Plantechno s.r.l, via Staffolo 60, 26041, Vicomoscano, Cremona, Italy.
Mol Biol Rep. 2013 Jul;40(7):4317-25. doi: 10.1007/s11033-013-2519-1. Epub 2013 May 5.
Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-β-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.
通过直接从林地土壤样本中回收的基因组 DNA 进行直接 PCR 扩增，获得了 8 个编码纤维素酶的基因。直接扩增是使用从可用的在线纤维素酶核苷酸序列设计的引物进行的。分离的基因彼此不同，与枯草芽孢杆菌的内切-β-1,4-葡聚糖酶同源。纤维素酶在大肠杆菌中进行功能表达，并在 37 和 60°C 下的可溶性底物上进行测试，表现出不同的纤维素酶活性。其中，重新命名为 CelWS6 的酶在较高温度下表现出良好的活性。对 CelWS6 的进一步分析表明，它在酸性环境（pH 值 4.0 到 6.0 之间）和高温下具有很高的性能，其最大活性在 pH 值 5.0 和 50°C。在最佳 pH 值下，它非常稳定，因为在 60°C 孵育 120 分钟后，其原始活性的 80%以上仍能保持。由于这些纤维素酶具有不同的纤维素酶活性，但氨基酸序列相似，因此可以评估序列与蛋白质功能之间的关系。
