The hydration reaction by microbial method is the crisis of the procedure of acrylamide production from acrylonitrile. This research studied the enzyme catalytic kinetics and de-active kinetics of nitrile hydratase in the type of free cell. Firstly, the effects of the concentration of cells, the temperature, pH value, the concentration of acrylonitrile and the concentration of acrylamide on the activity of nitrile hydratase was investigated. The result is that the temperature and the concentration of acrylamide are the most important among these factors. The activity of the nitrile hydratase was 5659 u/mL (broth) at 28 degrees C; the counterpart was only 663 u/mL (broth) at 5 degrees C. And the activity of NHase in solution of 45% acrylamide was just about half of that in solution of 5% acrylamide. After study on the relation of temperature and the reaction speed, It was found that the activation energy of the hydration of NHase was 65.57 kJ.mol-1. This paper studied the effects of concentration of cells, temperature, pH value, concentrations of acrylonitrile and acrylamide on the deactivation of Nhase, as well as the related enzyme de-active kinetics. The result also showed that the temperature and the concentration of acrylamide are the most important among these factors. In solution of 35% acrylamide, the residual activity was about 0% of the original value after 55 h; but in solution of 10% acrylamide, after the same period of time, the residual activity was 50% of the original one. It was also found that the concentration of acrylonitrile had little effect on the stability of NHase. The coefficient of deactivation at 28 degrees C was 21.77 times of the one at 5 degrees C. Correlating the temperature and the coefficient of deactivation, the activation energy of the de-active reaction was found to be 92.28 kJ.mol-1.
Acrylamide ; metabolism ; Acrylonitrile ; metabolism ; Catalysis ; Hydro-Lyases ; metabolism ; Hydrogen-Ion Concentration ; Kinetics ; Rhodococcus ; enzymology ; Temperature

Nitriles are an important type of synthetic intermediates in the production of fine chemicals because of their easy preparations and versatile transformations. The traditional chemical conversion of nitriles to carboxylic acids and amides is feasible but it requires relatively harsh conditions of heat, acid or alkali. Nitrile converting enzymes (nitrilase, nitrile hydratase and amidase) which are used as biocatalyst for the production of fine chemicals have attracted substantial interest because of their ability to convert readily available nitriles into the corresponding higher value amides or acids under mild conditions with excellent chemo-, regio- and stereo-selectivities. Many nitrile converting enzymes have been explored and widely used for the production of fine chemicals. In this paper, various examples of biocatalytic synthesis of pharmaceuticals and their intermediates, agrochemicals and their intermediates, food and feed additives, and other fine chemicals are presented. In the near future, an increasing number of novel nitrile converting enzymes will be screened and their potential in the production of useful fine chemicals will be further exploited.
Amides ; metabolism ; Amidohydrolases ; metabolism ; Aminohydrolases ; metabolism ; Carboxylic Acids ; metabolism ; Chemical Industry ; methods ; Hydro-Lyases ; metabolism ; Nitriles ; chemistry

The cultural conditions for the growth of Norcardia cell were studied in this paper. Controlling pH value, adding nutrient and optimizing the quantity of inducer during cultivation, the activity of nitrile hydratase reached 6567 u/mL (culture medium), which was the highest value appeared in native journals. In the farther hydratase experiments, no by-product, crylic acid, was detected. It showed that the activity of amidase was not promoted obviously while the activity of nitrile hydratase was increased greatly. The results set a strong foundation for the industrial application and the research on new technology.
Acrylamides ; metabolism ; Amidohydrolases ; metabolism ; Biotechnology ; methods ; Cell Culture Techniques ; Fermentation ; physiology ; Glucose ; metabolism ; Hydro-Lyases ; metabolism ; Hydrogen-Ion Concentration ; Nocardiaceae ; enzymology ; metabolism

