Genetic Engineering Reconstruction of Klebsiella pneumoniae Producing 1,3-propanediol by the Gene yqhD Encoding 1,3-propanediol Oxidoreductase Isoenzyme / 中国生物工程杂志

In the reductive branch, glycerol is first dehydrated to 3-hydroxypropionaldehyde that is then reduced to 1,3-PD under the consumption of reducing power (NADH). If over-expression of the gene dhaB encoding glycerol dehydrase is achieved,the reducing power will be scarce and 3-hydroxypropionaldehyde will be accumulated,which is disadvantage to produce 1,3-propanediol.The structure gene yqhD from E.coli encoding 1,3-propanediol oxidoreductase isoenzyme[under the consumption of reducing power (NADPH)]and the gene dhaB encoding glycerol dehydrase from Klebsiella pneumoniae was amplified using PCR method.The two gene were transferred into expression vector pEtac to construct a novel recombinant Klebsiella pneumoniae (pEtac-dhaB-tac-yqhD).Over-expression of yqhD and dhaB in Klebsiella pneumoniae was achieved with pEtac-dhaB-tac-yqhD.The fermentation result on aerobic conversion showed the increase of 20% of 1,3-propanediol yield by Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD) was obtained compared with Klebsiella pneumoniae.The main by-products,acetic acid and butanediol decreased estrogen receptors 30% obviously.

Synthesis of diacylglycerol using immoblized regiospecific lipase in continuously operated fixed bed reactors / 生物工程学报

Diacylglycerol, DAG, because of its multifunctional and nutritional properties, attracted considerable attention recently. Enzymatic synthesis of diacylglycerols from linoleic acid was investigated in a solvent-free reaction in a continuously operated fixed bed reactors containing Lipozyme RM IM. By appropriate manipulation of the fluid-residence time, the relative proportions of the various acylglycerols in the effluent stream can be controlled. In addition, the presence of excess glycerol is effective for the removal of water produced during the esterification reactions. Under the conditions of molar ratio of linoleic acid to glycerol of 0.5, the immoblized enzyme maintained high stability and allowed the reaction to continue for 10 days without significant deterioration in enzyme activity. It was determined that the conversion of fatty acid, content of 1,3-DAG and volume efficiency of reactor reached optima under the conditions: a packaged-bed reactor(with a ratio of packed length to inner diameter of 7.8), reacting temperature at 65 degrees C, molar ratio of linoleic acid to glycerol of 0.5, and feeding flow rate of 1.2 mL/min.

Expression and purification of thermostable alpha-glucuronidase from Thermotoga maritima / 生物工程学报

The xylanolytic enzymes found in Thermotoga maritima showed extremely high thermostability and considerable potential in industrial application. Yet expression level of the genes encoding these enzymes was very low. The alpha-glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The alpha-glucuronidase was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL with plasmid pET-28a(+), and made up about 20% of the total proteins present in the intracellular soluble fraction. The results proved the assumption that rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of aguA gene from hyperthermophilic bacterium T. maritima in E. coli. Purification procedure included two steps, heat treatment and immobilized metal affinity chromatography, and over 13.5mg of pure enzyme was obrained from 1L of induced culture. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 5.1 fold, and a yield of 55.1%. The optimum activity of recombinant alpha-glucuronidase was found at pH 6.0 and 85 degrees C, the enzyme retained 70% of its activity after 1 h of incubation at 85 degrees C. The induction conditions for expression of recombinant strain BL21-CodonPlus(DE3)-RIL/pET-28a-aguA were studied on induction time and duration by IPTG. The results showed that the activity of thermostable alpha-glucuronidase reach the maximum in 5-hour after inducted at the exponential phase (OD600 of 0.7 - 0.8).

Purification and characterization of glutamate dehydrogenase. from Corynebacterium glutamicum S9114 / 生物工程学报

Glutamate dehydrogenase (GDH) is a key enzyme in the biosynthesis of glutamate. The GDHs from Corynebacterium glutamicum S9114 the most commonly used strain in glutamate fermentation, were purified and their molecular structures and properties characterized. The coenzymes were also studied in the hope to increase glutamate production. Cells were harvested at mid-exponential phase by centrifugation and washed with Tris-HCl buffer containing DTT and EDTA (pH 7.5). The cells were then disrupted using a French pressure cell press and the supernatant was collected by centrifugation. The extract was concentrated by 70-fold using the AKTA-100 FPLC system employing a DEAE-cellulose ion exchange column, a hydrophobic interaction chromatography (HIC) and Sephadex G-200 gel filtration. The purified extracts contained NADPH-dependent GDH and NADH-dependent GDH. Both of the enzymes were highly specific for the coenzymes. The molecular masses of the NADPH-dependent GDH and its subunit were 188kD and 32kD respectively, suggesting the enzyme is a homo-hexamer. Our data reported for the first time the presence of NADH- dependent GDH in Corynebacterium glutamicum S9114, similar to other microorganisms containing both GDHs. The NADPH-dependent and NADH-dependent GDH in Corynebacterium glutamicum S9114 may participate in the assimilation and dissimilation of ammonia respectively. The absorptions of NADPH-dependent GDH was very weak at 280nm but very high at 215nm, suggesting a low phenylalanine and tyrosine content in the enzyme.