxygenases typically are highly chemo-, regio-, and/or enantioselective, making them attractive biocatalysts. In this review, an exclusive overview of known monooxygenases is presented, based on the type of cofactor that these enzymes require. This includes not only the cytochrome P450 and flavin-dependent monooxygenases, but also enzymes that utilize pterin, metal ions (copper or iron) or no cofactor at all. As most of these monooxygenases require nicotinamide coenzymes as electron donors, also an overview of current methods for coenzyme regeneration is given. This latter overview is of relevance for the biotechnological applications of these oxidative enzymes.

Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris.

Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.

Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from Rhodococcus erythropolis.

Nitrile hydratase and amidase from Rhodococcus erythropolis CIMB11540 were both cloned and expressed in Escherichia coli. Crude cell free extracts were used for the hydrolysis of different aromatic cyanohydrins. Nitrile hydratase expression was increased up to 5-fold by redesign of the expression cassette. The recombinant enzymes were successfully used for the conversion of several cyanohydrins to the corresponding alpha-hydroxy amides and acids while retaining enantiopurity.

Esterase EstE from Xanthomonas vesicatoria ( Xv_EstE) is an outer membrane protein capable of hydrolyzing long-chain polar esters.

A new esterase gene from Xanthomonas vesicatoria (formerly X. campestris) DSM 50861 was identified, cloned from a chromosomal gene library and overexpressed in Escherichia coli. The corresponding DNA fragment contains an ORF of 1,818 bp, encoding a hydrolase of the GDSL esterase family. A protein of about 67 kDa, named Xv_EstE, was expressed from this fragment. A N-terminal signal peptide was processed under low-expression conditions, yielding a 63-kDa mature protein. The predicted amino acid sequence showed distinct homology to esterases of the GDSL family. Based on homology, a catalytic triad Gly-Asp-Ser could be defined. Amino acid sequence alignments and computer-assisted structure prediction indicated the presence of a carboxyl-terminal beta-barrel membrane domain which might facilitate binding of Xv_EstE to the outer membrane. This could be verified by differential cell fractionation experiments, in which Xv_EstE was exclusively found in the outer membrane fraction. Xv_EstE showed preferential hydrolytic activity on short chain (up to C(8)) and para-substituted nitrophenylesters as substrates. However, only long-chain 1-hydroxy-pyrene-3,6,8-trisulfonic acid (HPTS)-fatty acid esters were hydrolyzed. Xv_EstE was also found to be active on a series of substrates of industrial interest, such as 1-methylprop-2-ynyl acetate, for which an enantioselectivity up to 93% ee could be recognized.

The hydroxynitrile lyase from almond: a lyase that looks like an oxidoreductase.

BACKGROUND: Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile lyase, a key enzyme of this process, cleaves a cyanohydrin into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in biocatalysis. Different classes of hydroxynitrile lyases have convergently evolved from FAD-dependent oxidoreductases, alpha/beta hydrolases, and alcohol dehydrogenases. The FAD-dependent hydroxynitrile lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis. RESULTS: We have determined the crystal structure of a 61 kDa hydroxynitrile lyase isoenzyme from Prunus amygdalus (PaHNL1) to 1.5 A resolution. Clear electron density originating from four glycosylation sites could be observed. As concerns the overall protein fold including the FAD cofactor, PaHNL1 belongs to the family of GMC oxidoreductases. The active site for the HNL reaction is probably at a very similar position as the active sites in homologous oxidases. CONCLUSIONS: There is strong evidence from the structure and the reaction product that FAD-dependent hydroxynitrile lyases have evolved from an aryl alcohol oxidizing precursor. Since key residues implicated in oxidoreductase activity are also present in PaHNL1, it is not obvious why this enzyme shows no oxidase activity. Similarly, features proposed to be relevant for hydroxy-nitrile lyase activity in other hydroxynitrile lyases, i.e., a general base and a positive charge to stabilize the cyanide, are not obviously present in the putative active site of PaHNL1. Therefore, the reason for its HNL activity is far from being well understood at this point.

Cloning and characterization of EstC from Burkholderia gladioli, a novel-type esterase related to plant enzymes.

By screening a genomic library of Burkholderia gladioli (formerly Pseudomonas marginata) for clones exhibiting esterolytic activity, the gene for a novel-type esterase (EstC) showing significant homology to plant enzymes could be isolated. High homology was found to two hydroxynitrile lyases originating from Hevea brasiliensis (tropical rubber tree) and Manihot esculenta (cassava), and to two proteins from Oryza sativa (rice) that are specifically induced upon infection by Pseudomonas syringae pv. syringae. The sequenced ORF encodes for a protein of 298 amino acids. The enzyme was efficiently overexpressed in Escherichia coli, purified and characterized with respect to enzymatic capabilities. The enzyme was able to hydrolyze a variety of esterase substrates of low to medium carbonic acid chain length, but no triglycerides were hydrolyzed. Despite the high sequence homology, no hydroxynitrile lyase activity could be recognized.