The valorization of starch-rich materials typically requires high-temperature gelatinization prior to enzymatic saccharification to convert the starch into usable sugars,which are subsequently transformed into biochem...The valorization of starch-rich materials typically requires high-temperature gelatinization prior to enzymatic saccharification to convert the starch into usable sugars,which are subsequently transformed into biochemical products.To bypass the energy-intensive gelatinization step,effective thermostable enzymes capable of degrading raw starch are essential.In this work,a raw starch-digesting amylolytic enzyme system was produced by a novel isolate,Priestia koreensis HL12 using sago starch as a carbon source at 30◦C for 48 h.The crude enzyme demonstrated thermophilic properties,exhibiting robust amylase activity across a broad temperature range(30-100◦C),with the highest amylase activity(168.05 U/mg protein)at 65◦C in 50 mM sodium phos-phate buffer pH 6 toward soluble starch.The enzyme efficiently hydrolyzed both raw and gelatinized starches,predominantly yielding maltooligosaccharides(DP3 and DP5)as valuable prebiotics,without producing glucose.The highest sugar yield was obtained from gelatinized potato starch,with 841.58 mg/g substrate(84%con-version),while cassava pulp hydrolysis below the gelatinization temperature achieved a 57.32%conversion,producing 434.65 mg/g maltopentaose as majority equivalent to 75.8%of product mixture.This non-thermal saccharification process without glucose byproduct has significant potential for prebiotic production,adding value to starch-rich agricultural byproducts andcontributing to a sustainable,zero-waste starch industry.展开更多
“Pinhão”, the seed of Araucaria angustifolia, is an important food, being part of the eating habits of Indigenous communities. In this study, we evaluated the oligosaccharide content, resistant starch and the g...“Pinhão”, the seed of Araucaria angustifolia, is an important food, being part of the eating habits of Indigenous communities. In this study, we evaluated the oligosaccharide content, resistant starch and the growth of probiotic bacteria. GF4 (1-fructofuranosylnystose) was the main fructo-oligosaccharides found, in higher contents compared to other food sources. Maltooligosaccharides (MOS) represented the main part of the oligosaccharides profile of Brazilian pine seeds. In descending order of importance was maltoheptaose (G7), maltohexose (G6) and maltotriose (G3). The starches from the variety Sanct josephi presented the highest amount of resistant starch that could stimulate probiotic strains, mainly B. breve and L. plantarum, and may have a prebiotic effect, potentially promoting health benefits. This study advances the understanding of the chemical composition of the main portion of the “pinhão” enhancing awareness of its potential as a healthy food source, contributing to different uses and indirectly with the species preservation.展开更多
α-Glucosidase hydrolyzesα-D-glucosides to produceα-D-glucose.Glycoside hydrolase family 13(GH13)containsα-glucosidases together with various amylolytic enzymes.GH13α-glucosidases fall into several distinct sub-fa...α-Glucosidase hydrolyzesα-D-glucosides to produceα-D-glucose.Glycoside hydrolase family 13(GH13)containsα-glucosidases together with various amylolytic enzymes.GH13α-glucosidases fall into several distinct sub-families,and subfamily 30(GH13_30)includes numerous Actinomycetesα-glucosidases.GH13_30α-glucosidase CmmB from Arthrobacter globiformis,specific to maltooligosaccharides,is involved in the intracellular meta-bolism of cyclobis-(1→6)-α-maltosyl.Herein,the function and structure of CmmB were investigated to advance understanding of the structure-function relationship.CmmB showed the highest k_(cat)/K_(m)for maltose of mal-tooligosaccharides,and k_(cat)/K_(m)drastically decreased with increasing substrate chain-length.The crystal struc-tures of CmmB in complex with a pseudodisaccharide(acarviosin)and pseudotetrasaccharide(acarbose)were determined at resolutions of 1.60 and 1.70Å,respectively.The overall structure of CmmB was typical of a GH13α-glucosidase.Most of the structure of theβ→αloop 7 of the catalytic(β/α)8-barrel domain was not determined in the acarbose complex,but the C-terminal side of this loop was modeled in the acarviosin complex.For binding to acarviosin,this loop took on a closed conformation,and I360 and R364 on this loop formed subsite+1 together with H224 and W280.Alanine substitution of these residues indicated that R364 was essential for the catalysis through a hydrogen bond with the O6 of the D-glucose residue in subsite+1.Theβ→αloop 7 is flexible upon binding to substrates,but I360 and R364 cannot participate in binding to maltooligosaccharides longer than maltose.The loss of interactions with these residues was concluded to result in the low preference for maltotriose and longer maltooligosaccharides.展开更多
