College of Pharmaceutical Science, Jilin University, Changchun, PR China.
College of Life Science, Jilin University, Changchun, PR China.
Int J Biol Macromol. 2019 Oct 1;138:528-535. doi: 10.1016/j.ijbiomac.2019.07.101. Epub 2019 Jul 16.
Subtilisins are a family of serine proteases used widely throughout the detergent, leather and food industries, with the identification and development of new enzymes holding much potential value. Thermus thermophilus HB8 was examined for serine proteases and found TTHA0724 gene. Sequence analysis of this putative serine protease placed it within the subtilisin family. To obtain active T. thermophilus HB8 subtilisins, three genes encoding prepro-subtilisin, pro-subtilisin and mature-subtilisin were cloned and expressed in Escherichia coli Transetta (DE3). Although direct expression of the mature-subtilisin gene was found to produce inactive inclusion bodies, expression of the pro-subtilisin gene resulted in active mature-subtilisin, indicating that the pro-sequence of translated pro-subtilisin underwent autoproteolysis. The resulting mature-subtilisin exhibited maximal activity between 65 and 85 °C at pH 7.5. The mature-subtilisin showed good stability, maintaining 50% activity after 48 h at 75 °C and >78% activity across the pH range 5.0-9.5. Furthermore, the mature-subtilisin demonstrated broad substrate specificity, with no requirement for the presence of metal ions which are essential for other subtilisin enzymes. Despite this Cu was able to increase enzyme activity, while Ca partially inhibited the activity. These properties suggest that T. thermophilus HB8 mature-subtilisin has potential value in its application in many industries.
枯草杆菌蛋白酶是一类广泛应用于洗涤剂、皮革和食品工业的丝氨酸蛋白酶，鉴定和开发新的酶具有很大的潜在价值。本研究对温泉热袍菌 HB8 进行了丝氨酸蛋白酶的研究，发现了 TTHA0724 基因。对该假定丝氨酸蛋白酶的序列分析将其置于枯草杆菌蛋白酶家族内。为了获得具有活性的温泉热袍菌 HB8 枯草杆菌蛋白酶，克隆并在大肠杆菌 Transetta（DE3）中表达了编码前体-枯草杆菌蛋白酶、原-枯草杆菌蛋白酶和成熟-枯草杆菌蛋白酶的三个基因。虽然直接表达成熟-枯草杆菌蛋白酶基因产生无活性的包涵体，但表达原-枯草杆菌蛋白酶基因会产生有活性的成熟-枯草杆菌蛋白酶，表明翻译的原-枯草杆菌蛋白酶的前导序列经历了自蛋白水解。所得成熟-枯草杆菌蛋白酶在 pH 7.5 时在 65-85°C 之间表现出最大活性。成熟-枯草杆菌蛋白酶具有良好的稳定性，在 75°C 下 48 小时后保持 50%的活性，在 pH 5.0-9.5 范围内保持 >78%的活性。此外，成熟-枯草杆菌蛋白酶表现出广泛的底物特异性，不需要存在金属离子，而金属离子是其他枯草杆菌蛋白酶所必需的。尽管如此，Cu 能够增加酶活性，而 Ca 则部分抑制了酶活性。这些特性表明温泉热袍菌 HB8 成熟-枯草杆菌蛋白酶在许多行业的应用中具有潜在价值。
