A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization.

In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography.

Heterologous protein expression in Trichoderma reesei using the cbhII promoter.

To express homologous or heterologous proteins in fungi, a protein expression system using the promoter of cellobiohydrolase II gene (cbhII) was constructed by generating an expression vector called pWEIIF00. The obtained vector possesses the left and right borders, a hygromycin phosphotransferase B selective marker and a strong promoter and terminator of cbhII from Trichoderma reesei. It can easily undergo random recombination.

Structural basis for cofactor and substrate selection by cyanobacterium succinic semialdehyde dehydrogenase.

Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of aldehydes to carboxylic acids. Cyanobacterium Synechococcus contains one ALDH enzyme (Sp2771), together with a novel 2-oxoglutarate decarboxylase, to complete a non-canonical tricarboxylic acid cycle. However, the molecular mechanisms for substrate selection and cofactor preference by Sp2771 are largely unknown.

Identification of Petriella setifera LH and characterization of its crude carboxymethyl cellulase for application in denim biostoning.

The phylogenetic tree of the partial elongation factor-1 alpha gene fits better than the partial 18S rDNA for generic classification. From the results of the molecular tree and analysis of morphological characters, Petriella setifera LH was identified. It can be induced to produce carboxymethyl cellulase (CMCase).

Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction.

Background: A nitrilase-mediated pathway has significant advantages in the production of optically pure (R)-(-)-mandelic acid. However, unwanted byproduct, low enantioselectivity, and specific activity reduce its value in practical applications. An ideal nitrilase that can efficiently hydrolyze mandelonitrile to optically pure (R)-(-)-mandelic acid without the unwanted byproduct is needed.

Enantioselective acylation of β-phenylalanine acid and its derivatives catalyzed by penicillin G acylase from Alcaligenes faecalis.

This study developed a simple, efficient method for producing racemic β-phenylalanine acid (BPA) and its derivatives via the enantioselective acylation catalyzed by the penicillin G acylase from Alcaligenes faecalis (Af-PGA). When the reaction was run at 25°C and pH 10 in an aqueous medium containing phenylacetamide and BPA in a molar ratio of 2:1, 8 U/mL enzyme and 0.1 M BPA, the maximum BPA conversion efficiency at 40 min only reached 36.

Three sulphated polysaccharides isolated from the mucilage of mud snail, Bullacta exarata philippi: characterization and antitumour activity.

Three sulphated polysaccharides, coded as BEMPA, BEMPB(1), BEMPB(2), were extracted from the mucilage of mud snail of Bullacta exarata and purified by DEAE-cellulose ion-exchange and size-exclusion chromatography. Structural analysis of purified polysaccharides by chemical and biochemical methods revealed BEMPA was a high (1→3,4)-linked mannose-containing polysaccharide with molecular weight of 22,977 Da. BEMPB(1), with molecular weight of 64,117 Da, was a high (1→3)-linked arabinose-containing polysaccharide.

Lactobacillus curieae sp. nov., isolated from stinky tofu brine.

A lactic acid bacterium, strain CCTCC M 2011381(T), isolated from the brine of the traditional Chinese snack, stinky tofu, was studied to determine its taxonomic position. It was a Gram-stain-positive, non-motile, facultatively anaerobic rod-shaped bacterium that did not exhibit catalase activity. The DNA G+C content of the strain was 44.

Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum.

Mycobacteria have been modified to transform sterols to produce valuable steroids. Here, we demonstrated that the oxidation of sterols to sterones is a rate-limiting step in the catabolic pathway of sterols in Mycobacterium neoaurum. Two cholesterol oxidases ChoM1 and ChoM2 involved in the step were identified in M.

A novel dextran dextrinase from Gluconobacter oxydans DSM-2003: purification and properties.

Dextran has already been widely applied in food, pharmaceutical, and chemical industries. In this study, a novel intracellular dextran dextrinase (DDase, EC 2.4.

In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E. coli.

Background: Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding.

Magnetic catechol-chitosan with bioinspired adhesive surface: preparation and immobilization of ω-transaminase.

The magnetic chitosan nanocomposites have been studied intensively and been used practically in various biomedical and biological applications including enzyme immobilization. However, the loading capacity and the remained activity of immobilized enzyme based on existing approaches are not satisfied. Simpler and more effective immobilization strategies are needed.

Impact of large aggregated uricases and PEG diol on accelerated blood clearance of PEGylated canine uricase.

Background: Uricase has proven therapeutic value in treating hyperuricemia but sufficient reduction of its immunogenicity may be the largest obstacle to its chronic use. In this study, canine uricase was modified with 5 kDa mPEG-SPA and the impact of large aggregated uricases and cross-linked conjugates induced by difunctional PEG diol on immunogenicity was investigated.

Methods And Findings: Recombinant canine uricase was first expressed and purified to homogeneity.

A novel technique for in situ aggregation of Gluconobacter oxydans using bio-adhesive magnetic nanoparticles.

