A Glycoside hydrolase(GH) typically contains one catalytic module and varied non-catalytic regions(NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding mod...A Glycoside hydrolase(GH) typically contains one catalytic module and varied non-catalytic regions(NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules(CBMs). Aga G4 is a GH16 endo-β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, aga G4-T140, in Escherichia coli. After purification and refolding, the truncated agarase r Aga G4-T140 retained the same catalytic temperature and p H value as r Aga G4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by r Aga G4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53:1 and a conversion ratio of approximately 70%, which were similar to those of r Aga G4. However, the truncated agarase r Aga G4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of r Aga G4-T140 was approximately 25 times the weight of that produced by equimolar r Aga G4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase Aga G4 and implies some new strategies to improve the properties of a GH enzyme.展开更多
Objective: Based on a partialsubtilisin-like protease, Pr1 genomic sequence of Pythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to th...Objective: Based on a partialsubtilisin-like protease, Pr1 genomic sequence of Pythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Pr1gene. Methods: The genomic DNA was firstly digested with BamHⅠ and then treated with calf intestinal alkaline phosphatase(CIAP). Next, a 5′ phosphorylated oligonucleotide was ligated to the 5′ ends of BamHⅠ-digested DNA. After denaturation, intrastrand annealing and polymerase extension, a pan with a handle was formed,and lastly the nested PCR was performed. Results: A 864 bp product was amplified,which was adjacent to the known sequence of Pr1 gene.The gene has been accessed by GenBank(Accession:JQ975036). Conclusion: Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes,which facilitates cloning full-length Pr1 gene.展开更多
基金financially supported by the Open Research Fund Program of Shandong Provincial Key Laboratory of Glycoscience&Glycotechnology(Ocean University of China)KLGG(OUC)201301the National Natural Science Foundation of China Grants 31300664 and 31130004the State Key Laboratory of Microbial Technology Grant(Shandong University)M2013-11
文摘A Glycoside hydrolase(GH) typically contains one catalytic module and varied non-catalytic regions(NCRs). However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules(CBMs). Aga G4 is a GH16 endo-β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, aga G4-T140, in Escherichia coli. After purification and refolding, the truncated agarase r Aga G4-T140 retained the same catalytic temperature and p H value as r Aga G4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by r Aga G4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53:1 and a conversion ratio of approximately 70%, which were similar to those of r Aga G4. However, the truncated agarase r Aga G4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of r Aga G4-T140 was approximately 25 times the weight of that produced by equimolar r Aga G4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase Aga G4 and implies some new strategies to improve the properties of a GH enzyme.
基金Supported by the Guangxi Department of education scientific research funds,China(No.200103YB154)
文摘Objective: Based on a partialsubtilisin-like protease, Pr1 genomic sequence of Pythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Pr1gene. Methods: The genomic DNA was firstly digested with BamHⅠ and then treated with calf intestinal alkaline phosphatase(CIAP). Next, a 5′ phosphorylated oligonucleotide was ligated to the 5′ ends of BamHⅠ-digested DNA. After denaturation, intrastrand annealing and polymerase extension, a pan with a handle was formed,and lastly the nested PCR was performed. Results: A 864 bp product was amplified,which was adjacent to the known sequence of Pr1 gene.The gene has been accessed by GenBank(Accession:JQ975036). Conclusion: Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes,which facilitates cloning full-length Pr1 gene.
