Department of Environmental Biology, Shandong Agricultural University, Tai'an, Shandong, 271018, PR China.
J Microbiol. 2011 Feb;49(1):121-9. doi: 10.1007/s12275-011-9355-6. Epub 2011 Mar 3.
The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494 amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases. The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting highest activity towards casein.
从嗜热真菌Thermoascus aurantiacus var. levisporus 中克隆、测序和表达了丝氨酸蛋白酶基因，并对重组蛋白进行了表征。全长 2592bp 的 cDNA 包含一个 1482bp 的 ORF，编码 494 个氨基酸。推导的氨基酸序列分析显示与枯草杆菌蛋白酶的高度同源性。该假定酶包含催化结构域，其活性位点由三个残基 Aspl83、His215 和 Ser384 形成。重组酶在毕赤酵母中的过表达后，其分子量估计为 59.1kDa。重组蛋白的活性为 115.58U/mg。该蛋白酶在 50°C 和 pH8.0 下表现出最大活性，在 60°C 下保持热稳定性，在 70°C 下 60 分钟后仍保留 60%的活性。该蛋白酶的活性被 PMSF 抑制，但不受 DTT 或 EDTA 抑制。该酶具有广泛的底物特异性，如明胶、酪蛋白和纯牛奶，并对酪蛋白表现出最高的活性。
