Met349 Mutations Enhance the Activity of 1,4-Î±-Glucan Branching

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Met349 mutations enhance the activity of 1,4-#-glucan branching enzyme from Geobacillus thermoglucosidans STB02 Yiting Liu, Xiaofeng Ban, Caiming Li, Zhengbiao Gu, Li Cheng, Yan Hong, and Zhaofeng Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on June 1, 2017

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Journal of Agricultural and Food Chemistry

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Title: Met349 mutations enhance the activity of 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02

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Authors: Yiting Liu b, Xiaofeng Ban b, Caiming Li b, Zhengbiao Gu a,b,c, Li Cheng a,b, Yan

5

Hong a,b,c and Zhaofeng Li a,b,c,*

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Affiliations：

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a

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China

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122,

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b

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

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c

Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi,

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Jiangsu 214122, China

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*Corresponding author:

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Address: School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122,

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People’s Republic of China. Tel/fax: +86-510-85329237

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E-mail address: [email protected] (Z. Li)

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1

ACS Paragon Plus Environment


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ABSTRACT: 1,4-α-Glucan branching enzyme (GBE, EC 2.4.1.18) is used to increase the

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number of α-1,6 branch points in starch and glycogen. Based on a multiple sequence

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alignment of the GBEs from a variety of bacteria, residue 349 (Geobacillus

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thermoglucosidans STB02 numbering) in region III is generally methionine in bacteria with

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higher identity, while it is threonine or serine in bacteria with lower identity. Four mutants

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(M349T, M349S, M349H, M349Y) were constructed by site-directed mutagenesis and

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characterized. M349T and M349S showed 24.5% and 21.1% increases in specific activity

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compared with that of wild-type GBE, respectively. In addition, M349T and M349S displayed

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24.2% and 17.6% enhancements in the α-1,6-glycosidic linkage ratio of potato starch samples,

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respectively. However, M349Y displayed a significant reduction in activity. Moreover, the

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mutations at M349 have a negligible effect on substrate specificity. Thus, M349T and M349S

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are more suitable for industrial applications than wild-type GBE.

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KEYWORDS: 1,4-α-glucan branching enzyme; starch; enzyme activity; Met349; mutation

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INTRODUCTION

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The formation of α-1,6-glucosidic linkages in bacterial glycogen is catalyzed by bacterial

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1,4-α-glucan branching enzymes (GBEs, EC 2.4.1.18), while the formation of these linkages

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in starch is catalyzed by plant GBEs. The reaction proceeds by cleaving an α-1,4-glycosidic

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bond and transferring the α-1,4-glucanopyranosyl chain to the C-6 hydroxyl position of a

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glucan chain.1-3 Previous studies have shown that this enzyme can be used in vitro to modify

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the structure of starch by increasing the number of the α-1,6-branch points.4 These

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enzyme-modified starches show potential application as novel functional foods by slowing

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digestion rate to attain extended glucose release.5-6 Corn starch modified using the GBE from

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G. thermoglucosidans STB02 showed reduced retrogradation.2,7 Jensen et al.8 demonstrated

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that GBE treatment could stabilize the granular structure of starch. Highly branched cyclic

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dextrin, a food ingredient, has been synthesized industrially using a thermostable GBE.9,10

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Compared with chemical processes, enzyme-catalyzed processes are more selective, more

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environmentally friendly, and have lower energy costs. GBEs have drawn worldwide attention

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for their potential use in industrial applications.11

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Thermostability, specific activity, and catalytic efficiency have always been considered

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among the critical factors determining the feasibility of using GBEs for industrial

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applications.12 Different strategies have been used to improve the enzymatic activity of GBEs,

50

including

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modification.16,17 One method of molecular modification, site-directed mutagenesis, has been

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used to modify the active center of GBE.3 This modification increased the enzyme’s specific

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activity, compared to wild-type GBE, suggesting that the mutation had created a more

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efficient enzyme.

heterologous

expression,13,14

fermentation

optimization,15

3


and

molecular


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Most GBEs belong to glycoside hydrolase family 13 (GH13), which also includes