Here, we present a novel technique to immobilize magnetic particles onto whole Gluconobacter oxydans in situ via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. Our approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine-coated nanoparticles to G. oxydans which resulted in cell aggregation.

Enhanced acetoin production by Serratia marcescens H32 with expression of a water-forming NADH oxidase.

Cofactor engineering was employed to enhance production of acetoin by Serratia marcescens H32. 2,3-Butanediol was a major byproduct of acetoin fermentation by S. marcescens H32.

Combining metabolic engineering and adaptive evolution to enhance the production of dihydroxyacetone from glycerol by Gluconobacter oxydans in a low-cost way.

Gluconobacter oxydans can rapidly and effectively transform glycerol to dihydroxyacetone (DHA) by membrane-bound quinoprotein sorbitol dehydrogenase (mSLDH). Two mutant strains of GDHE Δadh pBBR-PtufBsldAB and GDHE Δadh pBBR-sldAB derived from the GDHE strain were constructed for the enhancement of DHA production. Growth performances of both strains were largely improved after adaptively growing in the medium with glucose as the sole carbon source.

Construction of a genetically engineered microorganism for phenanthrene biodegradation.

The bacterium Pseudomonas sp. CGMCC2953, isolated from oil-polluted soil, was used as a recipient for a biodegradative gene encoding catechol 2,3-dioxygenase (C23O), which was successfully cloned into the plasmid pK4 derived from pRK415 with a broad host range. The apparent phenanthrene biodegradation parameters of the recombinant microorganism (Pseudomonas sp.

Characterization, efficacy, pharmacokinetics, and biodistribution of 5kDa mPEG modified tetrameric canine uricase variant.

PEGylated uricase is a promising anti-gout drug, but the only commercially marketed 10kDa mPEG modified porcine-like uricase (Pegloticase) can only be used for intravenous infusion. In this study, tetrameric canine uricase variant was modified by covalent conjugation of all accessible ɛ amino sites of lysine residues with a smaller 5kDa mPEG (mPEG-UHC). The average modification degree and PEGylation homogeneity were evaluated.

Structure-based characterization of canine-human chimeric uricases and its evolutionary implications.

Uricase was lost in hominoids during primate evolution, but the inactivation mechanism remains controversial. To investigate the inactivation process of hominoid uricase, chimeric constructions between canine and human uricase were employed to screen the target regions that may contain labile or inactivated mutations in deduced human uricase. Four chimeric uricases were constructed and showed different enzymatic characteristics.

A comparative study of β-1, 4-endoglucanase (possessing β-1, 4-exoglucanase activity) from Bacillus subtilis LH expressed in Pichia pastoris GS115 and Escherichia coli Rosetta (DE3).

β-1, 4-Endoglucanase (EG) from Bacillus subtilis LH was expressed in Escherichia coli Rosetta (DE3) and Pichia pastoris GS115, respectively. The CMCase activity of EG (EGE) from the cell lysate of DE3 reached 20,010U/ml, and that of EG (EGP) from the supernatant of GS115 was only 2008U/ml. EGE and EGP were bifunctional cellulases excluding β-1, 4-glucosidase (BGL).

Characterization and application of fusidane antibiotic biosynethsis enzyme 3-ketosteroid-∆1-dehydrogenase in steroid transformation.

Microbial ∆(1)-dehydrogenation is one of the most important transformations in the synthesis of steroid hormones. In this study, a 3-ketosteroid-∆(1)-dehydrogenase (kstD(F)) involved in fusidane antibiotic biosynthesis from Aspergillus fumigatus CICC 40167 was characterized for use in steroid transformation. KstD(F) encodes a polypeptide consisting of 637 amino acid residues.

The MrCYP52 cytochrome P450 monoxygenase gene of Metarhizium robertsii is important for utilizing insect epicuticular hydrocarbons.

Fungal pathogens of plants and insects infect their hosts by direct penetration of the cuticle. Plant and insect cuticles are covered by a hydrocarbon-rich waxy outer layer that represents the first barrier against infection. However, the fungal genes that underlie insect waxy layer degradation have received little attention.

Construction of two vectors for gene expression in Trichoderma reesei.

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei.

Biochemical and structural analysis of Gox2181, a new member of the SDR superfamily from Gluconobacter oxydans.

Gluconobacter oxydans enable to oxidize sugars and polyols incompletely to corresponding materials with potential industrial applications, containing around 75 putative dehydrogenases. One of these putative dehydrogenases, Gox2181, was cloned and expressed in Escherichia coli BL21 (DE3), and its X-ray crystal structure was determined to a resolution of 1.8 Å.

Characterization and regulation of the 2,3-butanediol pathway in Serratia marcescens.

Serratia marcescens has been proved to be a potential strain for industrial 2,3-butanediol production for its high yield, productivity, and other advantages. In this study, the genes slaA, slaB, slaC, and slaR were successfully cloned which were further confirmed to be encoding acetolactate decarboxylase, acetolactate synthase, 2,3-butanediol dehydrogenase, and a LysR-like regulator, respectively. Unlike in Klebsiella sp.