The current study deals with cloning and expressing a maltogenic α-amylase gene from thermophilic Bacillus stercoris YSP18(AmyYSP)in Escherichia coli BL21(DE3).AmyYSP belongs to the GH13_20 subfamily of Glycoside Hyd...The current study deals with cloning and expressing a maltogenic α-amylase gene from thermophilic Bacillus stercoris YSP18(AmyYSP)in Escherichia coli BL21(DE3).AmyYSP belongs to the GH13_20 subfamily of Glycoside Hydrolases and entails five conserved regions found in maltogenic α-amylases.As a monomer of 67 kDa,AmyYSP exhibits maximal activity at 80℃,pH 5.0 and retains>75%residual activity at 70-100℃ and pH 5.0-8.0.The kinetic and thermodynamic studies displayed that it has a high affinity for soluble starch(K_(m)=1.54±0.236 mgmL^(−1)),exhibits a longer half-life(38.5 h at 80℃ and 8.88 h at 100℃),and a higher EaD value of 3824±1.03 kJ mol^(−1).It was characterised as a Ca^(2+)-independent α-amylase,resistant to various denaturing additives.It hydrolyses soluble starch and raw corn starch efficiently,liberating glucose,maltose,maltotriose and maltotetraose as the main products.The thermostable and acid-stable,maltooligosaccharide forming AmyYSP is a versatile enzyme with prerequisites for successful application in starch-saccharification industries.展开更多
基金financially supported by Thammasat University Research Fund(Contract No.TUFT 4/2566)the 30st Science&Technology Research Grant from Thailand Toray Science Foundation.This work was also supported by the National Science,Research and Innovation Fund,Thailand Science Research and Innovation(TSRI)(Grant No.:FFB680075/0337).
文摘The valorization of starch-rich materials typically requires high-temperature gelatinization prior to enzymatic saccharification to convert the starch into usable sugars,which are subsequently transformed into biochemical products.To bypass the energy-intensive gelatinization step,effective thermostable enzymes capable of degrading raw starch are essential.In this work,a raw starch-digesting amylolytic enzyme system was produced by a novel isolate,Priestia koreensis HL12 using sago starch as a carbon source at 30◦C for 48 h.The crude enzyme demonstrated thermophilic properties,exhibiting robust amylase activity across a broad temperature range(30-100◦C),with the highest amylase activity(168.05 U/mg protein)at 65◦C in 50 mM sodium phos-phate buffer pH 6 toward soluble starch.The enzyme efficiently hydrolyzed both raw and gelatinized starches,predominantly yielding maltooligosaccharides(DP3 and DP5)as valuable prebiotics,without producing glucose.The highest sugar yield was obtained from gelatinized potato starch,with 841.58 mg/g substrate(84%con-version),while cassava pulp hydrolysis below the gelatinization temperature achieved a 57.32%conversion,producing 434.65 mg/g maltopentaose as majority equivalent to 75.8%of product mixture.This non-thermal saccharification process without glucose byproduct has significant potential for prebiotic production,adding value to starch-rich agricultural byproducts andcontributing to a sustainable,zero-waste starch industry.
文摘“Pinhão”, the seed of Araucaria angustifolia, is an important food, being part of the eating habits of Indigenous communities. In this study, we evaluated the oligosaccharide content, resistant starch and the growth of probiotic bacteria. GF4 (1-fructofuranosylnystose) was the main fructo-oligosaccharides found, in higher contents compared to other food sources. Maltooligosaccharides (MOS) represented the main part of the oligosaccharides profile of Brazilian pine seeds. In descending order of importance was maltoheptaose (G7), maltohexose (G6) and maltotriose (G3). The starches from the variety Sanct josephi presented the highest amount of resistant starch that could stimulate probiotic strains, mainly B. breve and L. plantarum, and may have a prebiotic effect, potentially promoting health benefits. This study advances the understanding of the chemical composition of the main portion of the “pinhão” enhancing awareness of its potential as a healthy food source, contributing to different uses and indirectly with the species preservation.
