ABSTRACT
The method for analysis and determination the cleavage of soybean sterol, in which the soybean sterol was degraded and the products androst-1,4-diene-,17-dione (ADD) and androst-4-ene-3,17-dion (AD) were developed by Liquid Chromatography-mass Spectrometry. The HPLC conditions adopted were: a All- tima ODS-2 column (250 mm?4.6 mm, 5 ?m), a mobile phase consisted of menthanol-water (70:30), a flow rate of 1.0 mL/min, a room column temperature. and the detective wavelength was 244 nm.The ZMD Micromass electrospray ionization (ESI)-mass spectrometer was employed. In such conditions the corre- sponding HPLC chromatogram and MS spectrum were obtained. The method has a linear ranger of 0.01 mg/mL ~ 0.09 mg/mL, R2 =0.9999, the recoveries of ADD and AD were 102.6% and 105.90%, the RSD of ADD and AD were 3.02%, 3.5% and 3.08%, 3.24%. This method showed high sensitivity, accuracyand easy to perform. It is suitable to analysis the process cleavage of soybean sterol as well as quality control of product.
ABSTRACT
The dhaD gene encoding glycerol dehydrogenase(GDH) from Klebsiella sp.was amplified,and was inserted into expression vector pET-28a(+),the plasmid pET-28a-dhaD was constructed and was transformed into Escherichia coli BL21(DE3).SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21.Then GDH was purified by Ni-NTA affinity chromatography,the results showed a single band about 39kDa on SDS-PAGE gel,and the specified activity was about 156U/mg.The special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%.The optimum reaction pH was 11.0,and the GDH activity have little changed when incubated in the buffer of pH7.0～11.0.The optimum reactive temperature was 30℃,and the GDH was more stable on the temperature of 25℃～45℃.The Km value was 0.54mmol/L and Vmax was 0.49 ?mol/ml?min in the glycerol.
ABSTRACT
Nicotiana tabacum is an important and classical model plant which can respond to the change of environmental conditions by accumulating osmoprotectants, such as glycerol and proline which contribute to the re-establishment of homeostasis when exposed to various adverse environmental stresses, such as drought, salinity, high and low temperatures. The optimization of ultrasonic extraction (UE) conditions of glycerol-3-phosphate dehydrogenase (GPDH) of tobacco leaf have been built by orthogonal test. It showed that optimum of the powers, treatment times, slot times and leaf-to-solvent ratios of UE was 75w, 2h, 2s, and 1[DK]∶12 g/ml, respectively. Under these conditions, the activity of GPDH has been tested as 0.3937U/mg protein, which was higher than other extraction methods such as liquid nitrogen and grinding on ice bath. According to investigation, it is the first description of determination of content of GPDH with ultrasonic in tobacco. It could provide basis for the further research in the relation of content of glycerol and osmotic pressure in tobacco.
ABSTRACT
A method to determine dihydroxyacetone (DHA) in fermentation broth was developed by high performance liquid chromatography (HPLC). DHA was separated on a Alltima C18(5?m,250?4.6mm). The mobile phase was 0.5% methanol solution (pH adjusted to 3.0 with H3PO4), the flow-rate was 1.0 ml/min and the detective wavelength was 200 nm. The detection limits of DHA was 0.1 g/L～10.0 g/L. 6.2 g/L DHA in the fermentation broth was detected by HPLC, which was in agreement with the result by spectrophotometric method.The method was applicable for DHA determination in the fermentation process.
ABSTRACT
Effect of organic acids on the synthesis of 1,3 propanediol was studied.The adsorption of organic acids from glycerol fermentation liquor by ion-exchange resins was investigated.The results showed that organic acid and 1,3 propanediol production was in negative relationship.The static adsorption showed that ion-exchange resin 005 had the best adsorption abilities of the organic acids in the glycerol fermentation liquor.It was showed that the yield of 1,3propanediol increased by 166% after the extraction of organic acids from glycerol fermentation liquor and the convertion rate increased by 34%.
