In this study,pea starch was modified with maltogenicα-amylase and then its physicochemical properties,chain length distribution,and in vitro digestibility were examined.An analysis of the starch products resulting f...In this study,pea starch was modified with maltogenicα-amylase and then its physicochemical properties,chain length distribution,and in vitro digestibility were examined.An analysis of the starch products resulting from the maltogenicα-amylase treatment revealed a decrease in the molecular weight,an increase in the proportion of short chains[degree of polymerization(DP)<13],a decrease in the proportion of long chains(DP>13),and a decrease in viscosity,resulting in slower digestion.The results indicated that maltogenicα-amylase can selectively hydrolyzeα-1,4-andα-1,6-glycosides to some extent.Cleaving theα-1,4 linkage of starch facilitated the transfer of the non-reducing D-glucosyl residues of maltose,thereby generating anα-1,6 branch linkage.The reaction between maltogenicα-amylase and pea starch was affected by the reaction time and enzyme concentration.The maximum slowly digestible starch content(38.5%)was obtained by digesting samples with 60.0 ppm maltogenicα-amylase for 4.0 h.These findings demonstrate that modifying pea starch using maltogenicα-amylase can generate starch products with new branching structures conducive to slow digestion.展开更多
A Lactobacillus sp.was screened from various cereal sourdoughs and was designated as Lactobacillus plantarum YXY418 based on the 16S rRNA gene analysis.A putative Lactobacillus plantarum maltogenic amylase,LpMA,was di...A Lactobacillus sp.was screened from various cereal sourdoughs and was designated as Lactobacillus plantarum YXY418 based on the 16S rRNA gene analysis.A putative Lactobacillus plantarum maltogenic amylase,LpMA,was discovered based on computer-aided analysis.Then,its encoding gene(lpma)was expressed in E.coli BL21(DE3).The expressed recombinant LpMA(reLpMA)was efficiently purified to 12.2-fold using the one-step nickel-nitrilotriacetic acid(Ni–NTA)affinity chromatography.The final recovery yield and specific activity of the purified reLpMA were 61%and 36.4 U/mg towards soluble starch,respectively.The purified reLpMA exhibited optimal amylolytic activity towards soluble starch at 45℃ and pH 6.0,with a good pH stability ranging from pH 5.0 to 8.0.Besides,the reLpMA also hydrolyzed soluble starch,β-CD and pullulan to maltose with specific activity of 96.4 SU/mL,78.2 CU/mL and 2.0 PU/mL,respectively.The reLpMA hydrolytic activity was increased in the presence of metal ions especiallyCa^(2+)andZn^(2+),which could be applied to different processing processes.Baking test indicated after 7-day storage,the reLpMA at a dosage of 2000 U/300 g could significantly reduce hardness and chewiness by 29.5%and 26.4%,respectively,compared with the control.Adding reLpMA improved bread quality,increased bread volume and decreased hardness during storage,thus extending its shelf life.展开更多
The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates.Here,the specific activity of a maltoge...The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates.Here,the specific activity of a maltogenic amylase from Lactobacillus rhamnosus YXY412(LrMA)toward soluble starch was exactly enhanced through hotspot-based research.Based on multiple sequence alignment,three-dimensional structure and existed literature,thirty-eight amino acid residues of LrMA were rationally selected for site-directed mutagenesis.After the screening of the mutants,LrMA^(D172A),LrMA^(G260A),LrMA^(K334A)and LrMA^(M477A)were selected with the activity accounted for 144-209% of that in wild-type.Among all the mutants,LrMA^(G260A) possessed the highest activity toward soluble starch,reached 133 U/mg,about twice as high as that in the wild-type.Its temperature for optimum activity still maintained at 60℃,while had no significant loss of thermal stability occurred.In addition,compared with the wild-type in pH stability,the mutant retained over 80% residual activity at a wider pH range of 4.5-8.5.Furthermore,the k_(cat)/K_(m)of LrMA^(G260A) was two times higher than that of the wild-type,indicating that the mutant had a better affinity and a higher conversion efficiency for soluble starch.展开更多
基金supported by the Science and Technology Program under Taizhou Municipal Government(Project No.TN202005)the Science and Technology Program of Jiangsu Agri-animal Husbandry Vocational College(NSF2021ZR04)the Taizhou Municipal 311 Personnel Training Program(2017-1-42),and the Jiangsu Provincial Qing Lan Project.
