As the utilization of maltodextrins in food,pharmaceuticals,and agriculture continues to expand,substantial research has been conducted to enhance their functionality.Among the methods presented,the one-pot approach f...As the utilization of maltodextrins in food,pharmaceuticals,and agriculture continues to expand,substantial research has been conducted to enhance their functionality.Among the methods presented,the one-pot approach for the synthesis of nonreducing maltoheptaose offers a novel solution to the challenge of maltodextrins with varying degrees of polymerization and reducing ends.Nevertheless,the key enzyme in this method was currently only expressed in Escherichia coli,which restricts the applicability of this method in the food and pharmaceutical industries.In this study,the food-grade expression of cyclomaltodextrinase(CDase,EC 3.2.1.54),one of the key enzymes,was achieved using Bacillus subtilis as a host.The enzymatic properties of the recombinant CDase were then investigated,and the extracellular secretion of the CDase was enhanced in order to make it more widely available for use in the food industry.The enzyme exhibited optimal activity at a pH of 8.0 and a temperature range of 35-45℃.After incubation at 25-35℃for 10 h,90%of the enzyme activity was retained.Additionally,the enzyme retained 80%of its initial activity after 24 h at pH 5.5-9.5.Finally,Cu2+completely inhibited the enzyme activity.The extracellular secretion efficiency of recombinant CDase was significantly increased by the addition of Mn^(2+)to the fermentation medium.The percentage of extracellular enzyme activity increased to 63.75%when the final concentration of Mn^(2+)in the fermentation medium was 5 mM,which was 5.3-fold higher than that of the unadded one.展开更多
Cyclomaltodextrinase (CDase, EC 3.2.1.54) is used in the preparation of maltooligosaccharides with a specific degree of polymerization by hydrolyzing cyclodextrins. Extracellular secretion of CDase is beneficial in pr...Cyclomaltodextrinase (CDase, EC 3.2.1.54) is used in the preparation of maltooligosaccharides with a specific degree of polymerization by hydrolyzing cyclodextrins. Extracellular secretion of CDase is beneficial in promoting its application potential. However, a major barrier is that proteins are difficult to cross the inner and outer membranes of cells. Herein, we made an effort to increase the permeability of cell membrane by the deletion of lipopolysaccharide synthesis-related gene (lpxM ). In addition, glycine and Ca^(2+) assisted in the extracellular secretion of recombinant CDase from Bacillus sphaericus E−244. The extracellular fermentation activity of CDase was 2.88 U/mL when ΔycjM -ΔmalS -ΔlpxM was cultured with 1 mM glycine and 5 mM Ca^(2+) for 20 h, accounting for 78.67% of the total fermentation activity, which was 6.26-fold and 6.45-fold higher than that of the parent strain, respectively. Glycine increased cell lysis and disrupted cell morphology, and Ca^(2+) repaired glycine-induced cell growth inhibition. Significantly, deletion of lpxM replaced the use of glycine to a considerable extent, and half the CDase extracellular secretion time.展开更多
文摘As the utilization of maltodextrins in food,pharmaceuticals,and agriculture continues to expand,substantial research has been conducted to enhance their functionality.Among the methods presented,the one-pot approach for the synthesis of nonreducing maltoheptaose offers a novel solution to the challenge of maltodextrins with varying degrees of polymerization and reducing ends.Nevertheless,the key enzyme in this method was currently only expressed in Escherichia coli,which restricts the applicability of this method in the food and pharmaceutical industries.In this study,the food-grade expression of cyclomaltodextrinase(CDase,EC 3.2.1.54),one of the key enzymes,was achieved using Bacillus subtilis as a host.The enzymatic properties of the recombinant CDase were then investigated,and the extracellular secretion of the CDase was enhanced in order to make it more widely available for use in the food industry.The enzyme exhibited optimal activity at a pH of 8.0 and a temperature range of 35-45℃.After incubation at 25-35℃for 10 h,90%of the enzyme activity was retained.Additionally,the enzyme retained 80%of its initial activity after 24 h at pH 5.5-9.5.Finally,Cu2+completely inhibited the enzyme activity.The extracellular secretion efficiency of recombinant CDase was significantly increased by the addition of Mn^(2+)to the fermentation medium.The percentage of extracellular enzyme activity increased to 63.75%when the final concentration of Mn^(2+)in the fermentation medium was 5 mM,which was 5.3-fold higher than that of the unadded one.
基金supported by the National Natural Science Foundation of China[grant number 31871745]Major Science and Technology Innovation Project of Shandong Province[grant number 2020CXGC010601].
文摘Cyclomaltodextrinase (CDase, EC 3.2.1.54) is used in the preparation of maltooligosaccharides with a specific degree of polymerization by hydrolyzing cyclodextrins. Extracellular secretion of CDase is beneficial in promoting its application potential. However, a major barrier is that proteins are difficult to cross the inner and outer membranes of cells. Herein, we made an effort to increase the permeability of cell membrane by the deletion of lipopolysaccharide synthesis-related gene (lpxM ). In addition, glycine and Ca^(2+) assisted in the extracellular secretion of recombinant CDase from Bacillus sphaericus E−244. The extracellular fermentation activity of CDase was 2.88 U/mL when ΔycjM -ΔmalS -ΔlpxM was cultured with 1 mM glycine and 5 mM Ca^(2+) for 20 h, accounting for 78.67% of the total fermentation activity, which was 6.26-fold and 6.45-fold higher than that of the parent strain, respectively. Glycine increased cell lysis and disrupted cell morphology, and Ca^(2+) repaired glycine-induced cell growth inhibition. Significantly, deletion of lpxM replaced the use of glycine to a considerable extent, and half the CDase extracellular secretion time.