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α-amylases (EC 3.2.1.1), pullulanases (EC 3.2.1.41), isoamylases (EC 3.2.1.68) and

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cyclodextrin glucanotransferases (EC 2.4.1.19) of similar structure.18,19 These enzymes have a

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common (α/β)8-barrel catalytic domain that contain the enzyme’s catalytic center, an

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N-terminal domain, and a C-terminal domain.20,21 Based on structural and biochemical

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information, Tyr300, Asp335, His340, Arg403, Asp405, Glu458, His525, and Asp526 (E. coli

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numbering) are highly conserved among the members of GH13. 22-24

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In a previous study, a GBE was obtained from G. thermoglucosidans STB02 and

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characterized for further research. This GBE displayed optimal activity at pH 7.5 and 50 °C.

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25

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numbering), a conserved amino acid located after the invariant Glu352, which is situated in

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conserved region III, have suggested that substitution of Glu459 with Asp (G459D) increases

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enzymatic activity, compared with that of the wild-type.3 Met349, which is also located in

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region III, is replaced by threonine or serine in enzymes that share lower sequence identity. A

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homology modeling analysis suggested that replacement of M349 with residues that create an

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extra hydrogen bonding interaction may increase the enzyme’s specific activity. To test this

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hypothesis and obtain GBE analogs with high specific activity, four GBE variants at M349

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(M349T, M349S, M349H, M349Y) were constructed and evaluated.

Limited site-directed mutagenesis has also been performed. Studies of Glu459 (E. coli

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MATERIALS AND METHODS

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Bacterial strains and plasmids. Escherichia coli JM109 was used for recombinant

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DNA manipulations. Plasmid pET-20b(+)/gbe, which contains the gbe gene from G.

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thermoglucosidans STB02 (GenBank accession no. KJ660983) was used as the template for 4


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site-directed mutagenesis, sequencing, and expression of the wild-type and mutant GBE

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proteins.25,26 E. coli BL21 (DE3) was used as the expression host for the wild-type and mutant

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GBEs produced in this study.

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Construction of site-directed mutants. Restriction endonucleases, prime STAR HS

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DNA polymerase, and PCR reagents were purchased from TaKaRa Shuzo (Otsu, Japan) and

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used in accordance with the manufacturer’s instructions. The desired mutations in GBE were

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introduced using a one-step PCR method with plasmid pET-20b(+)/gbe as the template. Sets

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of two complementary oligonucleotide primers were used for each mutation; their sequences

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are presented in Table 1. All PCR products were cloned separately into the pET-20b(+)/gbe

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vector using unique Nde I and Xho I restriction sites, resulting in plasmids with C-terminal

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His tags. The intended mutations were confirmed by DNA sequencing. The resulting mutant

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plasmids were used to transform E. coli BL21 (DE3) competent cells for expression studies.

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Production and purification of GBE (mutants) proteins. A single colony of E. coli

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BL21 (DE3) cells harboring a pET-20b(+)/gbe variant encoding a wild-type or mutant GBE

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was used to inoculate 50 mL of Luria−Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast

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extract, and 10 g/L NaCl, pH 7) supplemented with ampicillin (50 µg/mL), which was then

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incubated overnight, with shaking, at 37 °C. A 1 mL portion of this overnight culture was then

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diluted into 50 mL of Terrific Broth (TB) medium (12 g/L tryptone, 24 g/L yeast extract, 5 g/L

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glycerol, 17 mM KH2PO4, and 72 mM K2HPO4, pH 7.5) supplemented with ampicillin (50

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µg/mL) and incubated on a rotary shaker (200 rpm) at 37 °C until the absorbance of the

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culture at 600 nm (A600) reached approximately 0.6. At this point, the temperature was

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adjusted to 25 °C and expression of the recombinant enzyme was induced by the addition of

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0.01 mM isopropyl β-D-thiogalactopyranside (IPTG) (Sigma-Aldrich, St. Louis, MO, USA). 5



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After 24 h of induction, the cells were harvested by centrifugation at 10,000 g for 10 min,

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resuspended in 50 mM sodium phosphate buffer (pH 7.5), and disrupted by sonication. The

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enzyme was purified using His-tag affinity chromatography with a HiTrap ChelatingTM

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column (5 mL, GE Healthcare) and eluted with a linear gradient of 20-500 mM imidazole on

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an Akta purifier 10 purification system. Fractions containing the protein of interest were

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pooled and stored at -80 °C.