ABSTRACT
The fused gene (PTH-HSA) of parathyroid hormone (PTH) gene and Human Serum Albumin(HSA) gene was amplified without linker by Overlapping PCR technology. The spliced gene was clone into Pichia pastoris secretory vector pPIC9K. With the help of promoter AOX1 and mat ? signal peptide, the PTH-HSA gene was designed to secretory expression.Linearized by restriction enzyme SalI, The recombinant plasmid pPIC9K/PTH-HSA was transformed into Pichia pastoris KM71 by electroporation. The recombinant strains which were identified by G418 and PCR analysis were induced by methanol to express protein PTH-HSA. The target protein was expressed in fermentation supernatant. Western blot analysis of the fusion protein showed that the expressed fusion protein PTH-HSA had the antigenicity of HSA.adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis The specific activity of broth was about 318IU/ml.
ABSTRACT
The hom gene encoding for homoserine dehydrogenase was amplified from the genomic DNA of Corynebacterium glutamicum ATCC 13032.After the kanamycin-resistant gene(Km)cassette from plasmid pET28a was inserted into the center of hom,the hom::Km cassette was then electroporated into the competent cell of C.glutamicum ATCC 13032.And kanamycin-resistant clones were obtained.PCR was performed to confirm whether the Km gene was integrated into the hom gene of these clones and the recombinant strains of hom-disrupted were screened out.Fermentation results showed that the lysine yield of the hom-disrupted strain C.g-hom::Km-8 reached 4.7 g/L,which was 6.7 times that of C.glutamicum ATCC 13032.
ABSTRACT
The 1,3-propanediol oxidoreductase isoenzyme encoding gene (yqhD) from E. coli was amplified by PCR. yqhD was inserted in pEtac to yield the recombinant expression vector pEtac-yqhD. Over-expression of yqhD in E. coli JM109 was achieved with pEtac-yqhD. SDS-PAGE analysis showed an over-expressed recombinant product at about 43 kD, consistent with the molecular weight predicted from gene sequence. Compared with E. coli JM109 (pEtac), the 1,3-propanediol oxidoreductase isoenzyme activity of the recombinant E. coli (pEtac-yqhD) reached 120 u/mg protein under the induction of 1.0 mmol/L IPTG at 37 degrees C for 4 hours; at similar conditions, enzyme activity of E. coli JM109 (pEtac) was only 0.5 u/mg protein. The recombinant E. coli JM109 (pUCtac-dhaB, pEtac-yqhD) was constructed. After induction with 1.0 mmol/L IPTG, the recombinant strain could transform 50 g/L glycerol to 38 g/L 1,3-propanediol under aerobic conditions. This work demonstrated firstly that the 1,3-propanediol oxidoreductase isoenzyme could show high activity under aerobic conditions.
Subject(s)
Aerobiosis , Alcohol Dehydrogenase , Alcohol Oxidoreductases , Genetics , Metabolism , Aldehyde Reductase , Genetics , Metabolism , Escherichia coli , Genetics , Escherichia coli Proteins , Genetics , Metabolism , Genetic Engineering , Methods , Isoenzymes , Propylene Glycols , Metabolism , Recombinant Proteins , Genetics , MetabolismABSTRACT
More than 20 strains capable of producing dihydroxyacetone from glycerol were isolated from 4 different natural environment samples by using two detection methods. The strain 6-8 which could grow on medium containing glycerol as sole carbon source had a higher converting capability. Under a better culture, the highest DHA production of the strain 6?8 reached 6.4 g/L. In addition to general morphological and bio-chemical characteristics, the strain 6?8 was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of the strain 6-8 had similarity of 99.7% with Acinetobacter sp. suggesting that the strain 6-8 is one of subspecies of Acinetobacter sp.
ABSTRACT
In order to isolate genes related with the osmoadaptation and glycerol metabolism of Candida glycerinogenes, a transformation system based on the dominant selectable marker Zeocin and restriction enzyme-mediated integration (REMI) was established. Effects of seven restriction enzymes on transformation efficiency of C.glycerinogenes were tested. Transformation conditions were optimized in the presence of Hind III. Under the optimal conditions of OD_ 600 ≈1.3, voltage of 1.5 kV, 2.0?10~9 competent cells/mL, 100 units of Hind III added, the transformation efficiency was up to 129 trnaformants/?g DNA. 58% of transformants were stable on nonselective medium. These results suggest that REMI technique would be beneficial to the genetic transformation of C.glycerinogenes.