Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables

被引:31
|
作者
Liao, CH
Sullivan, J
Grady, J
Wong, LJC
机构
[1] UNIV MASSACHUSETTS, DEPT BIOL SCI, LOWELL, MA USA
[2] UNIV SO CALIF, CHILDRENS HOSP LOS ANGELES, DEPT PATHOL, LOS ANGELES, CA 90027 USA
关键词
D O I
10.1046/j.1365-2672.1997.00158.x
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
An improved method for purification of pectate Iyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca2+ concentration required for PLI and PLII activity was determined to be 0.5 mmol l(-1), The Ca2+ requirement could not be replaced by other metal cations such as Mg2+, Cu2+, Zn2+, Fe3+ and Co2+ Optimal pH for activity was determined to be between 8.5 and 9.0. The k(m) values for sodium polygalacturonate were 1.28 and 1.11 and ml(-1) for PLI and PLII, respectively. Both PLI and PLII were stable at ion temperatures (25 degrees C or below) for at least 1 month. However, at 37 degrees C, the activity decreased 50% in 36 h, Optimal temperatures for activity were estimated to be 46 degrees and 52 degrees C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48 degrees C or higher) increased when CaCl2 or a positively charged molecule such as poly lysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.
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收藏
页码:10 / 16
页数:7
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