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Enzyme activity assays. Iodine assay—The method was performed as previously

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described,1,27 with minor modifications. The substrate was 0.25% potato amylopectin type III

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(Sigma-Aldrich, St. Louis, MO, USA) or 0.1% potato amylose type III (Sigma-Aldrich)

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dissolved in 50 mM sodium phosphate buffer (pH 7.5). Suitably diluted enzyme (100 µg/mL,

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100 µL) was incubated with substrate solution (900 µL) at 50 °C for 15 min. The reaction was

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terminated by boiling for 15 min. A 5 mL aliquot of iodine reagent was added to the solution

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(300 µL), and the absorbance at 530 nm (A530) or 660 nm (A660) was measured. One unit of

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enzyme activity was defined as the amount of branching enzyme that decreased A530 or A660

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by 1% per min.

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Analysis

using

proton

nuclear

magnetic

resonance

spectroscopy.

The

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α-1,6-glycosidic linkage ratio of each starch sample was determined using proton nuclear

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magnetic resonance (1H NMR) spectroscopy with an Avance III 400 MHz spectrometer

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(Bruker Co., Germany). The analysis was performed according to a published method with

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some modifications.2 Potato starch was treated with GBE (wild-type or mutant) in 50 mM

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sodium phosphate buffer (pH 7.5) at 50 °C for 8 h. The reaction was stopped by heating at

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100 °C for 15 min. The samples were dried for 3 days using vacuum freeze-drying equipment.

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The control was potato starch treated in the absence of GBE. Starch samples were dissolved 6


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in deuterium oxide containg 0.2% (w/w) sodium deuteroxide (final starch concentration, 20

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mg/mL [w/v]) and then boiled, with stirring, for 30 min. The 1H NMR spectra were obtained

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at 60 °C. The α-1,6-glycosidic linkage ratio was quantified by dividing the area of the peak

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corresponding to the anomeric protons from the α-1,6-glycosidic linkages by the total area of

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the peaks corresponding to the anomeric protons of the α-1,6-glycosidic linkages and the

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α-1,4-glycosidic linkages.1,28

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Structure modeling of the (mutant) GBEs from various organisms. The X-ray crystal

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structures of the GBEs from E. coli, Mycobacterium tuberculosis H37Rv, Cyanothece sp.

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ATCC 51142 were obtained from the Research Collaboratory for Structural Bioinformatics

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(RCSB) Protein Data Bank (accession code IM7X, 3K1D, 5GQU). Homology models of the

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mutant GBEs were constructed using the SWISS-MODEL protein-modeling server

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(http://www.expasy.ch/swissmod/SWISS-MODEL.html). The PyMoL molecular Graphics

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System (http://www.pymol.org) was used to visualize and analyze the model structures

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generated.

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Statistical analysis. Statistical analyses were conducted with SPSS 17.0 software (SPSS

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Inc., Chicago, Illinois, USA). Values are presented as the mean ± standard deviation (SD) (n =

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3). Means with different letters within the same column are significantly different (p < 0.05).

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RESULTS

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Sequence alignment and structural analysis. A multiple sequence alignment of the

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branching enzymes from a variety of bacteria is shown in Table 2. These bacterial species

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share a range of sequence identities from 42% to 87%. Residue 349 (G. thermoglucosidans

146

STB02 GBE numbering) in region III is generally methionine in the bacteria with higher 7



147

sequence identity (42–87%), while it is threonine or serine in the bacteria with lower

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sequence identity (43–49%). The crystal structures of GBEs from different sources around

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position 349 were modeled (Figure 1). As seen in Fig. 1A, Met349 is located in an α-helix. It

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interacts with Tyr373 of the next α-helix and Asn372 of a nearby coil. When the residue

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corresponding to Met349 was changed to Thr, as in the GBE from E. coli (1M7X, 44%

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sequence identity), an additional interaction was built with Asn443 of a nearby β-sheet (Fig.

