Hanphakphoom Srisuda, Maneewong Narisara, Sukkhum Sukhumaporn, Tokuyama Shinji, Kitpreechavanich Vichien
Faculty of Science, Kasetsart University.
J Gen Appl Microbiol. 2014;60(1):13-22. doi: 10.2323/jgam.60.13.
Eleven strains of poly(L-lactide) (PLLA)-degrading thermophilic bacteria were isolated from forest soils and selected based on clear zone formation on an emulsified PLLA agar plate at 50°C. Among the isolates, strain LP175 showed the highest PLLA-degrading ability. It was closely related to Laceyella sacchari, with 99.9% similarity based on the 16S rRNA gene sequence. The PLLA-degrading enzyme produced by the strain was purified to homogeneity by 48.1% yield and specific activity of 328 U·mg-protein-1 with a 15.3-fold purity increase. The purified enzyme was strongly active against specific substrates such as casein and gelatin and weakly active against Suc-(Ala)₃-pNA. Optimum enzyme activity was exhibited at a temperature of 60°C with thermal stability up to 50°C and a pH of 9.0 with pH stability in a range of 8.5-10.5. Molecular weight of the enzyme was approximately 28.0 kDa, as determined by gel filtration and SDS-PAGE. The inhibitors phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetate (EDTA), and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) strongly inhibited enzyme activity, but the activity was not inhibited by 1 mM 1,10-phenanthroline (1,10-phen). The N-terminal amino acid sequences had 100% homology with thermostable serine protease (thermitase) from Thermoactinomyces vulgaris. The results obtained suggest that the PLLA-degrading enzyme produced by L. sacchari strain LP175 is serine protease.
从森林土壤中分离出11株聚（L-丙交酯）（PLLA）降解嗜热菌，并基于在50°C下乳化PLLA琼脂平板上形成的透明圈进行筛选。在这些分离株中，LP175菌株表现出最高的PLLA降解能力。基于16S rRNA基因序列，它与嗜糖拉氏菌密切相关，相似度为99.9%。该菌株产生的PLLA降解酶经纯化达到同质，产率为48.1%，比活性为328 U·mg-蛋白-1，纯度提高了15.3倍。纯化后的酶对酪蛋白和明胶等特定底物具有强活性，对Suc-(Ala)₃-pNA活性较弱。在温度为60°C时表现出最佳酶活性，热稳定性可达50°C，pH为9.0时在8.5-10.5范围内具有pH稳定性。通过凝胶过滤和SDS-PAGE测定，该酶的分子量约为28.0 kDa。抑制剂苯甲基磺酰氟（PMSF）、乙二胺四乙酸（EDTA）和乙二醇双（2-氨基乙醚）-N,N,N',N'-四乙酸（EGTA）强烈抑制酶活性，但1 mM 1,10-菲咯啉（1,10-phen）不抑制活性。N端氨基酸序列与普通嗜热放线菌的耐热丝氨酸蛋白酶（嗜热菌蛋白酶）具有100%同源性。所得结果表明，嗜糖拉氏菌LP175菌株产生的PLLA降解酶是丝氨酸蛋白酶。
