ABSTRACT
An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/minrespectively.
Uma lipase extracelular foi isolada e purificada a partir de um caldo de cultura de Pseudomonas aeruginosa SRT9 até homogeneidade visível empregando-se precipitação com sulfato de amônia, seguida de técnicas cromatográficas em colunas de fenil sefarose CL-4B e Mono Q HR 5/5, obtendo-se um fator de purificação de 98 vezes, e atividade especifica de 12307,8 U/mg. Por SDS_PAGE, estimou-se que o peso molecular da lipase purificada é 29kDa, com um ponto isoelétrico de 4,5. A lipase apresentou atividade máxima em uma ampla faixa de temperatura e pH, com ótimos a 55ºC e pH 6,9. A lípase foi mais ativa sobre triacilglicerois de cadeia longa (C14-C16). A lipase foi fortemente inibida por EDTA, o que sugere que a enzima pode ser uma metaloproteína. SDS e íons metálicos, como Hg2+, Zn2+,Cu2+, Ag2+ e Fe2+, diminuíram marcadamente a atividade da lipase. Sua grande estabilidade e atividade em solventes organicos sugerem que esta lípase pode ser uma excelente ferramenta tecnológica com várias aplicações como reações organosintéticas e preparação de produtos farmacêuticos enantiomericamente puros. Os valores de Km e Vmax para a enzima purificada na hidrólise de trioleina foram 1,11 mmol/L e 0,05 mmol/L/min, respectivamente.
Subject(s)
Ammonium Sulfate , Lipase/analysis , Metalloproteins/analysis , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Sepharose/analysis , Chromatography , Methods , MethodsABSTRACT
Objective To explore the effects of humulon on kinetic parameters of N-acetyltransferase-1(NAT1) of human gastric cancer SGC-7901.Methods Employing HPLC,using para-aminobenzoic acid(PABA) as substrate,in intact SGC-7901 cells and their cytoplasm,taking the speed of PABA being acetylated to Ac-PABA by NAT1 as the rate of NAT1,using double reciprocal plot,taking the reciprocal of concentration of PABA and reaction rate of NAT1 as coordinates,regression equation was obtainied and the Michaelis constant(Km) and maximum reaction velocity(Vmax) were calculated.Results Study on enzyme kinetics demonstrated,as for intact SGC-7901 cells,Km and Vmax of control group were(3.910?0.087) ?mol/L and(0.306 0?0.006 7) pmol/L(1?106 cells),respectively,Km and Vmax of the humulon group were(3.830?0.123) ?mol/L and(0.275 0?0.005 8) pmol(1?106 cells),respectively.As for the cytoplasm of SGC-7901 cells,Km and Vmax of control group were(760.2?210.2) ?mol/L and(0.191 0?0.043 7) pmol/(mg?min),Km and Vmax of the humulon group were(449.0?72.9) ?mol/L and(0.094 0?0.010 4) pmol/(mg?min).Statistically,as for intact SGC-7901 cells or their cytoplasm,there was no difference of the Km between control group and humulon group,but there was remarkable difference of Vmax between control group and humulon group,P