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1B). The residue corresponding to Met349 in the GBE from M. tuberculosis H37Rv (3K1D,

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43% sequence identity) is also Thr. This Thr made much stronger connections with Asn440

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and His444 of the adjacent β-sheet (Fig. 1C). In the GBE from Cyanothece sp. ATCC 51142

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(5GQU, 46% sequence identity), the residue corresponding to Met349 is Ser. This Ser

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interacted with the Asn492 of the adjacent β-sheet (Fig. 1D). In brief, the replacement of

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Met349 by Thr or Ser appears to be available to increase the stability of (α/β)8-barrel catalytic

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domain. This observation suggested that this residue is involved in catalytic activity. In order

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to probe the function and importance of this methionine residue in GBE, we used site-directed

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mutagenesis to replace Met349 of G. thermoglucosidans STB02 GBE with threonine, serine,

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histidine, or tyrosine to form mutants M349T, M349S, M349H, and M349Y, respectively.

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Expression and purification of wild-type and mutant GBEs. Genes encoding the four

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GBE mutants M349T, M349S, M349H and M349Y were successfully constructed through

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site-directed mutagenesis of the GBE expression plasmid pET-20b(+)/gbe. There were no

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obvious differences in expression level among the recombinant wild-type and mutant GBEs.

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The wild-type and mutant enzymes were expressed in E. coli BL21(DE3) and purified to

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apparent homogeneity. All the purified proteins appeared as a single band of about 75 kDa on

169

an SDS-PAGE gel when 1 µg was loaded on the gel (Figure S1). These purified wild-type and 8


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mutant GBEs were used for biochemical characterization.

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Activities of wild-type and mutant GBEs. The specific activities of the wild-type and

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mutant GBEs were determined using the iodine stain assay. The Km values of wild-type and

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mutant GBEs were measured using amylopectin as the substrate. The results are presented in

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Table 3. The specific activity of wild-type GBE was 299 ± 3 U/mg of protein when using

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amylose as the substrate. This was somewhat greater than the 63.75 U/mg obtained by Garg,

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et al.20 and much greater than the 36 U/mg obtained by Aga, et al.29 under similar conditions.

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When using amylopectin as the substrate, the M349T and M349S mutants showed 24.5% and

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21.1% improvements in specific activity, respectively, compared with that of wild-type GBE.

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In contrast, the M349H mutant showed a slight decrease in specific activity. The M349Y

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mutant displayed a 33.9% decrease in specific activity, compared with wild-type GBE. The

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wild-type and mutant GBEs had similar amylose to amylopectin activity ratios and Km values,

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indicating that the mutations at M349 had a negligible effect on substrate specificity (Table 3).

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Interestingly, the M349T mutant did not change the transfer pattern; rather, it enhanced chain

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transfer (Figure S3).

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The α-1,6-glycosidic linkage ratio of potato starch treated with the wild-type and

186

mutant GBEs. To further assess the utility of the mutant GBEs, each enzyme was assayed in

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a reaction mixture containing GBE protein (35 µg) and 5% ([w/v], wet basis) potato starch.

188

1

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shown in Fig. 2, the α-1,6 anomeric protons in potato starch were detected at 4.97 ppm, and

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the α-1,4 anomeric protons were detected at 5.37 ppm. These assignments were consistent

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with those determined in previous research.1 The α-1,6-glycosidic linkage ratios of potato

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starch samples treated with wild-type or mutant GBEs were shown in Fig. 3. After 8 h of

H NMR was used to determine the α-1,6-glycosidic linkage ratio of these starch samples. As

9



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incubation, the reaction product of potato starch with wild-type GBE revealed a degree of

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branching of 13.5%. Those of mutant M349T and M349S revealed greater degrees of

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branching (25.5% and 16.5%, respectively) than that of wild-type GBE. However, mutants

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M349H and M349Y showed 1.4% and 37.5% decreases, respectively, compared with that of

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wild-type GBE.

