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Abstract：
Considering the current growing interest in new and improved enzymes for use in a variety of applications, the present study aimed to characterize a novel detergent-stable serine alkaline protease from the extremophilic actinobacterium Microbacterium metallidurans TL13 (MmSP) using a combined in silico and experimental approach. The MmSP showed a close phylogenetic relationship with high molecular weight S8 peptidases of Microbacterium species. Moreover, its physical and chemical parameters computed using Expasy\'s ProtParam tool revealed that MmSP is hydrophilic, halophilic and thermo-alkali stable. 3D structure modelling and functional prediction of TL13 serine protease resulted in the detection of five characteristic domains: [catalytic subtilase domain, fibronectin (Fn) type-III domain, peptidase inhibitor I9, protease-associated (PA) domain and bacterial Ig-like domain (group 3)], as well as the three amino acid residues [aspartate (D182), histidine (H272) and serine (S604)] in the catalytic subtilase domain. The extremophilic strain TL13 was tested for protease production using agricultural wastes/by-products as carbon substrates. Maximum enzyme activity (390 U/gds) was obtained at 8th day fermentation on potato peel medium. Extracellular extract was concentrated and partially purified using ammonium sulfate precipitation methodology (1.58 folds purification fold). The optimal pH, temperature and salinity of MmSP were 9, 60 °C and 1 M NaCl, respectively. The MmSP protease showed broad pH stability, thermal stability, salt tolerance and detergent compatibility. In order to achieve the maximum stain removal efficacy by the TL 13 serine protease, the operation conditions were optimized using a Box-Behnken Design (BBD) with four variables, namely, time (15-75 min), temperature (30-60 °C), MmSP enzyme concentration (5-10 U/mL) and pH (7-11). The maximum stain removal yield (95 ± 4%) obtained under the optimal enzymatic operation conditions (treatment with 7.5 U/mL of MmSP during 30 min at 32 °C and pH9) was in good agreement with the value predicted by the regression model (98 ± %), which prove the validity of the fitted model. In conclusion, MmSP appears to be a good candidate for industrial applications, particularly in laundry detergent formulations, due to its high hydrophilicity, alkali-halo-stability, detergent compatibility and stain removal efficiency.
摘要：
考虑到目前对用于各种应用的新的和改进的酶越来越感兴趣，本研究旨在表征一种新型的去污剂稳定的丝氨酸碱性蛋白酶从极端放线菌微细菌金属duransTL13（MmSP）使用结合的计算机和实验方法。MmSP与微杆菌属的高分子量S8肽酶具有密切的系统发育关系。此外，使用Expasy的ProtParam工具计算的物理和化学参数表明MmSP是亲水的，嗜盐和热碱稳定。TL13丝氨酸蛋白酶的3D结构建模和功能预测导致五个特征结构域的检测：[催化枯草杆菌酶结构域，纤连蛋白（Fn）III型结构域，肽酶抑制剂I9，蛋白酶相关（PA）结构域和细菌Ig样结构域（第3组）]，以及三个氨基酸残基[天冬氨酸（D182），催化枯草杆菌酶结构域中的组氨酸(H272)和丝氨酸(S604)]。使用农业废物/副产物作为碳底物，测试了极端菌株TL13的蛋白酶生产。在马铃薯皮培养基上第8天发酵时获得了最大的酶活性（390U/gds）。浓缩细胞外提取物并使用硫酸铵沉淀法部分纯化(1.58倍纯化倍数)。最佳pH值,MmSP的温度和盐度分别为9、60°C和1MNaCl，分别。MmSP蛋白酶显示出广泛的pH稳定性，热稳定性，耐盐性和洗涤剂相容性。为了达到TL13丝氨酸蛋白酶的最大去污效果，使用具有四个变量的Box-Behnken设计(BBD)优化操作条件，即,时间（15-75分钟），温度（30-60°C），MmSP酶浓度(5-10U/mL)和pH(7-11)。在最佳酶操作条件下（在32°C和pH9下用7.5U/mL的MmSP处理30分钟）获得的最大去污率（95±4％）与回归模型预测的值（98±％）吻合良好，证明了拟合模型的有效性。总之,MMSP似乎是工业应用的良好候选者，特别是在洗衣洗涤剂配方中，由于其高亲水性，碱-卤-稳定性，洗涤剂相容性和去污效率。
