The thermal stability of New Zealand culture rabbit muscle aldolase was investigated by differential scanning calorimetry in the water content range 0.23-3.70 g water per g protein.The experimental results showed that...The thermal stability of New Zealand culture rabbit muscle aldolase was investigated by differential scanning calorimetry in the water content range 0.23-3.70 g water per g protein.The experimental results showed that at water contents below 0.47g/g,an endothermic peak was observed and at water contents above 0.57g/g,an endothermic and an exothermic peak were both observed on DSC thermogram.Thermal denaturation result of the enzyme and the relationship between two transition temperatures and water contents were first reported in this paper.Up to now we have not seen any similar reports concerning the exothermic transition.展开更多
Impact statement We have developed the GReedy Accumulated strategy for Protein Engineering(GRAPE)to improve enzyme stability across various applications,combining advanced computational methods with a unique clusterin...Impact statement We have developed the GReedy Accumulated strategy for Protein Engineering(GRAPE)to improve enzyme stability across various applications,combining advanced computational methods with a unique clustering and greedy accumulation approach to efficiently explore epistatic effects with minimal experimental effort.To make this strategy accessible to nonexperts,we introduced GRAPE‐WEB,an automated,user‐friendly web server that allows the design,inspection,and combination of stabilizing mutations without requiring extensive bioinformatics knowledge.展开更多
Mineralization catalyzed by carbonic anhydrase(CA)is one of the most promising technologies for capturing CO_(2).In this work,Escherichia coli BL21(DE3)was used as the host,and the N-terminus of ice nucleation protein...Mineralization catalyzed by carbonic anhydrase(CA)is one of the most promising technologies for capturing CO_(2).In this work,Escherichia coli BL21(DE3)was used as the host,and the N-terminus of ice nucleation protein(INPN)was used as the carrier protein.Different fusion patterns and vectors were used to construct CA surface display systems forα-carbonic anhydrase(HPCA)from Helicobacter pylori 26695 andα-carbonic anhydrase(SazCA)from Sulfurihydrogenibium azorense.The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity,while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system.The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA.Cell frac-tionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell’s outer membrane as a fusion protein.The enzyme activity of the surface display strain E-22b-I RL S(11.43 U⋅mL^(−1) OD 600−1)was significantly higher than that of the intracellular expression strain E-22b-S(8.355 U⋅mL^(−1) OD 600−1)under optimized induction conditions.Compared with free SazCA,E-22b-I RL S had higher thermal and pH stability.The long-term stability of SazCA was also significantly improved by surface display.When the engineered strain and free enzyme were used for CO_(2) mineralization,the amount of CaCO_(3) deposition catalyzed by the strain E-22b-I RL S on the surface(241 mg)was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S(173 mg).These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO_(2) capture and mineralization.展开更多
Chitin,the second most abundant biopolymer on Earth,poses significant hydrolysis challenges due to its intrinsic insolubility and the stringent conditions required for its conversion to chitin oligosaccharides(COSs),w...Chitin,the second most abundant biopolymer on Earth,poses significant hydrolysis challenges due to its intrinsic insolubility and the stringent conditions required for its conversion to chitin oligosaccharides(COSs),which hold considerable industrial and biomedical potential.This study pioneers the use of deep eutectic solvents(DESs)to enhance the hydrolysis of chitin by the engineered chitinase PoChi-FnIII-ChBD(PCFC)from Penicillium oxalicum.DES,composed of hydrogen bond donors and acceptors,are noted for their low toxicity,biodegradability,and ease of preparation.Specifically,a DES synthesized from choline chloride and thiourea(ChCl-THU)significantly improved the efficiency and stability of PCFC.Under optimal conditions(40℃,pH 5.5),PCFC’s activity in 5 vol%ChCl-THU increased by 29.25%,with a 1.68-fold increase in thermal stability and a specific activity of 19.96 U/mg against insoluble chitin.This integrated approach combines chitin solubilization with enzymatic conversion,streamlining industrial processes and demonstrating DESs as sustainable media for enhanced COSs production.展开更多
基金Supported by National Natural Science Fundation of China
文摘The thermal stability of New Zealand culture rabbit muscle aldolase was investigated by differential scanning calorimetry in the water content range 0.23-3.70 g water per g protein.The experimental results showed that at water contents below 0.47g/g,an endothermic peak was observed and at water contents above 0.57g/g,an endothermic and an exothermic peak were both observed on DSC thermogram.Thermal denaturation result of the enzyme and the relationship between two transition temperatures and water contents were first reported in this paper.Up to now we have not seen any similar reports concerning the exothermic transition.
