Crystal structure of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04

Xie, Xiaofang; Li, Yuelong; Ban, Xiaofeng; Zhang, Ziqian; Gu, Zhennan; Li, Caiming; Yan, Hong; Cheng, Li; Jin, Tengchuan; Li, Zhaofeng

doi:10.1016/j.ijbiomac.2019.07.104

Cited by 17 publications

(25 citation statements)

References 44 publications

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“…The inorganic salt optimization study showed the most obvious increase in amylase activity with the addition of Mn 2+ . It is reported that amylase is a kind of metal enzyme, which needs metal ions as an activator and accelerator (Xie et al., 2019). In addition, the addition of Zn 2+ inhibited the amylase activity during fermentation, which indicated that the Bacillus cereus strain has weak tolerance to certain ions, which was consistent with the results of other researchers (Wei et al., 2011; Wu, Wu, Xu, Kong, & Wu, 2020).…”

Section: Discussionmentioning

confidence: 99%

A high amylase‐producing bacterial strain exhibiting the phosphorus‐dissolving ability, isolated from freshwater aquaculture pond

et al. 2020

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Amylase‐producing bacteria could improve water quality contaminated by waste from feed residue and fish metabolism, thereby increasing the efficiency of aquaculture systems. The objective of this research was to screen and optimize fermentation conditions of a high amylase‐producing strain. Four amylase‐producing bacterial strains (named S458‐1, G05, H38 and B09) were isolated from a grass carp pond, and the strain S458‐1 showed the highest amylase‐producing ability, with 19.58 ± 0.38 mm hydrolysis circle diameter. The strain S458‐1 was identified as Bacillus cereus based on morphological identification, biochemical identification and 16S rDNA sequence analysis. The optimal culture medium formula included (in g/L) Ca2+ 0.8, Mg2+ 0.2, Mn2+ 0.4, Fe2+ 0.6, Al3+ 0.2, 1% soluble starch and 1% peptone. The optimal fermentation conditions were determined as initial pH 9, culture temperature 37°C, fermentation time of 60 hr and 2% inoculum. Under the optimal formula and condition, its enzyme activity increased from 32 U/ml to 173.01 U/ml, a 5.41‐fold increase. Surprisingly, our research found that the strain S458‐1 also had phosphorus degradation capabilities. Its phosphorus‐dissolving ability was both time‐ and concentration‐dependent. Thus, this study will make a contribution to the bacterial amylase based on the fermentation process and provide a theoretical basis for further research of aquatic probiotics.

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Section: Discussionmentioning

confidence: 99%

A high amylase‐producing bacterial strain exhibiting the phosphorus‐dissolving ability, isolated from freshwater aquaculture pond

et al. 2020

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“…The structural function of SBS has been validated when -amylase from Saccharomyces fibuligera R64 (Sfamy R64) was shown to exhibit low starch adsorptivity due to the absence of SBS ( Yusuf et al, 2017 ). In addition, an acarbose molecule which was a typical inhibitor of -amylases, was also observed to bind in the SBS of B. stearothermophilus STB04 (Bst-MFAse; PDB ID: 6ag0) located in the active site ( Xie et al, 2019a ). Importantly, a pair of aromatic amino acid residues was found at each of the three SBSs in the Halothermothrix orenii -amylase (AmyB), where the hydrophobic CH/-stacking interaction was prominently observed ( Tan et al, 2008 ).…”

Section: Survey Methodologymentioning

confidence: 99%

“…Despite the often-reported Ca 2+ -Na + -Ca 2+ linear metal triad ( Offen et al, 2015 ), Bst-MFAse had the unprecedented Ca 2+ - Ca 2+ - Ca 2+ triad ( Xie et al, 2019a ), where it had been used as the template to predict the 3D structures of both wild type and recombinant bacterial SR74 -amylases through homology modeling strategy ( Lim et al, 2020 ). However, NaII in Bli Amy replaced the calcium ion which had been reported to be located between Domain A and Domain C, where its role was not described ( Boi et al, 2020 ).…”

Section: Survey Methodologymentioning

confidence: 99%

Native to designed: microbial α-amylases for industrial applications

Lim

Oslan

2021

PeerJ

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Background α-amylases catalyze the endo-hydrolysis of α-1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, α-amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial α-amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work. Survey methodology and objectives A traditional review approach was taken to focus on the engineering of microbial α-amylases to enhance industrially favoured characteristics. The action mechanisms of α- and β-amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial α-amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100 °C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial α-amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries. Conclusions Microbial α-amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial α-amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

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“…707 (AmyG6) [44] (PDB 2D3N) overlaps with glucose residues 2 to 4 of the BliAmy tetraose structure. In the crystal structure of B. stearothermophilus STB04 (Bst-MFA) an acarbose molecule is bound at the SBS [45] (PDB code 6AG0) and has hydrophobic interactions with F4 (L3 in BliAmy). This acarbose molecule is shifted 2 glucose units to the N-terminus of the enzyme compared to the acarbose and by one glucose unit compared to the tetraose in BliAmy.…”

Section: Presence Of the Bliamy Sbs In Other Gh13_5 Amylasesmentioning

confidence: 99%

“…A maltose molecule is stacked on that tryptophan residue in BHA (Fig. 3C) (PDB 2GJP) [46], Bst-MFA (PDB 6AG0) [45] and AmyG6 (PDB 2D3N) (72, 66 and 69% seq. identity respectively).…”

Section: Other Surface Binding Sites In Gh13_5 Glycosidasesmentioning

confidence: 99%

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

Božić

Rozeboom

Lončar³

et al. 2020

International Journal of Biological Macromolecules

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Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

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Crystal structure of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04

Cited by 17 publications

References 44 publications

A high amylase‐producing bacterial strain exhibiting the phosphorus‐dissolving ability, isolated from freshwater aquaculture pond

A high amylase‐producing bacterial strain exhibiting the phosphorus‐dissolving ability, isolated from freshwater aquaculture pond

Native to designed: microbial α-amylases for industrial applications

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

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