ABSTRACT
The purification and the characteristics of an enzyme from Morganella morganii J-8, which could produce d-pseudoephedrine from 1-phenyl-2-methylamine-acetone, were performed in this study. In this research, first, cells were disrupted by ultrasonic treatment at 4 degrees C. The carbonyl enantioselective reductase was purified with a combination of ammonium precipitation, Phenyl Superose hydrophobic chromatography, DEAE anion exchange, and native polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme subunit was estimated to be 42.5kD on sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). The native molecular mass of the enzyme that was analyzed by high-performance liquid chromatography was found out to be 84.1 kD, which indicated that the enzyme was a dimmer. The purified enzyme was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the result showed that the purified enzyme had high homology with leucine dehydrogenase.
Subject(s)
Bacterial Proteins , Chemistry , Metabolism , Biocatalysis , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Leucine Dehydrogenase , Metabolism , Metals, Heavy , Pharmacology , Molecular Weight , Morganella morganii , Metabolism , Oxidoreductases , Chemistry , Metabolism , Pseudoephedrine , Chemistry , Metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Stereoisomerism , TemperatureABSTRACT
Enzymatic synthesis of monoglycerides by glycerolysis of oil and fats in microaqueous solvent-free media was investigated by using lipase from pseudomonus fluorescens (PFL). Initial eutectic point(IEP) was substituted for melt point of oil and fats in Critical Temperature Theory. By investigating the glycerolysis under different IEP, it is showed that there is a relationship between composition of the oils and the yield of monoglycerides: Y = -0.0006 X3 + 0.0592 X2 - 0.8909 X + 26.753(13% < X < 76.5%), here X is the contents(W/W) of saturated fatty acid residue (C16 + C18) in the oils, Y is the yield of monoglycerides at 40 degrees C. The optimum isothermal reaction conditions for a system which IEP is 40 degrees C are: 40 degrees C, 3%-4.5% (W/W) water in glycerol, dosage of lipase is 500 u/g oil when the mole ratio of glycerol to oil is 2.5:1. The highest yield of monoglycerides is 81.4% in 48 h by means of programming temperature reaction.
Subject(s)
Glycerides , Metabolism , Glycerol , Metabolism , Kinetics , Lipase , Metabolism , Palm Oil , Plant Oils , Metabolism , Pseudomonas fluorescens , Substrate Specificity , TemperatureABSTRACT
Enzymatic esterification by reacting caparic acid with glycerol in solvent-free system was studied. Lipases from Pseudomonas fluoresces(PFL), Mucor miehei(MML) and Candida antarictica(CAL) possessed good catalytic activity. The optimal reaction conditions to convert capric acid with CAL are: 60 degrees C, 20-100 u of CAL per gram capric acid, 12% (W/W) of initial water content in glycerol. CAL does not express its 1,3-specificity in final product. Mechanical fraying denatured CAL partly. 96.4% of catalytic activity of CAL recovered after 5 batches of reaction. Extraction with sodium carbonate solution can decrease acid value of product from 9.8 mg KOH/g to 0.68 mg KOH/g. Applying the enzymatic esterification in open system, under vacuum or dehydrating with molecular sieves all dehydrate effectively. Molar ratio of reactants does not influence the total conversion of capric acid but influences the yield of monoglyceride. With certain protocols, the reaction period could be shortened dramatically; conversion of capric acid reached 96.9% in 5 h.
Subject(s)
Catalysis , Decanoic Acids , Metabolism , Glycerol , Metabolism , Lipase , MetabolismABSTRACT
The recombinant plasmid pPIC-gpd-bgl-hyg was constructed, which contained GPD2 promotor and terminator from industrial yeasts Saccharomyces cerevisiae, ?-glucosidase gene (BGL1) from Sac-charomycopsis fibuligera and hyg from hygromycin as the selected marker. With the yeast’s high efficiency of homologous integrated, the BGL1 gene was successfully integrated into industrial yeasts S. cerevisiae. The recombined yeast could grow on the cultures with the cellobiose as a sole carbon source, and the ?-glucosidase activity achieved 0.764 U/mL after 48 hours’ cultivation. In the experiments of VHG ethanol fermentation, the cellobiose concentration in broth of recombined yeast was 80% lower than that of indus-trial yeast.
ABSTRACT
In gene manipulation, different selectable markers with various linkers are necessary. In order to get selectable markers directly, we constructed from pBlueScript SK(-) a series of particular plasmids, pSKsymKm, pSKsymBle, pSKsymEry, pSKsymHyg and pSKsymGm, each contains Kanamycin, Bleomycin, Erythromycin, Hygromycin or Gentamycin resistance cassette. By restriction enzyme digestion and gel extraction, any of five antibiotic resistance genes with specific ends can be conveniently obtained.