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
By using some intermediate metabolites in EMP pathway and TCA cycle as the control, the effects of amino acid supplements on glycerol production by Candida glycerinogenes in shake-flask fermentations with urea as nitrogen resource were investigated. The results showed that ten kinds of amino acids, including L-glutamic acid, L-glutamine, L-aspartic acid, L-asparagine, L-glycin, L-lysine, L-tyrosine, L-proline, L-histidine, and L-serine, had strong promotional effects on glycerol production; The optimal concentrations of these amino acids were 0.40, 0.45, 0.36, 0.35, 0.39, 0.36, 0.35, 0.45, 0.26, and 0.45 g/L, respectively. Accordingly the optimum contents of pyruvic acid, a-oxoglutarate, oxaloacetic acid, citrate, and succinate were 0.24, 0.42, 0.40, 0.37, and 0.38 g/L, respectively. The advantageous opportunities of supplement were as follows: L-lysine at the beginning of fermentation; pyruvic acid and oxaloacetic acid at the fourteenth hour; L-glutamic acid, L-glutamine, L-histidine, L-proline, L-aspartic acid, L-tyrosine, L-glycin, alpha-oxoglutarate, and succinate at the thirtieth hour; and L-asparagine, L-serine, and citrate at the forty-eighth hour. The addition of each stimulant at the optimal conditions could significantly promote glycerol production, with the glycerol yield on initial glucose and its increased speed exceeding 60% and 16%, respectively. The possible stimulatory mechanism due to the amino acid supplements is that the increased intermediate metabolites levels from amino acids degradation may have enhanced the flux through TCA cycle, which improve cell energetics. Meanwhile, the shift of carbon metabolism flux at the glyceraldhyde-3-phosphate node can result in the incremental flux through glycerol biosynthesis pathway.
Subject(s)
Amino Acids , Metabolism , Candida , Metabolism , Culture Media , Dose-Response Relationship, Drug , Fermentation , Glycerol , MetabolismABSTRACT
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
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.
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 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
In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.
Subject(s)
Bacillus , Genetics , Metabolism , Enzyme Stability , Fermentation , Genetic Engineering , Metals , Pharmacology , Recombinant Proteins , Serine Endopeptidases , Genetics , MetabolismABSTRACT
Two thermotolerant, ethanol-producing yeast cultures: THFY-4 and THFY-16 were isolated from 381 nature samples. THFY-4 can grow on 30% glucose plate at 51 ℃,while THFY-16 can grow on the same medium at 45℃.After preliminary ide ntification, THFY-4 was identified as Kluyveromyces sp. and THFY-16 belon gs to Saccharomyces genus. The ethanol fermentation experiment shows that T HFY-4 can only produce 4.88% (v/v) ethanol from 20% glucose after 60 hours, wh ile THFY-16 can produce 11.44% ethanol under the same condition. When using s accharified Canna edulis Ker wort as fermentation medium, 9.43%(v/v) ethanol we re produced from 16.1% glucose, which is 91.0% of the theoretical yield.
ABSTRACT
mutants which didnt produce red pigmen ts on malt extract agar plate were obtained.The 8 stable mutants were cultured on solid medium.Two samples wer e yellow,the others were white.The extracted samples were scanned in visible len gth.2 yellow samples showed only one absorptive peak at 370nm,the 6 white sample s showed no absorptive peak.The mutants producing only yellow pigments on solid medium were tested in liquid culture.The results indicated their ability to pro duce only yellow pigments were stable.
ABSTRACT
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.
ABSTRACT
Using chemically defined medium as the control, mechanism of corn steep liquor (CSL) in complex medium during glycerol production by Candida glycerinogenes was studied.The results showed that there were three key factors in CSL that had some great influences on glycerol fermentation of C.glycerinogenes, including phosphorus, nitrogen, and trace elements.The maximum glycerol yield of 53.44% was achieved at an optimal phosphorus concentration of 121.75mg/L, where the CSL concentration was 14g/L.Phosphorus in CSL could control the distribution of carbon metabolism flux between EMP pathway and HMP pathway.With the increase in CSL concentrations, superfluous phosphorus could restrain HMP pathway and activate EMP pathway, thus resulting in remarkable changes in various fermentation parameters of complex medium.Nitrogen in CSL could play a cooperative role in the regulative function of phosphorus.However, it was not a suitable nitrogen source for C.glycerinogenes.Trace elements in CSL could markedly improve the glucose consumption rate, accelerate the cell growth, and enhance the glycerol yield.