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Homology modeling of the mutant GBEs. A structural model of each of the mutant

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GBEs was built using the SWISS-MODEL protein-modeling server (Fig. 4). From the

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structure model of the wild-type GBE (Fig. 4A), Met349 could form hydrogen bonds with

201

Asn372 and Tyr373. Replacement of methionine with threonine or serine kept a neutral side

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chain at this position, but added an additional hydroxyl group and decreased the molecular

203

weight. Comparing the homology models of M349T (Fig. 4B) and M349S (Fig. 4C) with that

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of wild-type GBE suggested that the serine or threonine at this position could form an

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additional hydrogen bond with the highly conserved residue Asn337. Replacement of these

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neutral side chain with the positively charged imidazole of histidine (Fig. 4D) also introduced

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a hydrogen bond, but with Val340. Replacement of methionine with tyrosine (Fig. 4E) did not

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change the ionic character of the residue, but in this case, no additional hydrogen bonds were

209

predicted.

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DISCUSSION

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At the outset of this work, we speculated that residue 349 in conserved region III is

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connected with catalytic activity because of the results of a multiple sequence alignment of

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various 1,4-α-glucan branching enzymes sharing different sequence identities (42%–87%).

215

Structural analysis suggested that the replacement of Met349 by Thr or Ser contributed 10


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significantly to the stability of (α/β)8-barrel catalytic domain. Site-directed mutagenesis

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was used to investigate the function of this residue. Interestingly, mutants M349T and

218

M349S showed enhanced enzymatic activity, while mutants M349H and M349Y showed

219

decreased enzymatic activity. Thus, these GBE enzymes displayed specific activities in

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the order M349T > M349S > wild-type > M349H > M349Y.

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Homology modeling analysis suggested that the enhancement in enzyme activity

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was connected with a conformational stabilizing effect, since the mutants M349T and

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M349S, which showed a substantial increase in specific activity, were also predicted to

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have a newly introduced hydrogen bond. However, not all mutants with a newly

225

introduced hydrogen bond showed greater activity (Fig. 4 and Tables 3). The M349H

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mutant could bring an additional interaction with Val340 but result in lower activity.

227

There are two reasons for this. For one thing, previous studies have revealed the residues

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(Thr455, Thr452, Ser504) corresponding to Met349 in GBE from different source all

229

interact with Asn of the adjacent β-sheet (Figure 1). A multiple sequence alignment of

230

the branching enzymes in GH13 showed that Asn337 is highly conserved but Val340 is

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not. For another thing, residue 349 was situated in the active center such that its side

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chain could mediate solvent effects. The M349H mutation would increase the polarity of

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the environment surrounding Asp309 and Glu352, which could drastically decrease their

234

interaction (Table S1). The M349Y mutant, of which the polar side chain introduced

235

without forming additonal hydrogen bonds, decreased enzyme activity compared with

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wild-type GBE. These results suggested that enhancing the contact with conserved

237

residue Asn337 and maintaining the polarity of the environment enhanced enzyme

238

activity of GBE. 11



239

Under the same reaction conditions used for the modification of starch, the enzymes used

240

in this study produced α-1,6 branch points in the order M349T > M349S > wild-type >

241

M349H > M349Y. The most promising of these mutants, M349T, did not change the optimal

242

pH, temperature, substrate preference, or transfer pattern (Figures S2, S3). Thus, mutants

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M349T and M349S have substantial potential for use in industrial applications.

244

In conclusion, Met349, located in the central (α/β)8 barrel catalytic domain (region III) of

245

the GBE from G. thermoglucosidans STB02, plays a key role in branching enzyme activity.

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The M349T and M349S mutants displayed 24.5% and 21.1% increases in branching activity,

247

compared with that of wild-type GBE, respectively. In contrast, mutants M349H and M349Y

248

displayed decreased activity. More importantly, the M349T and M349S mutants also

249

increased the number of α-1,6 branch points. Thus, the two mutants are more suitable for the

250

enzymatic modification of starch than the wild-type enzyme. This mutation site has never

251

been reported before, and the results of the structure–function analysis at residue 349 obtained

252

in this study may be helpful and provide theoretical reference for further study.

