2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l−1 DHB with a molar yield of 0.15.
MS/MS techniques are well customized now for proteomic analysis, even for non-sequenced organisms, since peptide sequences obtained by these methods can be matched with those found in databases from closely related sequenced organisms. We used this approach to characterize the protein content of the "Rovabio Excel", an enzymatic cocktail produced by Penicillium funiculosum that is used as feed additive in animal nutrition. Protein separation by bi-dimensional electrophoresis yielded more than 100 spots, from which 37 proteins were unambiguously assigned from peptide sequences. By one-dimensional SDS-gel electrophoresis, 34 proteins were identified among which 8 were not found in the 2-DE analysis. A third method, termed 'peptidic shotgun', which consists in a direct treatment of the cocktail by trypsin followed by separation of the peptides on two-dimensional liquid chromatography, resulted in the identification of two additional proteins not found by the two other methods. Altogether, more than 50 proteins, among which several glycosylhydrolytic, hemicellulolytic and proteolytic enzymes, were identified by combining three separation methods in this enzymatic cocktail. This work confirmed the power of proteome analysis to explore the genome expression of a non-sequenced fungus by taking advantage of sequences from phylogenetically related filamentous fungi and pave the way for further functional analysis of P. funiculosum.
The filamentous fungus Talaromyces versatilis produces a wide range of cellulolytic and hemicellulolytic enzymes such as xylanases. The recent accessibility to the T. versatilis genome allows identifying two new genes, xynE and xynF, encoding glycoside-hydrolases from family GH11. Both genes were cloned and expressed in the methylotrophic yeast Pichia pastoris in order to compare these new xylanases with two other GH11 xylanases from T. versatilis (XynB and XynC) that were previously reported. High-level expression of recombinant enzymes was obtained for the four enzymes that were purified to homogeneity. The XynB, XynC, XynE and XynF enzymes have molecular masses of 34, 22, 45 and 23 kDa, an optimal pH between 3.5 and 4.5 and an optimal temperature between 50 °C and 60 °C. Interestingly, XynF has shown the best thermal stability at 50 °C for at least 180 min with a weak loss of activity. The four xylanases catalysed hydrolysis of low viscosity arabinoxylan (LVAX) with K m(app) between 11.5 and 23.0 mg.mL(-1) and k cat/K m(app) 170 and 3,963 s(-1) mg(-1).mL. Further investigations on the rate and pattern of hydrolysis of the four enzymes on LVAX showed the predominant production of xylose, xylobiose and some (arabino)xylo-oligosaccharides as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing degree of polymerisation of oligomer up to 6, suggesting that the specificity region of XynE and XynF spans at least six xylose residues. Because of their attractive properties, T. versatilis xylanases might be considered for biotechnological applications.
An amidase with a wide activity spectrum was purified from the Brevibacterium sp. R 312 and studied. The purification was performed by precipitating the proteins of the supernatant fraction obtained after centrifugation of sonicated cells. The resulting proteins were submitted to a gel filtration step followed by DEAE-Sephadex ion exchange chromatography and then by another gel filtration process. The purified amidase had a molecular weight of 180,000 and was composed of four subunits of the same molecular weight (43,000 2,000). Its isoelectric point was 3.5. This enzyme was able to hydrolyze a large number of amides into their corresponding organic acids and it also possessed an acyl transferase activity.Amidases, and especially microbial amidases, have been extensively studied (CLARKE 1970). These enzymes seem t o be very wide spread among protists: bacteria (CLARKB The Brevibacterium sp. R 312 strain is able to transform all sufficiently water-soluble iiitriles into the corresponding organic acids with their amides as an intermediary step (ARNAUD et al. 1976 a, b and c).Two mutant strains were isolated from the Brevibacterium sp. R 312 wild type strain:Brevibacterium sp. 19 is no longer able to hydrate nitriles into amides; it retains, however, an amidase activity similar to the wild type (BUI et al. 1984a); Brevibacterium sp. A4 has a nitrile-hydratase activity identical to that of the R 312 strain, but does no longer hydrolyze most amides into corresponding organic acids ( JALLAGEAS et al. . The isolation of these two mutant strains conclusively demonstrated that two wide spectrum enzymes are involved in the metabolism of nitriles by the Brevibacterium sp.
R 312 strain:-a nitrile-hydratase which transforms nitriles into amides.-an amidase which hydrolyzes amides into their corresponding organic acids.Besides the Brevibacteriurn sp. R 312, Arthrobacter sp. J-1 (YAMADA et al. , ASANO et al. 1980, 1982a was reported recently as one of the very few microorganisms possessing both kinds of enzymes.
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