Wahab Walaa A Abdel, Saleh Shireen A A, Elzairy Nermeen H, Ahmed Samia A, Zaki Eman R, Salama Walaa H, Mostafa Faten A
Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, Cairo, Egypt.
Molecular Biology Department, National Research Centre, Dokki, Cairo, Egypt.
Microb Cell Fact. 2024 Dec 17;23(1):330. doi: 10.1186/s12934-024-02600-0.
The main obstacle facing the utilization of microbial enzymes in industrial applications is the high cost of production substrates. As a result of the mentioned different wastes (coffee powder waste, dates nawah powder, molokhia stems, pea peels, lemon peels, and corn cobs) were investigated as low-cost nutritional substrates for the production of microbial β-galactosidase in this study. The purification of the enzyme and its kinetic and thermodynamics were investigated.
β-galactosidase was effectively produced by Aspergillus foetidus utilizing lemon peels and coffee powder waste by solid-state fermentation technique. The production yield was improved through Plackett-Burman Design declaring the significant effect of lemon peels and coffee waste powder, and beef extract quantities on A. foetidus β-galactosidase production. Followed by Central Composite Design investigating each factor with five levels resulting in 37363.1 U.ml production. The enzyme was fully purified by gel filtration technique through Sephadex G-150 giving one band with a molecular weight 40 KDa on SDS-PAGE gel. The maximal β-galactosidase activity was obtained at 50 °C with 0.4% ONPG. Cu, Fe, and Hg showed severe inhibitory effect on pure enzyme activity. Energy required for enzyme activation (E) and denaturation (E) were determined to be 17.40, and 43.86 KJ.mol, respectively. Parameters reflecting β-galactosidase thermal stability at 40, 45, and 50 °C as T and D-values values were determined to be 283.92, 209.43, and 168.56 min, and 943.34, 695.84, and 560.06 min, respectively.
微生物酶在工业应用中面临的主要障碍是生产底物成本高昂。因此，本研究考察了不同的废弃物（咖啡粉废料、椰枣纳瓦粉、锦葵茎、豌豆皮、柠檬皮和玉米芯）作为生产微生物β-半乳糖苷酶的低成本营养底物。对该酶的纯化及其动力学和热力学进行了研究。
通过固态发酵技术，恶臭曲霉利用柠檬皮和咖啡粉废料有效地生产了β-半乳糖苷酶。通过Plackett-Burman设计提高了产量，该设计表明柠檬皮、咖啡废料粉和牛肉提取物的量对恶臭曲霉β-半乳糖苷酶的生产有显著影响。随后通过中心复合设计对每个因素进行五个水平的研究，产量达到37363.1 U.ml。通过Sephadex G-150凝胶过滤技术对该酶进行了完全纯化，在SDS-PAGE凝胶上得到一条分子量为40 kDa的条带。在50°C和0.4%邻硝基苯-β-D-半乳糖苷（ONPG）条件下获得了最大β-半乳糖苷酶活性。铜、铁和汞对纯酶活性表现出严重的抑制作用。酶激活所需能量（E）和变性所需能量（E）分别测定为17.40和43.86 KJ.mol。反映β-半乳糖苷酶在40、45和50°C下热稳定性的参数T和D值分别测定为283.92、209.43和168.56分钟，以及943.34、695.84和560.06分钟。
