Agarases are hydrolytic enzymes that act on the hydrolysis of agar and have a broad range of applications in food,cosmetics and pharmaceutical industries. In this study, a glycerol feeding strategy based on induction ...Agarases are hydrolytic enzymes that act on the hydrolysis of agar and have a broad range of applications in food,cosmetics and pharmaceutical industries. In this study, a glycerol feeding strategy based on induction mode optimization for high cell density and β-agarase production was established, which could effectively control acetate yield. First, exponential feeding strategy of glycerol with different overall specific growth rates（μ） was applied in the pre-induction phase. The results showed that the low μ（μ=0.2） was suggested to be the optimal for cell growth and β-agarase production. Second, the effects of induction temperature and the inducer concentration on cell growth and β-agarase production were investigated in the post-induction phase. When induced by isopropyl-β-d-thiogalactoside（IPTG）, the strategy of 0.8 mmol/L IPTG induction at 20℃ was found to be optimal for β-agarase production. When cultivation was induced by continuous lactose feeding strategy of 1.0 g/（L·h）, the β-agarase activity reached 112.5 U/mL, which represented the highest β-agarase production to date.Furthermore, the β-agarase was capable of degrading G. lemaneiformis powder directly to produce neoagarooligosaccharide, and the hydrolysates were neoagarotetraose（NA4） and neoagarohexaose（NA6）. The overall research may be useful for the industrial production and application of β-agarase.展开更多
Neoagarooligosaccharides(NAOS),derived from the hydrolysis of agarose by β-agarases,have been found to have various biological activities.Herein,AgaDcat,originated from marine agar-degrading bacterium Zobellia galact...Neoagarooligosaccharides(NAOS),derived from the hydrolysis of agarose by β-agarases,have been found to have various biological activities.Herein,AgaDcat,originated from marine agar-degrading bacterium Zobellia galactanivorans,was for the first time successfully recombined and expressed in a eukaryotic(Pichia pastoris)system.Compared with other endo-β-agarase,AgaDcat has potential applications in producing neoagarotetraose(NA4)and neoagarohexaose(NA6)effectively because of its product specificity.The optimal temperature and pH of AgaDcat expressed in P.pastoris were 50℃ and 7.0,respectively.The maximum hydrolysis of agarose was achieved after 8h reaction at 45℃ and pH 7.0 using 750 U/g β-agarase.Through regulating hydrolysis conditions,NAOS with controllable degrees of polymerization(mainly NA4 and NA6)could be obtained.The Km value of AgaDcat expressed in P.pastoris was 1.377 mg/mL,which was significantly lower than that of AgaDcat expressed in Escherichia coli(6.358 mg/mL).Moreover,the melting temperature of AgaDcat expressed in P.pastoris(53.1℃)was 4.5℃ higher than that of AgaDcat expressed in E.coli.AgaDcat expressed in P.pastoris exhibited higher thermal stability and substrate affinity than that expressed in E.coli,which could be ascribed to the glycosylation of proteins during translation modification period in the yeast expression system.展开更多
基金The Public Science and Technology Research Funds Projects of Ocean under contract No.201505026the Fujian Province Natural Science Foundation under contract Nos 2016J01160 and 2017N0015the Scientific Research Foundation of Third Institute of Oceanography,SOA under contract No.2016038
文摘Agarases are hydrolytic enzymes that act on the hydrolysis of agar and have a broad range of applications in food,cosmetics and pharmaceutical industries. In this study, a glycerol feeding strategy based on induction mode optimization for high cell density and β-agarase production was established, which could effectively control acetate yield. First, exponential feeding strategy of glycerol with different overall specific growth rates（μ） was applied in the pre-induction phase. The results showed that the low μ（μ=0.2） was suggested to be the optimal for cell growth and β-agarase production. Second, the effects of induction temperature and the inducer concentration on cell growth and β-agarase production were investigated in the post-induction phase. When induced by isopropyl-β-d-thiogalactoside（IPTG）, the strategy of 0.8 mmol/L IPTG induction at 20℃ was found to be optimal for β-agarase production. When cultivation was induced by continuous lactose feeding strategy of 1.0 g/（L·h）, the β-agarase activity reached 112.5 U/mL, which represented the highest β-agarase production to date.Furthermore, the β-agarase was capable of degrading G. lemaneiformis powder directly to produce neoagarooligosaccharide, and the hydrolysates were neoagarotetraose（NA4） and neoagarohexaose（NA6）. The overall research may be useful for the industrial production and application of β-agarase.
文摘Neoagarooligosaccharides(NAOS),derived from the hydrolysis of agarose by β-agarases,have been found to have various biological activities.Herein,AgaDcat,originated from marine agar-degrading bacterium Zobellia galactanivorans,was for the first time successfully recombined and expressed in a eukaryotic(Pichia pastoris)system.Compared with other endo-β-agarase,AgaDcat has potential applications in producing neoagarotetraose(NA4)and neoagarohexaose(NA6)effectively because of its product specificity.The optimal temperature and pH of AgaDcat expressed in P.pastoris were 50℃ and 7.0,respectively.The maximum hydrolysis of agarose was achieved after 8h reaction at 45℃ and pH 7.0 using 750 U/g β-agarase.Through regulating hydrolysis conditions,NAOS with controllable degrees of polymerization(mainly NA4 and NA6)could be obtained.The Km value of AgaDcat expressed in P.pastoris was 1.377 mg/mL,which was significantly lower than that of AgaDcat expressed in Escherichia coli(6.358 mg/mL).Moreover,the melting temperature of AgaDcat expressed in P.pastoris(53.1℃)was 4.5℃ higher than that of AgaDcat expressed in E.coli.AgaDcat expressed in P.pastoris exhibited higher thermal stability and substrate affinity than that expressed in E.coli,which could be ascribed to the glycosylation of proteins during translation modification period in the yeast expression system.
