Gödde C, Liebergesell M, Steinbüchel A
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Germany.
FEMS Microbiol Lett. 1999 Apr 15;173(2):365-72. doi: 10.1111/j.1574-6968.1999.tb13527.x.
Several bacteria were isolated which were able to utilize poly(beta-L-malic acid) as sole carbon source for growth. The poly(beta-L-malic acid) hydrolyzing enzyme of Comamonas acidovorans strain 7789 was detected in the membrane fraction. The enzyme was purified by isolation of crude cell membranes by ultracentrifugation of disrupted cells, solubilization of the membrane fraction with octylglucoside, selective precipitation with 50% saturated ammonium sulfate and preparative isolectric focusing. SDS-PAGE analysis revealed a M(r) of 43,000. The pH optimum was 8.1 and the Km was 0.13 microM (in terms of monomeric units) and 0.0021 microM poly(beta-L-malic acid) at pH 8.1 (100 mM glycylglycine buffer). Addition of NaCl, KCl, CaCl2 or MgCl2 (from 25 to 100 mM) decreased the hydrolase activity, whereas EDTA or polymethane sulfonic acid fluoride had no influence on the enzyme. The depolymerization of poly(beta-L-malic acid) proceeded from the ends of the polyester resulting in the formation of L-malate. Esterase activity was not detectable with p-nitrophenyl acetate or p-nitrophenyl butyrate, which is used to determine for example poly(3-hydroxybutyric acid) depolymerase activity.
分离出了几种能够利用聚（β-L-苹果酸）作为唯一碳源进行生长的细菌。在食酸丛毛单胞菌7789菌株的膜部分检测到了聚（β-L-苹果酸）水解酶。通过对破碎细胞进行超速离心分离粗细胞膜、用辛基葡糖苷溶解膜部分、用50%饱和度的硫酸铵进行选择性沉淀以及制备性等电聚焦来纯化该酶。SDS-PAGE分析显示其分子量为43,000。最适pH为8.1，在pH 8.1（100 mM甘氨酰甘氨酸缓冲液）下，Km值为0.13 μM（以单体单元计）和0.0021 μM聚（β-L-苹果酸）。添加NaCl、KCl、CaCl2或MgCl2（25至100 mM）会降低水解酶活性，而EDTA或聚甲磺酸氟化物对该酶没有影响。聚（β-L-苹果酸）的解聚从聚酯的末端开始，导致L-苹果酸的形成。用对硝基苯乙酸或对硝基苯丁酸检测不到酯酶活性，而对硝基苯乙酸或对硝基苯丁酸可用于例如测定聚（3-羟基丁酸）解聚酶活性。
