用于生物技术目的的洋葱伯克霍尔德氏菌脂肪酶：纯化、结晶及表征

Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

作者信息

Bornscheuer U, Reif O W, Lausch R, Freitag R, Scheper T, Kolisis F N, Menge U

机构信息

School of Agricultural Sciences, Department of Applied Biological Sciences, Nagoya, Japan.

出版信息

Biochim Biophys Acta. 1994 Sep 28;1201(1):55-60. doi: 10.1016/0304-4165(94)90151-1.

DOI:10.1016/0304-4165(94)90151-1

PMID:7522571

Abstract

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

摘要

洋葱假单胞菌（天野株）的商业脂肪酶（三酰基甘油脂肪酶，EC 3.1.1.3）通过在苯基琼脂糖上进行单次色谱法纯化至同质。洗脱的脂肪酶在4℃下于含有58 - 69%异丙醇的洗脱液中自发结晶。脂肪酶的产率为87 - 100%，在三油精水解过程中的比活性为5800 U/mg蛋白质。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）分析，该蛋白质的分子量为34.1 kDa。通过SDS - PAGE和毛细管区带电泳测定其纯度≥99%。固定在琼脂糖上增加了其在有机溶剂中的稳定性。洋葱假单胞菌的这种脂肪酶在底物特异性和结晶过程方面与其他假单胞菌菌株的脂肪酶不同。它在有机溶剂和超临界二氧化碳中表现出高稳定性。