基金supported by the National Key R&D Program of China(grant no.2021YFC2103600)the National Natural Science Foundation of China(31822002,32170033,and 32422001)+2 种基金the Key Research Program of Frontier Sciences(ZDBS‐LY‐SM014)the Biological Resources Program(KFJBRP‐009 and KFJ‐BRP‐017‐58)from the Chinese Academy of Sciences,the Informatization Plan of Chinese Academy of Sciences(CAS‐WX2021SF‐0111)the Youth Innovation Promotion Association CAS(2022086).
文摘Impact statement We have developed the GReedy Accumulated strategy for Protein Engineering(GRAPE)to improve enzyme stability across various applications,combining advanced computational methods with a unique clustering and greedy accumulation approach to efficiently explore epistatic effects with minimal experimental effort.To make this strategy accessible to nonexperts,we introduced GRAPE‐WEB,an automated,user‐friendly web server that allows the design,inspection,and combination of stabilizing mutations without requiring extensive bioinformatics knowledge.
基金the financial support provided by the National Key Research and Development Program of China(Project No.2018YFA0902100)the National Natural Science Foundation of China(No.22178262,No.21576197).
文摘Mineralization catalyzed by carbonic anhydrase(CA)is one of the most promising technologies for capturing CO_(2).In this work,Escherichia coli BL21(DE3)was used as the host,and the N-terminus of ice nucleation protein(INPN)was used as the carrier protein.Different fusion patterns and vectors were used to construct CA surface display systems forα-carbonic anhydrase(HPCA)from Helicobacter pylori 26695 andα-carbonic anhydrase(SazCA)from Sulfurihydrogenibium azorense.The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity,while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system.The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA.Cell frac-tionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell’s outer membrane as a fusion protein.The enzyme activity of the surface display strain E-22b-I RL S(11.43 U⋅mL^(−1) OD 600−1)was significantly higher than that of the intracellular expression strain E-22b-S(8.355 U⋅mL^(−1) OD 600−1)under optimized induction conditions.Compared with free SazCA,E-22b-I RL S had higher thermal and pH stability.The long-term stability of SazCA was also significantly improved by surface display.When the engineered strain and free enzyme were used for CO_(2) mineralization,the amount of CaCO_(3) deposition catalyzed by the strain E-22b-I RL S on the surface(241 mg)was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S(173 mg).These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO_(2) capture and mineralization.
基金supported by the Project of the Natural Science Foundation of Hunan Province(project number:2022JJ30299)the Project of the Natural Science Foundation of Hunan Province(project number:2022JJ30290).
文摘Chitin,the second most abundant biopolymer on Earth,poses significant hydrolysis challenges due to its intrinsic insolubility and the stringent conditions required for its conversion to chitin oligosaccharides(COSs),which hold considerable industrial and biomedical potential.This study pioneers the use of deep eutectic solvents(DESs)to enhance the hydrolysis of chitin by the engineered chitinase PoChi-FnIII-ChBD(PCFC)from Penicillium oxalicum.DES,composed of hydrogen bond donors and acceptors,are noted for their low toxicity,biodegradability,and ease of preparation.Specifically,a DES synthesized from choline chloride and thiourea(ChCl-THU)significantly improved the efficiency and stability of PCFC.Under optimal conditions(40℃,pH 5.5),PCFC’s activity in 5 vol%ChCl-THU increased by 29.25%,with a 1.68-fold increase in thermal stability and a specific activity of 19.96 U/mg against insoluble chitin.This integrated approach combines chitin solubilization with enzymatic conversion,streamlining industrial processes and demonstrating DESs as sustainable media for enhanced COSs production.
