Selective immobilization of Bacillus subtilis lipase A from cell culture supernatant: Improving catalytic performance and thermal resistance |
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| Affiliation: | 1. Deparment of Chemistry and Chemical Processing Technologies, Mersin University, 33343, Mersin, Turkey;2. Deparment of Chemistry and Chemical Processing Technologies, Dicle University, 21280, Diyarbakır, Turkey;3. Food Processing Programme, Mersin University, 33343, Mersin, Turkey;4. Deparment of Chemistry and Chemical Processing Technologies, Siirt University, 56100, Siirt, Turkey;5. Chemistry Department, Sciences Faculty, Erciyes University, 38039, Kayseri, Turkey;6. Technology Research & Application Center (TAUM), Erciyes University, 38039, Kayseri, Turkey;1. College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou, 310015, PR China;2. Institute of Quality and Standard for Agriculture Products, Zhejiang Academy of Agricultural Science, Hangzhou, 310021, PR China;3. School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China;1. Izmir Biomedicine and Genome Center, 35340, İzmir, Turkey;2. Department of Chemistry, The Graduate School of Natural and Applied Sciences, Dokuz Eylul University, 35390, İzmir, Turkey;3. Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, 35340, İzmir, Turkey;4. Department of Chemistry, Division of Biochemistry, Faculty of Science, Dokuz Eylul University, 35390, İzmir, Turkey;1. University of Campinas, School of Chemical Engineering, Campinas, SP, Brazil;2. Federal University of São Carlos, Center of Agricultural Sciences, Araras, SP, Brazil;1. Department of Chemical Engineering, Institute of Chemical Technology, Matunga, Mumbai, 400 019, India;2. Department of Microbiology, Tripura University, Suryamaninagar, Tripura, 799 022, India;3. Bhabha Atomic Research Centre, Trombay, Mumbai, 400 085, India |
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| Abstract: | Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50 °C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns. |
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| Keywords: | Porous glyoxyl silica Immobilization Enzymatic improvement Thermal resistance |
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