Tanio T, Fukui T, Shirakura Y, Saito T, Tomita K, Kaiho T, Masamune S
Eur J Biochem. 1982 May;124(1):71-7. doi: 10.1111/j.1432-1033.1982.tb05907.x.
A strain of Alcaligenes faecalis T1, which was isolated from activated sludge, excreted an extracellular poly(3-hydroxybutyrate) depolymerase as it grew in a medium containing poly(3-hydroxybutyrate) as the sole carbon source. The molecular weight of the enzyme, purified from the culture medium to electrophoretic homogeneity, was 48 000 as determined by Sephadex G-100 filtration, and 50 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The pH optimum for the enzyme reaction was 7.5. The purified enzyme depolymerized poly(3-hydroxybutyrate) purified from Zoogloea ramigera 1-16-M, but did not attack the bacterial native poly(3-hydroxybutyrate)-containing granules. Km values were 13.3 micrograms/ml (= 0.78 microM, based on an estimated average molecular weight of 17 000) for poly(3-hydroxybutyrate) and 5.4 mM for the trimeric ester of D(--)-3-hydroxybutyric acid. Analysis of hydrolytic products of poly(3-hydroxybutyrate), several oligomeric esters of D(--)-3-hydroxybutyric acid, and the methyl ester of the trimeric ester indicated that the enzyme hydrolyzed these substrates from the free hydroxyl terminus, releasing D(--)-3-hydroxybutyrate dimer units one at a time.
从活性污泥中分离出的一株粪产碱菌T1，在以聚（3-羟基丁酸酯）作为唯一碳源的培养基中生长时，会分泌一种细胞外聚（3-羟基丁酸酯）解聚酶。通过葡聚糖G-100过滤法测定，从培养基中纯化至电泳纯的该酶分子量为48000，在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳测定其分子量为50000。该酶反应的最适pH为7.5。纯化后的酶能解聚从生枝动胶菌1-16-M中纯化得到的聚（3-羟基丁酸酯），但不作用于细菌含天然聚（3-羟基丁酸酯）的颗粒。聚（3-羟基丁酸酯）的Km值为13.3微克/毫升（基于估计平均分子量17000，相当于0.78微摩尔），D（-）-3-羟基丁酸的三聚体酯的Km值为5.4毫摩尔。对聚（3-羟基丁酸酯）、几种D（-）-3-羟基丁酸的低聚体酯以及三聚体酯的甲酯的水解产物分析表明，该酶从游离羟基末端水解这些底物，一次释放一个D（-）-3-羟基丁酸二聚体单元。
