During the fermentation process of starchy foods, the low pH environment poses a severe challenge to the stability of maltogenic amylase.At present, most maltogenic amylase is sensitive to acidic conditions and rapidly inactivates in low pH environments, which limits its application in bread making.This research studied the maltogenic amylase derived from Lactobacillus paralimentarius and designed mutation sites for acid-resistant modification from both the pKa value of the catalytic center and surface charge.Through single-point mutation and combined mutation, the best forward acid-resistant mutant K322D/K361D was successfully screened.Its optimal pH dropped from 5.0 to 4.5, and the hydrolysis activity at pH 4.5 increased to 433.23 U/mg, which was 2.27 times higher than that of the wild type.Its stability was greater than 68% in the pH range of 4.0-5.0.Structural and electrostatic potential energy analysis showed that the change in the net charge on the surface of the mutant helped to improve the conformational state of the enzyme, enhanced the ability to adjust the flexible structure, and thereby improving pH stability.This study provides a theoretical basis for improving the anti-aging effect of maltogenic amylase in flour products and shows good application prospects.
HOU Qian
,
HUANG Zongxiao
,
LI Ning
,
XU Yan
,
MU Xiaoqing
. Modification of acid resistance of Lactobacillus paralimentarius maltogenic amylase[J]. Food and Fermentation Industries, 2025
, 51(9)
: 9
-16
.
DOI: 10.13995/j.cnki.11-1802/ts.039322
[1] LEE H S, KIM M S, CHO H S, et al.Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other[J].Journal of Biological Chemistry, 2002, 277(24):21891-21897.
[2] JONES A, LAMSA M, FRANDSEN T P, et al.Directed evolution of a maltogenic α-amylase from Bacillus sp.TS-25[J].Journal of Biotechnology, 2008, 134(3-4):325-333.
[3] JI H Y, LI X X, JIANG T, et al.A novel amylolytic enzyme from Palaeococcus ferrophilus with malto-oligosaccharide forming ability belonging to subfamily GH13_20[J].Food Bioscience, 2022, 45:101498.
[4] ZHOU J, LI Z K, ZHANG H, et al.Novel maltogenic amylase CoMA from Corallococcus sp.strain EGB catalyzes the conversion of maltooligosaccharides and soluble starch to maltose[J].Applied and Environmental Microbiology, 2018, 84(14):e00152-18.
[5] CHANG R R, LI M, WANG Y F, et al.Retrogradation behavior of debranched starch with different degrees of polymerization[J].Food Chemistry, 2019, 297:125001.
[6] ZHANG W H, WANG J, GUO P P, et al.Study on the retrogradation behavior of starch by asymmetrical flow field-flow fractionation coupled with multiple detectors[J].Food Chemistry, 2019, 277:674-681.
[7] 郭玲玲, 张巍, 史铁嘉.酶制剂在面包品质改良方面的研究进展[J].农业科技与装备, 2010(7):37-39.
GUO L L, ZHANG W, SHI T J.Studies on the action mechanism of enzymic preparations to improve the baking quality of bread[J].Agricultural Science&Technology and Equipment, 2010(7):37-39.
[8] VAN KERREBROECK S, MAES D, DE VUYST L.Sourdoughs as a function of their species diversity and process conditions, a meta-analysis[J].Trends in Food Science & Technology, 2017, 68:152-159.
[9] CHA H J, YOON H G, KIM Y W, et al.Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose[J].European Journal of Biochemistry, 1998, 253(1):251-262.
[10] KIM I C, CHA J H, KIM J R, et al.Catalytic properties of the cloned amylase from Bacillus licheniformis[J].Journal of Biological Chemistry, 1992, 267(31):22108-22114.
[11] CHO H Y, KIM Y W, KIM T J, et al.Molecular characterization of a dimeric intracellular maltogenic amylase of Bacillus subtilis SUH4-2[J].Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2000, 1478(2):333-340.
[12] KIM T J, KIM M J, KIM B C, et al.Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain[J].Applied and Environmental Microbiology, 1999, 65(4):1644-1651.
[13] NIELSEN J E, MCCAMMON J A.Calculating pKa values in enzyme active sites[J].Protein Science, 2003, 12(9):1894-1901.
[14] JOSHI M D, SIDHU G, POT I, et al.Hydrogen bonding and catalysis:A novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase[J].Journal of Molecular Biology, 2000, 299(1):255-279.
[15] LI Z X, NIU C T, YANG X H, et al.Enhanced acidic resistance ability and catalytic properties of Bacillus 1, 3-1, 4-β-glucanases by sequence alignment and surface charge engineering[J].International Journal of Biological Macromolecules, 2021, 192:426-434.
[16] YANG H Q, LIU L, SHIN H D, et al.Structure-based engineering of histidine residues in the catalytic domain of α-amylase from Bacillus subtilis for improved protein stability and catalytic efficiency under acidic conditions[J].Journal of Biotechnology, 2013, 164(1):59-66.
[17] TURUNEN O, VUORIO M, FENEL F, et al.Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1, 4-β-xylanase Ⅱ increases the thermotolerance and shifts the pH optimum towards alkaline pH[J].Protein Engineering, 2002, 15(2):141-145.
[18] CHEN A N, XU T T, GE Y, et al.Hydrogen-bond-based protein engineering for the acidic adaptation of Bacillus acidopullulyticus pullulanase[J].Enzyme and Microbial Technology, 2019, 124:79-83.
[19] LEE S J, LEE D W, CHOE E A, et al.Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius:Role of Lys-269 in pH optimum[J].Applied and Environmental Microbiology, 2005, 71(12):7888-7896.
[20] NIELSEN J E, BEIER L, OTZEN D, et al.Electrostatics in the active site of an α-amylase[J].European Journal of Biochemistry, 1999, 264(3):816-824.
[21] SINNOTT M L.Catalytic mechanism of enzymic glycosyl transfer[J].Chemical Reviews, 1990, 90(7):1171-1202.
[22] HARRIS T K, TURNER G J.Structural basis of perturbed pKa values of catalytic groups in enzyme active sites[J].IUBMB Life, 2002, 53(2):85-98.
