Asitok Atim, Ekpenyong Maurice, Takon Iquo, Antai Sylvester, Ogarekpe Nkpa, Antigha Richard, Edet Philomena, Ben Ubong, Akpan Anthony, Antai Agnes, Essien Joseph
Environmental Microbiology and Biotechnology Unit, Department of Microbiology, Faculty of Biological Sciences, University of Calabar, Nigeria.
University of Calabar Collection of Microorganisms (UCCM), Department of Microbiology, University of Calabar, Nigeria.
Biotechnol Rep (Amst). 2022 Jun 5;35:e00746. doi: 10.1016/j.btre.2022.e00746. eCollection 2022 Sep.
A strain of , isolated from fermenting bean-processing wastewater, produced alkaline protease in pretreated cassava waste-stream, but with low yield. Strain improvement by alternate combinatorial random mutagenesis and bioprocess optimization using comparative statistical and neural network methods enhanced yield by 17.8-fold in mutant kGy-04-UV-25. Kinetics of production by selected mutant modeled by logistic and modified Gompertz functions revealed higher specific growth rate in mutant than in the parent strain, likewise volumetric and specific productivities. Purification by PEG/Na citrate aqueous two-phase system recovered 73.87% yield and 52.55-fold of protease. Its activity was stable at 5-35% NaCl, 45-75°C, and was significantly enhanced by 1-15 mM sodium dodecyl sulfate (SDS). The protease was inhibited by low concentrations of phenyl-methyl-sulfonyl fluoride but was activated by 1-5 mM Mn suggesting a manganese-dependent serine‑protease. The 45.7 kDa thermo-halo-stable alkaline protease demonstrated keratinolytic and blood-stain removal potentials showing prospects in textile and detergent industries, respectively.
从发酵豆制品加工废水中分离出的一株菌株，在预处理的木薯废水中能产生碱性蛋白酶，但产量较低。通过交替组合随机诱变和使用比较统计及神经网络方法进行生物工艺优化来改良菌株，使突变体kGy-04-UV-25的产量提高了17.8倍。用逻辑函数和修正的Gompertz函数对所选突变体的生产动力学进行建模，结果显示突变体的比生长速率高于亲本菌株，体积产率和比产率也是如此。通过聚乙二醇/柠檬酸钠双水相系统纯化，蛋白酶的回收率为73.87%，纯化倍数为52.55倍。其活性在5-35%氯化钠、45-75°C条件下稳定，并且在1-15 mM十二烷基硫酸钠（SDS）作用下显著增强。该蛋白酶受到低浓度苯甲基磺酰氟的抑制，但在1-5 mM锰的作用下被激活，表明它是一种依赖锰的丝氨酸蛋白酶。这种45.7 kDa的热-卤-稳定碱性蛋白酶具有角蛋白分解和去除血渍的潜力，分别在纺织和洗涤剂行业展现出应用前景。