There is growing interest in biodiesel and this results in the accumulation of glycerol. The exploitation and application of glycerol has attracted more and more attention. In the current study, glycerol was biotransformed to produce 3-hydroxypropionaldehyde by genetic engineering bacteria. It is known that 3-hydroxypopionaldehyde has been widely used as an important intermediate for chemicals, effective antimicrobial agent, and fix agent for tissues. A pair of primers was designed on the basis of the sequence of both NH2-terminus and the amino acid sequence of glycerol dehydratase reported by NCBI, and a fragment about 1.6 kb was obtained by PCR amplification using the total genome DNA of Lactobacillus reuteri as template, then the fragment was cloned to the pMD18-T vector and sequenced. Two specific primers were designed according to the obtained sequence, and a fragment with length of 1674 bp was amplified using PCR with these two specific primers. Consequently, the resulting products were digested with EcoR I and Hind III and ligated using T4 DNA ligase to the pET28b vector digested with the same enzymes. The recombinant plasmid, named pET28b-dhaB, was transformed into E. coli BL21. The positive clones were induced with IPTG and the expression products were further analyzed by SDS-PAGE, indicating that protein with a molecule weight of around 65 kD was obtained. Furthermore, the glycerol dehydratase activity was evaluated and compared with the wild type strain as well.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glyceraldehyde ; analogs & derivatives ; chemistry ; metabolism ; Hydro-Lyases ; biosynthesis ; genetics ; Lactobacillus reuteri ; enzymology ; genetics ; Propane ; chemistry ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

The dhaB gene encoding glycerol dehydratase and dhaG dhaF gene encoding glycerol dehydratase reactivating factor from Citrobacterfreundii were amplified by PCR. The temperature control expression vector pHsh harboring yqhD, dhaB, dhaG and dhaF gene was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-dhaG-dhaF-yqhD). The results from SDS-PAGE analysis show that the recombinant product was consistent with the molecular weight predicted from gene sequence. The fermentation result show that the yield of 1,3-propanediol was increased by 28% compared with E. coli JM109(pHsh-dhaB-yqhD).
Bacterial Proteins ; biosynthesis ; genetics ; Citrobacter freundii ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Hydro-Lyases ; biosynthesis ; genetics ; Propylene Glycols ; metabolism ; Transformation, Bacterial ; genetics

JadH is a bifunctional hydoxylase/dehydrase involved in jadomycin biosynthesis; it catalyzes a post-PKS modification reaction to convert 2,3-dehydro-UWM6 to dehydrorabelomycin. To identify the key residues involved in substrate-binding and catalysis, structural modeling and multiple sequence alignments of JadH homologs were performed to predict nine residues at the proximity of substrate. Site-directed mutagenesis of the corresponding residues and in vitro evaluation of the activities of the mutant enzymes, indicate these mutations severely reduced JadH activity. Our results indicate these residues are specifically involved in substrate-binding or catalysis in JadH.
Amino Acid Sequence ; Catalysis ; Hydro-Lyases ; genetics ; metabolism ; Isoquinolines ; metabolism ; Mixed Function Oxygenases ; genetics ; metabolism ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutant Proteins ; metabolism ; Naphthoquinones ; metabolism ; Streptomyces ; metabolism ; Substrate Specificity

1,3-propanediol production by microbial fermentation has become the research hot spot for its amiability with the environment. Here the molecular mechanism of glycerol bioconversion to 1,3-propanediol was outlined by elucidating the fermentation strains, metabolic pathways, regulon and key enzymes. Of enzymes, glycerol dehydrogenase, the velocity-limiting enzyme in glycerol reductive pathway, was emphatically discussed with regard to its molecular structure and reactivating factors. This paper aims to provide the basis for genetic modification of fermentation strains.
Bacteria ; enzymology ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biosynthetic Pathways ; Fermentation ; Gene Order ; Glycerol ; metabolism ; Hydro-Lyases ; genetics ; metabolism ; Industrial Microbiology ; methods ; trends ; Propylene Glycols ; metabolism ; Sugar Alcohol Dehydrogenases ; genetics ; metabolism

