This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using erro...This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, two mutants of H260L and D84A/H260L showed enhanced thermal stability than the wild-type predecessor (WT). Site-directed mutagenesis demonstrated that only amino acid residue at Position 260 plays an important role in the thermostability of P. carrageenovora arylsulfatase. Thermal inactivation analysis showed that the half-life (t1/2) values at 55°C for H260L, H260I, H260Q, H260F and H260R were 40.6, 48.4, 30.9, 29.1 and 34.5 min, respectively, while that of WT was 9.1 min. Structure modeling demonstrated that the additional hydrogen bonds and/or optimization of surface charge-charge interactions could be responsible for the increased thermostability imparted by H260L, H260I, H260Q, H260F and H260R.展开更多
By using the methods of literature analysis,field investigation,exploration and research,experience summary,comparative inductive analysis,etc.,through the search of the latest literature and policy materials,producti...By using the methods of literature analysis,field investigation,exploration and research,experience summary,comparative inductive analysis,etc.,through the search of the latest literature and policy materials,production enterprises and domestic application-based undergraduate universities with related majors have been investigated on the spot.By analyzing the failed cases and drawing lessons from the successful experience,this paper explores the transformation and practical construction of the biopharmaceutical training base in application-based colleges and universities.This paper innovates and designs a new construction mode of biopharmaceutical training base in application-based universities,which provides reliable experience and valuable research results for the construction or transformation of biopharmaceutical training base in application-based colleges and universities.It has a very important potential value for promoting the social and economic benefits.展开更多
An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was express...An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.展开更多
基金The National Natural Science Foundation of China under contract No.31401632the Program for New Century Excellent Talents in Fujian Province University,China under contract No.B15139
文摘This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, two mutants of H260L and D84A/H260L showed enhanced thermal stability than the wild-type predecessor (WT). Site-directed mutagenesis demonstrated that only amino acid residue at Position 260 plays an important role in the thermostability of P. carrageenovora arylsulfatase. Thermal inactivation analysis showed that the half-life (t1/2) values at 55°C for H260L, H260I, H260Q, H260F and H260R were 40.6, 48.4, 30.9, 29.1 and 34.5 min, respectively, while that of WT was 9.1 min. Structure modeling demonstrated that the additional hydrogen bonds and/or optimization of surface charge-charge interactions could be responsible for the increased thermostability imparted by H260L, H260I, H260Q, H260F and H260R.
基金2020 New Engineering Research and Reform Practice Project in Fujian Province[(MinJiaoGao(2020)4-59)]Research Project on General Education and Teaching Reform of Universities in Fujian Province(FBJG20190201,FBJG20200178)+1 种基金the 13 th Five-Year Plan of Education and Science in Xiamen City(1926)Education and Teaching Reform Research Project of Xiamen Medical College(XBGL2019002,XBJG2020002,XBGL2019003,XBGL2019005).
文摘By using the methods of literature analysis,field investigation,exploration and research,experience summary,comparative inductive analysis,etc.,through the search of the latest literature and policy materials,production enterprises and domestic application-based undergraduate universities with related majors have been investigated on the spot.By analyzing the failed cases and drawing lessons from the successful experience,this paper explores the transformation and practical construction of the biopharmaceutical training base in application-based colleges and universities.This paper innovates and designs a new construction mode of biopharmaceutical training base in application-based universities,which provides reliable experience and valuable research results for the construction or transformation of biopharmaceutical training base in application-based colleges and universities.It has a very important potential value for promoting the social and economic benefits.
基金The Natural Science Foundation of Fujian Province of China under contract No.2016J01162the Program for New Century Excellent Talents in Fujian Province University,China under contract No.B15139
文摘An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.