Heterologous Expression Of Maltogenic Amylase LpMA And Its Application In Bread Making

Maltogenic amylase（MAase,EC 3.2.1.133）is also called raw maltoseα-amylase belongs to hydrolases and transferases.Maltogenic amylase mainly hydrolyzesα-（1,4）-glycosidic bonds from the non-reducing ends of amylose molecules,the product of complete hydrolysis is mainlyα-maltose.Besides,maltogenic amylase can also hydrolyze randomly from the inside of starch moleculeα-（1,4）-glycosidic bonds andα-（1,6）-glycosidic bonds.When applied to the bread making process,the product of starch hydrolyzed by maltose amylase:α-maltose,glucose,oligosaccharide DP2-9,amylodextrin and amylopectin with smaller moleculars and shorter branches.They are beneficial for extending the shelf life of baked goods,making maltogenic amylase valuable in the field of food baking and flour modification.In this study,several strains of lactic acid bacteria（Lactobacillus sp.）preserved in our laboratory were used as starting strains.After strain identification,the new maltogenic amylase gene lpma was excavated from them and expressed in E.coli.After determining the enzymatic characteristics of recombinant maltogenic amylase（reLpMA）,it was applied to bread baking to study its effect on improving bread quality and anti-aging of bread during storage.The purpose of this study is to explore a new maltogenic amylase with good effect from Lactobacillus sp.for the bread baking industry.（1）By comparing the 16S r DNA of the strains,the conserved strains were identified and named as Lactobacillus plantarum YXY418 and Lactobacillus rhamnosus YXY412.The genomic DNA were extracted as templates to clone the putative maltogenic amylase genes lpma and lrma.By using bioinformatics analysis techniques,they were compared with the previously reported maltogenic amylase sequence.The results showed that the two polypeptides were maltogenic amylase,and were named LpMA and Lr MA.（2）Using lpma as the research object,a recombinant expression plasmid p ET-28a（+）/lpma was constructed and LpMA was expressed in E.coli BL21.The activity of reLpMA was 36.4U·mg^-1 for soluble amylase,and it can also hydrolyze cyclodextrin（β-CD）and pullulan polysaccharides.Using soluble starch as the substrate,the enzymatic characteristic parameters of purified reLpMA were measured.The optimum temperature and p H were 45°C and 6.0,and the reLpMA had good stability between p H 5.0-8.0.Re LpMA has a strong resistance to metal ions,and it can be widely used in industrial processing.The kinetic parameters V_max and k_cat/K_mof reLpMA for soluble starch andβ-CD were 52.3 m M,22.1 m M and 3.28 m M^-1s^-1 and 3.98m M^-1s^-1,respectively.The results showed that the catalytic efficiency ofβ-CD is higher than soluble starch.（3）The optimum amount of reLpMA in bread baking was studied by adding three different gradients of reLpMA to bread making.The measurement results of bread properties showed that compared with the blank control group,the specific volume of bread added with 2000 U reLpMA slightly increased from 6.4 to 6.7.After 7 days of storage,the hardness and chewability of bread decreased by 29.5%and 26.4%respectively.The effects of two maltose amylases（reLpMA and Angel MAM）on bread baking quality were compared.Compared with the control group,the anti-aging effect of reLpMA added with the same amount of enzyme（2000 U）in bread was slightly inferior to Angel MAM.After 7 days of storage,the hardness of bread was significantly reduced by 29.5%and 33.2%,and the chewability was reduced by 26.5%and31.9%,respectively.The results showed that reLpMA can achieve similar effect with MAM,which could improve the quality of bread and prolong the shelf life of bread.（4）The compound of reLpMA andα-amylase was used in the production of wheat bread to further improve the quality of bread baking.The result showed that reLpMA andα-amylase played a synergistic role,after 7 days of storage,the hardness and chewiness of bread significantly decreased by 39.0%and 36.3%,respectively.The results of LF-NMR showed that the complex enzyme can delay the loss of moisture in bread during storage,improve the anti-aging ability of bread,and correspondingly extend the shelf life of bread.