Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester.

Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library.

Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli.

Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E.

Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197.

A novel gene encoding a thermostable esterase (designated as Est-gela) was isolated from the moderate thermophile Bacillus gelatini KACC 12197. The open reading frame of this gene (1170 bp) encodes 389 amino acid residues, and the molecular weight of Est-gela is approximately 42 kDa. The protein sequence of Est-gela shows similarity with β-lactamases and esterases (⩽ 43%).

Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases.

Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen.

Complete genome sequence of Klebsiella oxytoca M1, isolated from Manripo area of South Korea.

Here we report the full genome sequence of Klesiella oxytoca M1, isolated from Manripo area of South Korea. The strain K. oxytoca M1 is able to produce either 2,3-butanediol or acetoin selectively by controlling the pH and temperature.

Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4.

Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH(4))(2)SO(4) on 1,3-PD production by K.

Role of osmotic and salt stress in the expression of erythrose reductase in Candida magnoliae.

The osmotolerant yeast, Candida magnoliae, which was isolated from honeycomb, produces erythritol from sugars such as fructose, glucose, and sucrose. Erythrose reductase in C. magnoliae (CmER) reduces erythrose to erythritol with concomitant oxidation of NAD(P)H.

Structural and functional characterization of S-adenosylmethionine (SAM) synthetase from Pichia ciferrii.

S-adenosylmethionine synthetase (SAM-s) catalyzes the synthesis of S-adenosylmethionine (SAM), which is essential for methylation, transcription, proliferation, and production of secondary metabolites. Here SAM-s from Pichia ciferrii were selectively cloned using RNA CapFishing and rapid amplification of cDNA ends (RACE). The putative full-length cDNA of SAM-s encoded a 383 amino acid protein (42.

Optimization of culture medium for enhanced production of exopolysaccharide from Aureobasidium pullulans.

Polysaccharides produced by microorganisms are utilized for a variety of purposes, including the use in cosmetics and as food additives. More recently, polysaccharides have been exploited by the medical and pharmaceutical industries, and those originated from many species of mushrooms have been especially useful in industrial applications; however, the production and synthesis of these compounds is costly and time consuming. In this study, we developed a method for low-cost production of exopolysaccharide (EPS) that effectively screens components and optimizes medium composition using statistical methods (Plackett-Burman and Box-Behnken design).

Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110.

Background: Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts.

Characterization of a novel oligomeric SGNH-arylesterase from Sinorhizobium meliloti 1021.

A novel oligomeric SGNH-arylesterase (Sm23) from Sinorhizobium meliloti 1021 was characterized using biochemical and biophysical methods. A sequence comparison of Sm23 with other SGNH members confirmed the presence of catalytic triad (Ser(10), Asp(187), and His(190)) and oxyanion holes (Ser(10)-Gly(50)-Asn(90)). The wild type enzyme was able to hydrolyze p-nitrophenyl acetate, alpha- and beta-naphthyl acetate, while S10A mutant completely lost its activity.

Identification and characterization of a novel oligomeric decaprenyl diphosphate synthase.

A novel oligomeric decaprenyl pyrophosphate synthase was identified. Circular dichroism measurements indicated that it is alpha-helical, and stable against pH changes and denaturants. Three peptides corresponding to the conserved regions were synthesized and metal affinities were investigated.

Synergistic effects of chromosomal ispB deletion and dxs overexpression on coenzyme Q(10) production in recombinant Escherichia coli expressing Agrobacterium tumefaciens dps gene.

For biotechnological production of coenzyme Q(10) (CoQ(10)) in recombinant Escherichia coli, three genetic manipulations were performed: heterologous expression of decaprenyl diphosphate synthase (Dps) from Agrobacterium tumefaciens, deletion of endogenous octaprenyl diphosphate synthase (IspB), and overexpression of 1-deoxy-d-xylulose synthase (Dxs). Expression of the dps gene and deletion of the ispB gene in E. coli BL21(DE3)DeltaispB/pAP1 allowed production of CoQ(10) only.

Purification, crystallization and preliminary crystallographic analysis of Est-Y29: a novel oligomeric beta-lactamase.

beta-Lactam antibiotics such as penicillins and cephalosporins have a four-atom ring as a common element in their structure. The beta-lactamases, which catalyze the inactivation of these antibiotics, are of great interest because of their high incidence in pathogenic bacteria. A novel oligomeric class C beta-lactamase (Est-Y29) from a metagenomic library was expressed, purified and crystallized.

Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenase.

Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species.

Selective utilization of fructose to glucose by Candida magnoliae, an erythritol producer.

Candida magnoliae isolated from honeycomb is an industrially important yeast with high erythritol-producing ability. Erythritol has been used as functional sugar substitute for various foods. In order to analyze the physiological properties of C.

Cloning and characterization of thermostable esterase from Archaeoglobus fulgidus.

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa.

Proteomic analysis of fructophilic properties of osmotolerant Candida magnoliae.

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae.

Two threonine residues required for role of AfsKav in controlling morphogenesis and avermectin production in Streptomyces avermitilis.

AfsKav is a eukaryotic-type serine/threonine protein kinase, required for sporulation and avermectin production in Streptomyces avermitilis. In terms of their ability to complement SJW4001 (DeltaafsK-av), afsK-av mutants T165A and T168A were not functional, whereas mutants T165D and T168D retained their ability, indicating that Thr-165 and Thr-168 are the phosphorylation sites required for the role of AfsKav. Expression of the S-adenosylmethione synthetase gene promoted avermectin production in the wild-type S.

Purification, crystallization and preliminary crystallographic analysis of Est25: a ketoprofen-specific hormone-sensitive lipase.

Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form.

A short C-terminal region of alpha-synuclein protects a (R/S)-non-specific esterase from Archaeglobus fulgidus.

We show that alpha-synuclein could assist the molecular activity of a ketoprofen-(R/S) nonspecific esterase from Archaeglobus Fulgidus. Specifically, several synthetic peptides from alpha-synuclein, each having random coil conformation in far-UV spectra, could protect the enzyme activity against stress conditions such as heat and organic solvents.

Identification and characterization of a novel (S)-ketoprofen-specific esterase.

A new (S)-ketoprofen specific esterase (EST-Y29) was identified from a metagenome library from environmental samples, which showed homologies with class C-beta lactamase, penicillin binding protein, and other lipases/esterases. In order to investigate the biochemical and biophysical properties, the recombinant protein was overexpressed, purified to homogeneity, and characterized. This EST-Y29 has high catalytic activity against p-nitrophenyl esters of short fatty acids (C(2) and C(4)) and alpha-naphthyl acetate with activation energy of 30.

Selective utilization of fructose to glucose by Candida magnoliae, an erythritol producer.

Candida magnoliae isolated from honeycomb is an industrially important yeast with high erythritol-producing ability. Erythritol has been used as functional sugar substitute for various foods. In order to analyze the physiological properties of C.

Chaperone-like activities of alpha-synuclein: alpha-synuclein assists enzyme activities of esterases.

Alpha-synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of alpha-synuclein has not yet been known. Here we have shown that alpha-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of alpha-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity.

Amplification of 1-deoxy-D-xyluose 5-phosphate (DXP) synthase level increases coenzyme Q10 production in recombinant Escherichia coli.

For the enhancement of coenzyme Q(10) (CoQ(10)) production, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase of Pseudomonas aeruginosa was constitutively coexpressed in a recombinant Escherichia coli strain, which harbors the ddsA gene from Gluconobacter suboxydans encoding decaprenyl diphosphate synthase. It was found that the expression of the ddsA gene caused depletion of the isopentenyl diphosphate (IPP) pool in E. coli.