High coenzyme affinity chimeric amine dehydrogenase based on domain engineering.

NADH-dependent phenylalanine amine dehydrogenase (F-AmDH) engineered from phenylalanine dehydrogenase (PheDH) catalyzes the synthesis of aromatic chiral amines from prochiral ketone substrates. However, its low coenzyme affinity and catalytic efficiency limit its industrial application. Here, we developed a chimeric amine dehydrogenase, cFLF-AmDH, based on the relative independence of the structure at the domain level, combined with a substrate-binding domain from F-AmDH and a high-affinity cofactor-binding domain from leucine amine dehydrogenase (L-AmDH).

A Sustainable Approach for Synthesizing ()-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase.

In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added ()-4-aminopentanoic acid using an engineered glutamate dehydrogenase from (GDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of GDH. After targeted saturation mutagenesis, the mutant GDH, which was active toward LA, was identified.

Transamination-Like Reaction Catalyzed by Leucine Dehydrogenase for Efficient Co-Synthesis of α-Amino Acids and α-Keto Acids.

α-Amino acids and α-keto acids are versatile building blocks for the synthesis of several commercially valuable products in the food, agricultural, and pharmaceutical industries. In this study, a novel transamination-like reaction catalyzed by leucine dehydrogenase was successfully constructed for the efficient enzymatic co-synthesis of α-amino acids and α-keto acids. In this reaction mode, the α-keto acid substrate was reduced and the α-amino acid substrate was oxidized simultaneously by the enzyme, without the need for an additional coenzyme regeneration system.

Optimization and Validation of a Headspace Solid-Phase Microextraction with Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometric Detection for Quantification of Trace Aroma Compounds in Chinese Liquor (Baijiu).

The detection of trace aroma compounds in samples with complex matrices such as Chinese liquor (Baijiu) requires a combination of several methods, which makes the analysis process very complicated. Therefore, a headspace solid-phase microextraction (HS-SPME) method coupled with two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) was developed for the quantitation of a large number of trace compounds in Baijiu. Optimization of extraction conditions via a series of experiments revealed that dilution of the alcohol content of 8 mL of Baijiu to 5%, followed by the addition of 3.

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient L-homophenylalanine synthesis.

Background: Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block L-homophenylalanine (L-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading.

Results: In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient L-HPA synthesis.

Enhanced catalytic efficiency and coenzyme affinity of leucine dehydrogenase by comprehensive screening strategy for L-tert-leucine synthesis.

L-tert-leucine (L-Tle) is widely used as vital chiral intermediate for pharmaceuticals and as chiral auxiliarie for organocatalysis. L-Tle is generally prepared via the asymmetric reduction of trimethylpyruvate (TMP) catalyzed by NAD-dependent leucine dehydrogenase (LeuDH). To improve the catalytic efficiency and coenzyme affinity of LeuDH from Bacillus cereus, mutation libraries constructed by error-prone PCR and iterative saturation mutation were screened by two kinds of high-throughput methods.

Improving the production of NAD via multi-strategy metabolic engineering in Escherichia coli.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in numerous physiological processes. As an attractive product in the industrial field, NAD also plays an important role in oxidoreductase-catalyzed reactions, drug synthesis, and the treatment of diseases, such as dementia, diabetes, and vascular dysfunction. Currently, although the biotechnology to construct NAD-overproducing strains has been developed, limited regulation and low productivity still hamper its use on large scales.

[High efficient co-expression of leucine dehydrogenase and glucose dehydrogenase in Escherichia coli].

Objective: Different co-expression strategies to express leucine dehydrogenase and glucose dehydrogenase in E. coli were done to observe the effect of expression of different enzyme. A recombinant strain with two high enzyme activities was built for efficiently asymmetric synthesis of L-tert-leucine.

Disorder prediction-based construct optimization improves activity and catalytic efficiency of Bacillus naganoensis pullulanase.

Pullulanase is a well-known starch-debranching enzyme. However, the production level of pullulanase is yet low in both wide-type strains and heterologous expression systems. We predicted the disorder propensities of Bacillus naganoensis pullulanase (PUL) using the bioinformatics tool, Disorder Prediction Meta-Server.

Auto-induction-based rapid evaluation of extracellular enzyme expression from lac operator-involved recombinant Escherichia coli.

Degeneration of engineered strains and decreased production of target gene products were often observed during recombinant bioprocess. Although several strategies have been developed to ensure high levels of target gene products, these methods seemed to be complex and laborious. By investigating possible factors contributing to the decreased yield, degeneration of host cells was identified as the main cause.

Breeding of a cyclic imide-assimilating bacterium, Pseudomonas putida s52, for high efficiency production of pyruvate.

A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction.

Integration of newly isolated biocatalyst and resin-based in situ product removal technique for the asymmetric synthesis of (R)-methyl mandelate.

The enantioselective reduction of methyl benzoylformate to (R)-methyl mandelate, an important pharmaceutical intermediate and a versatile resolving agent, was investigated in this study. After minimizing the reaction-specific constraints (constraints dependent on the nature of the substrate and product) by preliminary selection of the reaction parameters, an effective whole cell biocatalyst (Saccharomyces cerevisiae AS2.1392) was obtained by simple screening procedures.

Enhancement of substrate concentration in microbial stereoinversion through one-pot oxidation and reduction by aqueous two-phase system.

An extractive biocatalytic method of aqueous two-phase system was employed for stereoinversing (R)-1-phenyl-1,2-ethanediol into (S)-1-phenyl-1,2-ethanediol by Candida parapsilosis CCTCC M203011. It was observed that substrate and product inhibitions in microbial stereoinversion through one-pot oxidation and reduction were removed efficiently by extractive biocatalysis in aqueous two-phase system with PEG 4000/phosphate potassium system, and that the substrate concentration was enhanced from 15 to 30 g/L with product optical purity of 99.02% e.

Crystallization and preliminary X-ray crystallographic analysis of a carbonyl reductase from Candida parapsilosis.

A novel short-chain NADPH-dependent (S)-1-phenyl-1,2-ethanediol dehydrogenase (SCR) has been crystallized. Two distinct but related crystal forms of SCR were obtained using the hanging-drop vapour-diffusion method and a reservoir solution consisting of 18%(w/v) polyethylene glycol 2000 monomethyl ether and 8%(v/v) 2-propanol as the precipitant. The crystals were rhomboid in shape with average dimensions of 0.