Deletion of a Non-Catalytic Region Increases the Enzymatic Activity of a β-Agarase from Flammeovirga sp. MY04

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.

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）. AgaG4 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, agaG4-T140, in Escherichia coli. After purification and refolding, the trtmcated agarase rAgaG4-T140 retained the same catalytic temperature and pH value as rAgaG4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by rAgaG4-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 rAgaG4. However, the truncated agarase rAgaG4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of rAgaG4-T140 was approximately 25 times the weight of that produced by equimolar rAgaG4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase AgaG4 and implies some new strategies to improve the properties of a GH enzyme.