253 254

ABBREVIATIONS

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GBE, 1,4-α-Glucan branching enzyme; Met, methionine; Glu, glutamic acid; Asp, aspartic

256

acid; Thr, threonine; Ser, serine; Tyr, tyrosine; His, histidine; GH13, glycoside hydrolase

257

family 13; LB, Luria−Bertani; TB, Terrific Broth; IPTG, isopropyl β-D-thiogalactoside; 1H

258

NMR , proton nuclear magnetic resonance

12


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ACKNOWLEDGMENTS

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Financial support for this work was received from the Science and Technology Support

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(Agriculture) program of Jiangsu Province (No. BE2014305), the China Postdoctoral Science

262

Foundation (No. 2014M560394, 2016T90420), the Jiangsu Planned Projects for Postdoctoral

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Research Funds (No. 1401100C), and the program of "Collaborative innovation center of food

264

safety and quality control in Jiangsu Province".

265 266

SUPPORTING INFORMATION

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Table S1 Van der Waals volumes and hydropathy indexes of the amino acid side chains.

268

Figure S1 SDS-PAGE analysis of the purified mutant and wild-type GBEs.

269

Figure S2 Optimal temperature (A) and pH (B) of wild-type GBE and its M439T mutant.

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Figure S3 Chain length distribution analysis of amylopectin treated with wild-type GBE and

271

its M439T mutant.

13



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catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis. Biochim.

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2010, 92, 360-369.

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13. Wu, S.; Liu, Y.; Yan, Q.; Jiang, Z. Gene cloning, functional expression and

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characterisation of a novel glycogen branching enzyme from Rhizomucor miehei and its

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application in wheat breadmaking. Food Chem. 2014, 159, 85-94.

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14. Ko, Y. T.; Chung, P. S.; Shih, Y. C.; Chang, J. W. Cloning, characterization, and

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expression of mungbean (Vigna radiata L.) starch branching enzyme II cDNA in Escherichia

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coli. J. Agric. Food Chem. 2009, 57, 871-879.

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15. Ding, R.; Li, Z.; Chen, S.; Wu, D.; Wu, J.; Chen, J. Enhanced secretion of recombinant

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alpha-cyclodextrin glucosyltransferase from E. coli by medium additives. Process Biochem.

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2010, 45, 880-886. 15



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16. Huang, M.; Li, C.; Gu, Z.; Cheng, L.; Hong, Y.; Li, Z. Mutations in cyclodextrin

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glycosyltransferase

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beta-cyclodextrin production. J. Agric. Food Chem. 2014, 62, 11209-11214.

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17. Li, Z.; Zhang, J.; Wang, M.; Gu, Z.; Du, G.; Li, J.; Wu, J.; Chen, J. Mutations at subsite-3

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in

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alpha-cyclodextrin specificity. Appl. Microbiol. Biot. 2009, 83, 483-490.

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18. Romeo, T.; Kumar, A.; Preiss, J. Analysis of the Escherichia coli glycogen gene cluster

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suggests that catabolic enzymes are encoded among the biosynthetic genes. Gene 1988, 70,

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363-76.

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19. Suzuki, E.; Suzuki, R. Distribution of glucan-branching enzymes among prokaryotes.

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Cell. Mol. Life Sci. 2016, 73, 2643-2660.

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20. Pal, K.; Kumar, S.; Sharma, S.; Garg, S. K.; Alam, M. S.; Xu, H. E.; Agrawal, P.;

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Swaminathan, K. Crystal structure of full-length Mycobacterium tuberculosis H37Rv

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glycogen branching enzyme: insights of N-terminal beta-sandwich in substrate specificity and

333

enzymatic activity. J. Biol. Chem. 2010, 285, 20897-903.

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21. Macgregor, E. A.; Janecek, S.; Svensson, B. Relationship of sequence and structure to

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specificity in the alpha-amylase family of enzymes. Biochim. Biophys. Acta 2001, 1546, 1-20.

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22. Uitdehaag, J. C.; Alebeek, G. J.; Veen, B. A.; Dijkhuizen, L.; Dijkstra, B. W. Structures of

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maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase

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give insight into the mechanisms of transglycosylation activity and cyclodextrin size

339

specificity. Biochemistry 2000, 39, 7772-80.

