Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic.
Folia Microbiol (Praha). 2012 Jul;57(4):335-9. doi: 10.1007/s12223-012-0137-2. Epub 2012 Apr 20.
A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-D: -glucosaminide, N,N´-diacetyl-β-D: -chitobiose, or N,N´,N˝-triacetyl-β-D: -chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.
从人粪便中分离到一株严格厌氧嗜温的几丁质分解细菌，鉴定为梭菌。该菌对各种类型的生长基质作出反应，产生了一系列几丁质分解酶，这些酶是 J4 菌株分泌组的重要组成部分。通过在含有胶体几丁质的 M-10 培养基中培养来估计内切几丁质酶、外切几丁质酶和 N-乙酰氨基葡萄糖苷酶的酶活性，从而对分泌的活性蛋白进行了表征。在含有葡萄糖的 M-10 培养基中培养的 J4 菌株产生的酶活性显著降低。通过 SDS-PAGE 分离细胞外分泌的蛋白质谱。在复性 SDS-PAGE 的酶谱上证实了几丁质酶的变异性。用 N-乙酰-β-D：-氨基葡萄糖苷、N，N´-二乙酰-β-D：-壳二糖或 N，N´，N˝-三乙酰-β-D：-壳三糖的 4-甲基伞形酮衍生物分别作为 β-N-乙酰氨基葡萄糖苷酶、壳二糖酶或内切几丁质酶的活性，在紫外线下可视化酶。通过二维凝胶电泳分析分离分泌组的蛋白质成分。然后，使用 MALDI-TOF/TOF 串联质谱对不同的蛋白条带进行分离、鉴定和表征。根据数据库搜索的序列同源性进行最终鉴定。
