Shalaby Mohab M, Samir Reham, Goma Fatma Al-Zahraa M, Rammadan Mohammed A
Microbiology & Immunology Department, Faculty of pharmacy, Egyptian Russian University, Cairo, Egypt.
Microbiology & Immunology Department, Faculty of pharmacy, Cairo University, Cairo, Egypt.
Biotechnol Rep (Amst). 2021 Apr 18;30:e00620. doi: 10.1016/j.btre.2021.e00620. eCollection 2021 Jun.
The expanding interest in bioremediation of poorly degradable wastes has led to the discovery of many microbial enzymes capable of degrading recalcitrant substances such as keratinaceous wastes that are produced in vast quantities on daily basis. Such enzymes don't only work as a bioremediation tool but also have multiple beneficial applications. Hence, environmental samples were collected from sewage water, soils, animal bodies and feces in order to isolate keratinase producing organisms. Keratinolytic isolates were isolated from sewage water; soils; animal bodies; animal feces, and identified both traditionally and molecularly through 16S-rRNA sequencing to be strain. Produced keratinase was purified by centrifugation, ammonium sulfate precipitation, and HPLC, then assayed using Azokeratine based analysis. keratinase quantification yielded a 420 ± 1.63 U/mL. Optimum production was obtained at 40 °C, pH 7, 3 days incubation, 0.5 % substrate, 0.4 /l magnesium ion, 2% v/v inoculum, 0.5 /l NaCl, 0.4 /l KHPO, and 0.3 /l KHPO. Production was increased by 1.9 fold after acclimatization to reach 809 ± 2.49 U/mL in only 2 days. Thermal and pH stability testing revealed the effectiveness of the isolated keratinase over a wide range of temperatures at neutral pH. Finally, isolated keratinase enhanced fusidic acid topical penetration to treat induced deep skin bacterial infection in mice. A 1.4 fold decrease in treatment period and a 2 log cycle reduction in the viable count of were noticed in keratinase/fusidic acid treated mice compared to mice treated with fusidic acid alone. This study shed some light on a simple keratinase production optimization technique and suggested a promising medical application of this enzyme as a drug delivery agent.
对难降解废物生物修复的兴趣不断扩大，促使人们发现了许多能够降解顽固性物质的微生物酶，如每天大量产生的角蛋白类废物。这类酶不仅可作为生物修复工具，还具有多种有益应用。因此，从污水、土壤、动物体和粪便中采集环境样本，以分离产角蛋白酶的生物。从污水、土壤、动物体、动物粪便中分离出角蛋白分解菌，并通过16S - rRNA测序进行传统和分子鉴定，确定为菌株。通过离心、硫酸铵沉淀和高效液相色谱法纯化产生的角蛋白酶，然后使用基于偶氮角蛋白的分析方法进行测定。角蛋白酶定量结果为420±1.63 U/mL。在40℃、pH 7、培养3天、底物浓度0.5%、镁离子浓度0.4 mmol/L、接种量2% v/v、氯化钠浓度0.5 mmol/L、磷酸氢二钾浓度0.4 mmol/L和磷酸二氢钾浓度0.3 mmol/L的条件下可获得最佳产量。驯化后产量提高了1.9倍，仅在2天内就达到809±2.49 U/mL。热稳定性和pH稳定性测试表明，分离出的角蛋白酶在中性pH的广泛温度范围内均有效。最后，分离出的角蛋白酶增强了夫西地酸的局部渗透作用，用于治疗小鼠诱导的深部皮肤细菌感染。与仅用夫西地酸治疗的小鼠相比，用角蛋白酶/夫西地酸治疗的小鼠治疗期缩短了1.4倍，活菌数减少了2个对数周期。本研究揭示了一种简单的角蛋白酶生产优化技术，并表明该酶作为药物递送剂具有广阔的医学应用前景。