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23. Brzozowski, A. M. B.; Davies, G. J. Structure of the Aspergillus oryzae α-amylase

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complexed with the inhibitor acarbose at 2.0 Å resolution. Biochemistry 1997, 36, 10837-45.

cyclodextrin

from

Bacillus

circulans

glycosyltransferase

from

enhance

beta-cyclization

Paenibacillus

16


macerans

activity

and

enhancing

Page 17 of 26


342

24. Abad, M. C.; Binderup, K.; Rios-Steiner, J.; Arni, R. K.; Preiss, J.; Geiger, J. H. The

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X-ray crystallographic structure of Escherichia coli branching enzyme. J. Biol. Chem. 2002,

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277, 42164-42170.

345

25. Ban, X.; Li, C.; Gu, Z.; Bao, C.; Qiu, Y.; Hong, Y.; Cheng, L.; Li, Z. Expression and

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biochemical characterization of a thermostable branching enzyme from Geobacillus

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thermoglucosidans. J. Mol. Microbiol. Biotechnol. 2016, 26, 303-311.

348

26. Liu, Y.; Li, C.; Gu, Z.; Xin, C.; Li, C.; Yan, H.; Li, Z. Alanine 310 is important for the

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activity of 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02. Int.

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J. Biol. Macromol. 2017, 97, 156.

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27. Vu, N. T.; Shimada, H.; Kakuta, Y.; Nakashima, T.; Ida, H.; Omori, T.; Nishi, A.; Satoh,

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H.; Kimura, M. Biochemical and crystallographic characterization of the starch branching

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enzyme I (BEI) from Oryza sativa L. Biosci. Biotech. Bioch. 2008, 72, 2858-66.

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28. Lee, B. H.; Yan, L.; Phillips, R. J.; Reuhs, B. L.; Jones, K.; Rose, D. R.; Nichols, B. L.;

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Quezada-Calvillo, R.; Yoo, S. H.; Hamaker, B. R. Enzyme-synthesized highly branched

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maltodextrins have slow glucose generation at the mucosal alpha-glucosidase level and are

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Sslowly digestible in vivo. PloS one 2013, 8.

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29. Aga, H.; Okamoto, I.; Taniguchi, M.; Kawashima, A.; Abe, H.; Chaen, H.; Fukuda, S.

359

Improved yields of cyclic nigerosylnigerose from starch by pretreatment with a thermostable

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branching enzyme. J. Biosci. Bioeng. 2010, 109, 381-387.

361

30. Darby, N. J.; Creighton, T. E. Protein structure. IRL Press at Oxford University Press:

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Oxford [etc.], 1993.

363

31. Kyte, J.; Doolittle, R. F. A simple method for displaying the hydropathic character of a

364

protein. J. Mol. Biol. 1982, 157, 105-132. 17



365

FIGURE CAPTIONS

366

Figure 1. Three-dimensional models of the GBEs from various organisms. A, GBE from G.

367

thermoglucosidans STB02; B, GBE from E. coli (PDB ID=1M7X); C, GBE from M.

368

tuberculosis H37Rv (PDB ID=3K1D); D, GBE from Cyanothece sp. ATCC 51142 (PDB

369

ID=5GQU). The atoms of amino acids 443, 455, 478, and 479 (E. coli GBE numbering); 440,

370

444, 452, 475, and 476 (M. tuberculosis H37Rv GBE numbering); 492, 504, 527, and 528

371

(Cyanothece sp. ATCC 51142 GBE numbering); and 337, 340, 349, 372 and 373 (G.

372

thermoglucosidans STB02 GBE numbering) are displayed with stick representations.

373 374

Figure 2. Representative 1H NMR analysis of potato starch treated with GBE. The peak

375

corresponding to the anomeric protons of the α-1,6-glycosidic linkages can be seen at 4.97

376

ppm. The peak corresponding to the anomeric protons of the α-1,4-glycosidic linkages can be

377

seen at 5.37 ppm.