文摘In this study,pea starch was modified with maltogenicα-amylase and then its physicochemical properties,chain length distribution,and in vitro digestibility were examined.An analysis of the starch products resulting from the maltogenicα-amylase treatment revealed a decrease in the molecular weight,an increase in the proportion of short chains[degree of polymerization(DP)<13],a decrease in the proportion of long chains(DP>13),and a decrease in viscosity,resulting in slower digestion.The results indicated that maltogenicα-amylase can selectively hydrolyzeα-1,4-andα-1,6-glycosides to some extent.Cleaving theα-1,4 linkage of starch facilitated the transfer of the non-reducing D-glucosyl residues of maltose,thereby generating anα-1,6 branch linkage.The reaction between maltogenicα-amylase and pea starch was affected by the reaction time and enzyme concentration.The maximum slowly digestible starch content(38.5%)was obtained by digesting samples with 60.0 ppm maltogenicα-amylase for 4.0 h.These findings demonstrate that modifying pea starch using maltogenicα-amylase can generate starch products with new branching structures conducive to slow digestion.
基金supported by the National Natural Science Foundation of China(21676117)the Natural Science Foundation of Jiangsu Province for Youth of China(No.BK20180622).
文摘A Lactobacillus sp.was screened from various cereal sourdoughs and was designated as Lactobacillus plantarum YXY418 based on the 16S rRNA gene analysis.A putative Lactobacillus plantarum maltogenic amylase,LpMA,was discovered based on computer-aided analysis.Then,its encoding gene(lpma)was expressed in E.coli BL21(DE3).The expressed recombinant LpMA(reLpMA)was efficiently purified to 12.2-fold using the one-step nickel-nitrilotriacetic acid(Ni–NTA)affinity chromatography.The final recovery yield and specific activity of the purified reLpMA were 61%and 36.4 U/mg towards soluble starch,respectively.The purified reLpMA exhibited optimal amylolytic activity towards soluble starch at 45℃ and pH 6.0,with a good pH stability ranging from pH 5.0 to 8.0.Besides,the reLpMA also hydrolyzed soluble starch,β-CD and pullulan to maltose with specific activity of 96.4 SU/mL,78.2 CU/mL and 2.0 PU/mL,respectively.The reLpMA hydrolytic activity was increased in the presence of metal ions especiallyCa^(2+)andZn^(2+),which could be applied to different processing processes.Baking test indicated after 7-day storage,the reLpMA at a dosage of 2000 U/300 g could significantly reduce hardness and chewiness by 29.5%and 26.4%,respectively,compared with the control.Adding reLpMA improved bread quality,increased bread volume and decreased hardness during storage,thus extending its shelf life.
基金supported by the Postdoctoral Science Foundation of China(2021M691278)the National Key Special Project for the 13th National 5-Year Plan Program of China(2016YFD0400500).
文摘The semi-rational design of enzymes has become a popular and effective modification method to improve their hydrolytic activity and/or thermal stability toward target substrates.Here,the specific activity of a maltogenic amylase from Lactobacillus rhamnosus YXY412(LrMA)toward soluble starch was exactly enhanced through hotspot-based research.Based on multiple sequence alignment,three-dimensional structure and existed literature,thirty-eight amino acid residues of LrMA were rationally selected for site-directed mutagenesis.After the screening of the mutants,LrMA^(D172A),LrMA^(G260A),LrMA^(K334A)and LrMA^(M477A)were selected with the activity accounted for 144-209% of that in wild-type.Among all the mutants,LrMA^(G260A) possessed the highest activity toward soluble starch,reached 133 U/mg,about twice as high as that in the wild-type.Its temperature for optimum activity still maintained at 60℃,while had no significant loss of thermal stability occurred.In addition,compared with the wild-type in pH stability,the mutant retained over 80% residual activity at a wider pH range of 4.5-8.5.Furthermore,the k_(cat)/K_(m)of LrMA^(G260A) was two times higher than that of the wild-type,indicating that the mutant had a better affinity and a higher conversion efficiency for soluble starch.