基金The synchrotron radiation experiments were performed at the BL45XU of SPring-8 and BL-5A of Photon Factory with the approval of the Japan Synchrotron Radiation Research Institute(JASRI)(Proposal No.2020A2555 and 2021A2561 for SPring-8and 2023G101 for Photon Factory)supported in part by the Platform Project for Supporting Drug Discovery and Life Science Research[Basis for Supporting Innovative Drug Discovery and Life Science Research(BINDS)](to M.Y.)from the Japan Agency for Medical Research and Development(AMED)under Grant Number JP18am0101071(support number 0058)and JP19am0101083.
文摘α-Glucosidase hydrolyzesα-D-glucosides to produceα-D-glucose.Glycoside hydrolase family 13(GH13)containsα-glucosidases together with various amylolytic enzymes.GH13α-glucosidases fall into several distinct sub-families,and subfamily 30(GH13_30)includes numerous Actinomycetesα-glucosidases.GH13_30α-glucosidase CmmB from Arthrobacter globiformis,specific to maltooligosaccharides,is involved in the intracellular meta-bolism of cyclobis-(1→6)-α-maltosyl.Herein,the function and structure of CmmB were investigated to advance understanding of the structure-function relationship.CmmB showed the highest k_(cat)/K_(m)for maltose of mal-tooligosaccharides,and k_(cat)/K_(m)drastically decreased with increasing substrate chain-length.The crystal struc-tures of CmmB in complex with a pseudodisaccharide(acarviosin)and pseudotetrasaccharide(acarbose)were determined at resolutions of 1.60 and 1.70Å,respectively.The overall structure of CmmB was typical of a GH13α-glucosidase.Most of the structure of theβ→αloop 7 of the catalytic(β/α)8-barrel domain was not determined in the acarbose complex,but the C-terminal side of this loop was modeled in the acarviosin complex.For binding to acarviosin,this loop took on a closed conformation,and I360 and R364 on this loop formed subsite+1 together with H224 and W280.Alanine substitution of these residues indicated that R364 was essential for the catalysis through a hydrogen bond with the O6 of the D-glucose residue in subsite+1.Theβ→αloop 7 is flexible upon binding to substrates,but I360 and R364 cannot participate in binding to maltooligosaccharides longer than maltose.The loss of interactions with these residues was concluded to result in the low preference for maltotriose and longer maltooligosaccharides.
基金the Department of Biotechnology,Ministry of Science and Technology,India for providing a project(BT/PR25092/NER/95/1009/2017)the Council of Scientific and Industrial Research(CSIR),Govt.of India for providing the CSIR-NET JRF fellowship.
文摘The current study deals with cloning and expressing a maltogenic α-amylase gene from thermophilic Bacillus stercoris YSP18(AmyYSP)in Escherichia coli BL21(DE3).AmyYSP belongs to the GH13_20 subfamily of Glycoside Hydrolases and entails five conserved regions found in maltogenic α-amylases.As a monomer of 67 kDa,AmyYSP exhibits maximal activity at 80℃,pH 5.0 and retains>75%residual activity at 70-100℃ and pH 5.0-8.0.The kinetic and thermodynamic studies displayed that it has a high affinity for soluble starch(K_(m)=1.54±0.236 mgmL^(−1)),exhibits a longer half-life(38.5 h at 80℃ and 8.88 h at 100℃),and a higher EaD value of 3824±1.03 kJ mol^(−1).It was characterised as a Ca^(2+)-independent α-amylase,resistant to various denaturing additives.It hydrolyses soluble starch and raw corn starch efficiently,liberating glucose,maltose,maltotriose and maltotetraose as the main products.The thermostable and acid-stable,maltooligosaccharide forming AmyYSP is a versatile enzyme with prerequisites for successful application in starch-saccharification industries.
