Abstract
Pullulanase (EC 3.2.1.41) plays an important role in the specific hydrolysis of branch points in amylopectin. Enhancing its thermostability is required for its industrial application. In this study, rational protein design was used to improve the thermostability of PulB from Bacillus naganoensis (AB231790.1), which has strong enzymatic properties. Three positive single-site mutants (PulB-D328H, PulB-N387D, and PulB-A414P) were selected from six mutants. After incubation at 65°C for 5 min, the residual activities of PulB-D328H, PulB-N387D, and PulB-A414P were 4.5-, 1.7-, and 1.47-fold higher than PulB-WT, and their Tm values (the temperature at which half protein molecule denature) were 1.8°C, 0.4°C, and 0.9°C higher than PulB-WT, respectively. Then the final combined mutant PulB-328/387/414 was constructed. The t1/2 of it was 12.9-fold longer than that of PulB-WT at 65°C and the total increase in Tm of it (5.0°C) was almost 60% greater than the sum of individual increases (3.1°C). In addition, kinetic studies revealed that the kcat and the kcat/Km of PulB-328/387/414 increased by 38.8% and 12.9%. The remarkable improvement in thermostability and the high catalytic efficiency of PulB-328/387/414 make it suitable for industrial applications.
Highlights
Natural starch is one of the most familiar carbohydrates and is abundant in foods such as potatoes, corn, cassava, and wheat
Starch is mainly composed of α-1,4-glycosidic bonds and α1,6-glycosidic bonds, with α-1,6-glycosidic bonds forming amylopectin [1]
Because most amylolytic enzymes are specific for α-1,4-glucosidic bonds, the α-1,6-glucosidic bonds must be effectively cleaved for complete utilization of amylopectin
Summary
Natural starch is one of the most familiar carbohydrates and is abundant in foods such as potatoes, corn, cassava, and wheat. Starch is mainly composed of α-1,4-glycosidic bonds and α1,6-glycosidic bonds, with α-1,6-glycosidic bonds forming amylopectin [1]. Starch contains 20–25% amylose and 75–80% amylopectin. The starch in waxy maize and glutinous rice is composed almost entirely of amylopectin. Because most amylolytic enzymes are specific for α-1,4-glucosidic bonds, the α-1,6-glucosidic bonds must be effectively cleaved for complete utilization of amylopectin. Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme that has the specific ability to hydrolyze α-1,6–glycosidic bonds in amylopectin to obtain amylose [1,2]. It belongs to glycosyl hydrolase (GHase) family 13 ( known as the α-amylase family). Pullulanase has PLOS ONE | DOI:10.1371/journal.pone.0165006 October 20, 2016
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