Met349 Mutations Enhance the Activity of 1,4-α-Glucan Branching Enzyme from Geobacillus thermoglucosidans STB02

Abstract

1,4-α-Glucan branching enzyme (GBE, EC 2.4.1.18) is used to increase the number of α-1,6 branch points in starch and glycogen. On the basis of a multiple sequence alignment of the GBEs from a variety of bacteria, residue 349 (Geobacillus thermoglucosidans STB02 numbering) in region III is generally methionine in bacteria with higher identity, while it is threonine or serine in bacteria with lower identity. Four mutants (M349T, M349S, M349H, and M349Y) were constructed by site-directed mutagenesis and characterized. M349T and M349S showed 24.5% and 21.1% increases in specific activity compared with that of wild-type GBE, respectively. In addition, M349T and M349S displayed 24.2% and 17.6% enhancements in the α-1,6-glycosidic linkage ratio of potato starch samples, respectively. However, M349Y displayed a significant reduction in activity. Moreover, the mutations at M349 have a negligible effect on substrate specificity. Thus, M349T and M349S are more suitable for industrial applications than wild-type GBE.

How To Produce Methane Precursor in the Upper Ocean by An Untypical Non‐Heme Fe‐Dependent Methylphosphonate Synthase?

“…As noted in the Introduction, 2‐His‐1‐carboxylate (Glu or Asp) or 3‐His motif binding to ferrous ion is a typical coordination mode for MNFe enzymes, while in the active site of MPnS the facial 2‐His‐1‐Gln triad motif is composed of a glutamine residue (Gln152) along with two histidines (His148 and His190) . Considering that the effect of this difference is difficult to predict without any evidences because a mutation of a residue may reduce or increase the activity of an enzyme, it is necessary to perform the calculations to examine it. We mutated the Gln152 to a glutamate (Gln152E), a histidine (Gln152H), and an aspartate (Gln152D) respectively, and calculated the corresponding barriers of the rate‐limiting step in the MPnS reaction (i. e., the hydrogen abstraction of formate by C1 radical, from Int6 to Int7a in Scheme , described in the previous section).…”

Mutations in Amino Acid Residues of Limosilactobacillus reuteri 121 GtfB 4,6-α-Glucanotransferase that Affect Reaction and Product Specificity

“…One-dimensional 1 H nuclear magnetic resonance (NMR) spectra of the product mixtures were recorded on an Avance III 400 MHz spectrometer (Bruker Co., Germany), with D 2 O as a solvent and a temperature of 338 K for the probe. The analysis was performed according to a previously published method, with some modifications. , Prior to analysis, the samples (20 mg) were exchanged twice with 0.5 mL of D 2 O (99 atom % D) (Aladdin, Shanghai, China) and then boiled, with stirring, for 1 h. After that, the samples were dissolved in 0.5 mL of D 2 O containing 0.03% TMSP acetone as an internal standard. Relative amounts (percentages) of the different linkages were estimated by integrating the respective signal peak areas.…”

An Innovative Short-Clustered Maltodextrin as Starch Substitute for Ameliorating Postprandial Glucose Homeostasis

“…Pullulanase M1 (700 U/mL) from Klebsiella planticola was obtained from Megazyme International Ltd. (County Wicklow, Ireland). Recombinant GBE from G. thermoglucosidans STB02 (200 U/mL) was produced as previously described . All other chemicals were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China).…”