To identify the anti-bacterial compound(s) from Streptomyces hygroscopicus 17997, a geldanamycin producer, silica gel thin layer chromatography (TLC) TLC was used to separate the secondary metabolites of S. hygroscopicus 17997. Compound(s) from the silica gel TLC with anti-Gram positive bacteria activity and becoming red upon color reaction by 2.0 mol/L NaOH was analyzed by HPLC. The UV absorption profile and the retention time of a peak of HPLC were identical to those of authentic elaiophylin. A conserved region of dTDP-glucose-4,6-dehydratase (Tgd) gene was amplified by PCR from the genomic DNA of Streptomyces hygroscopicus 17997. DNA sequence analysis of the amplified DNA fragment indicated that it should be the tgd gene of elaiophylin biosynthetic gene cluster. These results implied that the compound in the peak of HPLC was elaiophylin, a macrodiolide antibiotic. The compound was then confirmed to be elaiophylin by LC-(+)-ESI-MS, which revealed that Streptomyces hygroscopicus 17997 was an elaiophylin producer. At the same time, a fast procedure, which consisted of silica gel TLC, color reaction, HPLC, PCR detection and DNA sequence analysis of tgd gene, and LC-(+)-ESI-MS, was established for rapid identification of elaiophylin and its producer.
Benzoquinones ; metabolism ; Chromatography, Liquid ; methods ; DNA, Bacterial ; genetics ; Hydro-Lyases ; genetics ; Lactams, Macrocyclic ; metabolism ; Macrolides ; analysis ; isolation & purification ; metabolism ; Mass Spectrometry ; methods ; Sequence Analysis, DNA ; Streptomyces ; genetics ; isolation & purification ; metabolism

The key and crucial step of metabolic engineering during quinic acid biosynthesize using shikimic acid pathway is high expression of quinate 5-dehydrogenase. The gene qa-3 which code quinate 5-dehydrogenase from Neurospora crassa doesn't express in Escherichia coli. By contrast with codon usage in Escherichia coli, there are 27 rare codons in qa-3, including eight AGG/AGA (Arg) and nine GGG (Gly). Two AGG are joined together (called box R) and some GGG codons are relative concentrate (called box G). Along with the secondary structure of mRNA analysed in computer, the free energy of mRNA changes a lot from -374.3 kJ/mol to least -80.5 kJ/mol when some bases in the end of qa-3 were transformed, and moreover, the change of free energy is quite small when only some bases in the box G and box R transformed. After the change of rare codon and optimization of some bases in the end, qa-3 was expression in E. coli and also the enzyme activity of quinate 5-dehydrogenase can be surveyed accurately. All the work above benefit the further research on producing quinic acid engineering bacterium.
Alcohol Oxidoreductases ; biosynthesis ; genetics ; Base Sequence ; Codon ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Hydro-Lyases ; genetics ; Molecular Sequence Data ; Neurospora crassa ; enzymology ; genetics ; RNA, Messenger ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Shikimic Acid ; metabolism ; Transformation, Bacterial

The gdrA, gdrB gene coding glycerol dehydratase reactivase factor were amplified by using the genomic DNA of Klebsiella pneumoniae as the template. The gdrA and gdrB were inserted in pMD-18T to yield the recombinant cloning vector pMD-gdrAB. After the DNA sequence was determined, the gdrAB gene was subcloned into expression vector pET-28a(+) to yield the recombinant expression vector pET-28gdrAB. Under screening pressure by ampicillin and kanamycin simultaneously, the activity of glycerol dehydratase reactivase was characterized by coexpression of pET-32gldABC, which carry the gldABC gene encoding glycerol dehydratase, and pET-28gdrAB in E. coli BL21(DE3).
Bacterial Proteins ; genetics ; isolation & purification ; metabolism ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; genetics ; Hydro-Lyases ; genetics ; isolation & purification ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction