Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine.

Background: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene.

Food-grade γ-aminobutyric acid production by immobilized glutamate decarboxylase from Lactobacillus plantarum in rice vinegar and monosodium glutamate system.

Objectives: γ-amino butyric acid (GABA) is a non-protein amino acid, considered a potent bioactive compound. This study focused on biosynthesis of food-grade GABA by immobilized glutamate decarboxylase (GAD) from Lactobacillus plantarum in the rice vinegar and monosodium glutamate (MSG) reaction system.

Results: The gene encoding glutamate decarboxylase (GadB) from L.

Improving the Thermostability and Activity of Transaminase From by Charge-Charge Interaction.

Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of ()-selective amine transaminase from (-ATA) is its weak thermostability, with a half-life ( ) of only 6.9 min at 40°C.

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase.

Glutamate decarboxylase (GAD; EC 4.1.1.

A Single Mutation Increases the Thermostability and Activity of Amine Transaminase.

Enhancing the thermostability of ()-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis.

Biosynthesis of γ-aminobutyrate by engineered Lactobacillus brevis cells immobilized in gellan gum gel beads.

γ-Aminobutyrate (GABA) is an important chemical in pharmaceutical field. The use of lactic acid bacteria as biocatalysts for the conversion of l-monosodium glutamate (MSG) into GABA opens interesting perspectives for the production of this functional compound. In this work, an engineered GABA high-producing strain Lactobacillus brevis GadA was constructed by overexpressing a C-terminally truncated GadA mutant, which is active in expanded pH range.

Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis.

Amine transaminases are a class of efficient and industrially-desired biocatalysts for the production of chiral amines. In this study, stabilized variants of the (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) were constructed by consensus mutagenesis. Using Consensus Finder (http://cbs-kazlab.

Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis.

Glutamate decarboxylase (GAD), which is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme, can catalyze α-decarboxylation of l-glutamate (L-Glu) to γ-aminobutyrate (GABA). The crystal structure of GAD in complex with PLP from Lactobacillus brevis CGMCC 1306 was successfully solved by molecular-replacement, and refined at 2.2 Å resolution to an R factor of 18.

Improving thermostability of (R)-selective amine transaminase from Aspergillus terreus through introduction of disulfide bonds.

To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design and Modelling of Disulfide Bonds in Proteins to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilized mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.

Effects of hydroxypropyl degree on physiochemical activities of chitosan from squid pens.

Chitosan was prepared by alkaline N-deacetylation of β-chitin and hydroxypropyl derivatives with different degrees of substitution (DS) were synthesized. It was characterized by Fourier transform infrared (FT-IR) and elemental analysis. The DS of hydroxypropyl chitosan (HPCS) calculated by an element analyzer were 0.

Biochemical activities of 6-carboxy β-chitin derived from squid pens.

TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated 6-carboxy β-chitin derivatives (T-chitin) with different carboxylate content were successfully synthesized by controlling the addition level of NaClO as the primary oxidant. The structural and biochemical properties of the derivatives were investigated. The carboxylate contents of the derivatives calculated by electrical conductivity titration were 1.

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase.

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of L-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction.

Cytochrome P450 BM-3 evolved by random and saturation mutagenesis as an effective indole-hydroxylating catalyst.

Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate.

Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation.

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored.

Optimization of fermentation conditions for P450 BM-3 monooxygenase production by hybrid design methodology.

Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E. coli. Operational conditions for maximum production were determined with twelve parameters under consideration: the concentration of FeCl(3), induction at OD(578) (optical density measured at 578 nm), induction time and inoculum concentration.

Application of artificial neural network coupling particle swarm optimization algorithm to biocatalytic production of GABA.

The biotransformation of L-sodium glutamate (L-MSG) to gamma-aminobutyric acid (GABA) catalyzed by the cells of Lactobacillus brevis with higher glutamate decarboxylase activity was investigated. The results showed that pH, temperature, and FeSO(4) x 7H(2)O concentration had significantly positive effect on GABA yield. The individual and interactive effects of pH, temperature, and FeSO(4) x 7H(2)O concentration were further optimized in terms of GABA yield.

Gelation conditions and transport properties of hollow calcium alginate capsules.

The diameter, membrane thickness, and compression intensity of hollow Ca-alginate capsules were measured at different gelation conditions, such as the reactant concentration, dropping velocity, and gelation time. The optimum operation conditions for preparing capsules were determined at 100 g/L CaCl(2), 10 g/L sodium alginate (Na-alginate), a dropping velocity of 150 droplets/min, and a gelation time of 10 min. Diffusion of some saccharide and amino acid from bulk solution into capsules was investigated, and the diffusion coefficients were calculated by the developed mathematical model.