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▼