378 379

Figure 3. The α-1,6-glycosidic linkage ratios of potato starch samples treated with wild-type

380

or mutant GBEs for 8 h. The heights of bars annotated with different letters are significantly

381

different (p < 0.05).

382 383

Figure 4. Three-dimensional models of the GBEs from G. thermoglucosidans STB02 and its

384

mutants. A, Wild-type GBE; B mutant M349T; C mutant M349S; D mutant M349H; E

385

mutant M349Y. The atoms of amino acids 349, 372, 373, 337, and 340 are displayed with

386

stick representations.

387 18


Page 18 of 26

Page 19 of 26


TABLES Table 1. Primers used for site-directed mutagenesis Primer sequence (5’–3’)a

Desired mutation

CCGGGCATATTGACGATTGCCGAAG M349T CTTCGGCAATCGTCAATATGCCCGG CCGGGCATATTGAGCATTGCCGAAG M349S CTTCGGCAATGCTCAATATGCCCGG CCGGGCATATTGCACATTGCCGAAG M349H CTTCGGCAATGTGCAATATGCCCGG CCGGGCATATTGTATATTGCCGAAG M349Y CTTCGGCAATATACAATATGCCCGG a

Nucleotide sequences corresponding to the mutated amino acids are underlined.

19



Page 20 of 26

Table 2. Partial sequence alignment of several branching enzymes from various organisms Region IIIa

Source

Identity (%)

GenBank Accession Number

G. thermoglucosidans STB02

348

LMIAED

100

AID62090.1

Parageobacillus toebii

319

LMIAED

87

KYD31961.1

Bacillus stearothermophilus

348

LMIAED

83

BAH85872.1

Anoxybacillus flavithermus

348

LMTAED

73

OAO82534.1

Bacillus cereus

348

LMTAED

64

BAE96028.1

Streptococcus mutans

351

44

KZM63769.1

Deinococcus radiodurans

356

LMIAEE

42

AAF11402.1

Acidothermus cellulolyticus 11B

457

VTVAEE

45

ABK52449.1

Mycobacterium tuberculosis H37Rv

451

VTIAEE

43

3K1D_Ab

VTMAEE

44

ACI76450.1

QTIAEE

49

WP_010880434.1

MTIAEE

47

BAB69858.1

Escherichia coli

MMIAEE

454

Aquifex aeolicus

358

Rhodothermus marinus

352

Cyanothece sp. ATCC 51142

503

LSIAEE

46

5GQU_Ab

Hydrococcus rivularis

483

LSIAEE

45

WP_073598221.1

Myxosarcina sp. GI1

482

LSIAEE

45

WP_036479403.1

Nostoc calcicola

481

LSIAEE

45

WP_073641013.1

a

superscript identifies the residue number of the first residue in the Region II sequence.

b

this is an RCSB Protein Data Bank identification number.

20


Page 21 of 26


Table 3. Specific activities of wild-type and mutant GBEs Specific activity (U/mg) Enzyme

Ratio

Km (mg/mL)

384 ± 2c

0.779

1.07 ± 0.05b

404 ± 2e

478 ± 8e

0.845

1.02 ± 0.04b

M349S

396 ± 3d

465 ± 2d

0.852

0.884 ± 0.04a

M349H

311 ± 3c

375 ± 2b

0.829

0.903 ± 0.05a

M349Y

203 ± 2a

254 ± 2a

0.799

1.23 ± 0.04c

Amylose

Amylopectin

Wild-type

299 ± 3b

M349T

Each value represents the mean of three independent measurements, and means with different letters within the same column are significantly different (p < 0.05).

21 ACS Paragon Plus Environment


FIGURE GRAPHICS Figure 1.


Page 22 of 26

Page 23 of 26


Figure 2.



Page 24 of 26

Figure 3. 12

e

α-1,6 linkages Ratio (%)

10

d

c

c

8 6

b

a

4 2 0 control

Wild-type M349T M349S Sample

M349H


M349Y

Page 25 of 26


Figure 4.



TOC Graphic


Page 26 of 26

Met349 Mutations Enhance the Activity of 1,4-Î±-Glucan Branching

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