Functional and cooperative stabilization of a two-metal (Ca, Zn) center in α-amylase derived from Flavobacteriaceae species

Yin, Haidi; Zhou, Yang; Nie, Xinyu; Li, Shannan; Sun, Xuyang; Gao, Chao; Wang, Zenghang; Zhou, Guangming; Xu, Ping; Yang, Chunyu

doi:10.1038/s41598-017-18085-4

Cited by 17 publications

(6 citation statements)

References 37 publications

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“…SPR results (Figure 3b-d Fallal, 2017;Kuddus, 2017;Yin et al, 2017). Lower concentration salt may improve the stability of α-amylase activity.…”

Section: Effect Of Ph and Salt On The Interaction Between Three Flavo...mentioning

confidence: 95%

“…I G U R E 3 SPR response for the interaction of three flavonoids (200 μM) and α-amylase (300 μM) with various pH values (a), various concentrations of KCl (b), various concentrations of MgCl 2 (c), and various concentrations of CaCl 2 (d) at room temperature is 0.150 M. Similar results are also observed from Figure 3d, the optimal CaCl 2 concentration is 0.100 M for the binding of acacetin, buddleoside with α-amylase. On the other hand, many studies have confirmed that some metal ions (K + , Mg 2+ , and Ca 2+ ) can maintain the maximum reactivity of the enzyme (El-Sayed,Abou-Dobara, & El- Fallal, 2017;Kuddus, 2017;Yin et al, 2017). On the one hand, α-amylase is a metalloenzyme (Gupta, Gigras, Mohapatra, Goswami, & Chauhan, 2003),and Cl − is activator of the α-amylase(Kuriki & Imanaka, 1999).…”

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confidence: 99%

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An assessment of the interaction for three Chrysanthemum indicum flavonoids and α‐amylase by surface plasmon resonance

Huang

Yin

et al. 2019

Food Science & Nutrition

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This study evaluated the interaction of Chrysanthemum indicum (CI) flavonoids (luteolin, acacetin, and buddleoside) with α‐amylase. Surface plasmon resonance (SPR) assay showed their equilibrium dissociation constants (KD) are 1.9695 ± 0.12, 2.9240 ± 0.20, and 3.2966 ± 0.08 mM at pH 6.0, respectively. Furthermore, their binding affinities were influenced by KCl, MgCl2, and CaCl2. Enzymatic kinetic studies revealed that three flavonoids exhibited noncompetitive α‐amylase inhibitory activity. The inhibitory sequence is luteolin > acacetin > buddleoside, which was in accordance with the results of binding affinity from SPR. 1,1‐diphenyl‐2‐picryl hydrazyl radical assay demonstrated that antioxidant activities of three flavonoids were inhibited significantly with α‐amylase. Meanwhile, the study reveals that hydroxyl on C′‐4, C′‐5, and C‐7 of flavonoids play an important role on the interaction of three flavonoids with α‐amylase. Also, SPR could be used as sensor for rapid screening inhibitors of α‐amylase and provide useful information for the application of C. indicum flavonoids in food and pharmaceutical area.

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“…SPR results (Figure 3b-d Fallal, 2017;Kuddus, 2017;Yin et al, 2017). Lower concentration salt may improve the stability of α-amylase activity.…”

Section: Effect Of Ph and Salt On The Interaction Between Three Flavo...mentioning

confidence: 95%

mentioning

confidence: 99%

An assessment of the interaction for three Chrysanthemum indicum flavonoids and α‐amylase by surface plasmon resonance

Huang

Yin

et al. 2019

Food Science & Nutrition

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“…In spite of the increased activity (3.2%) detected by introducing a new disulphide linkage in FSA at E200C and H201C, enhanced ligand-dependent thermostability i.e., Ca 2+ and Zn 2+ was detected after the incubation at 50C for 30 min when E204G and C214S mutations were performed ( Yin et al, 2017 ). Nonetheless, the improvement in thermostability ( t 12 at 95C) at 26-fold (Ca 2+ -absent) and 5-fold (Ca 2+ -present) was observed when the loop deletion of I181 and G182 (IG) coupled with N193F and S242A were performed on the maltohexaose-forming -amylase from B. stearothermophilus (AmyMH) ( Li, Duan & Wu, 2016 ).…”

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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“…These metals could be bridged by a residue which is specific to the enzyme [15]. Yin and coauthors [31] [32]. Babu & Satyanarayana [33] also reported that while calcium inhibited the activity of the α-amylase from Bacillus coagulans B49, Mn 2+ slightly stimulated the amylase activity.…”

Section: Reactivation Of Rsda With Different Metal Cationsmentioning

confidence: 99%

Impact of Metal Ion Substitution on the Activity and Stability of Saccharifying Raw Starch Digesting Amylase from Aspergillus carbonarius

Nwagu

Aoyagi

Okolo

2020

JABB

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Though raw starch digesting amylases can be utilized in numerous bioprocesses, poor activity and stability remain a limiting factor. In this study, the effect of metal ion substitution on the activity and stability of the RSDA from Aspergillus carbonarius was investigated. The amylase was inactivated using the chelating agent ethylene di aminotetraacetic acid (EDTA) and reactivated using different metal ions. The effect of different metal ions on the reactivation of the amylase activity was investigated. Impact of the metal ions on the stability of the amylase was also studied. Kinetic constants of the native enzyme were compared to the metal reactivated holoenzyme. Most efficient was 5 mM concentration of Co2+ with 94.6% activity recovery. Others included 5 mM Zn2+ (77.7%) and 5 mM Ca2+ (68.7%). Incubating the Co2+ activated amylase in 10 mM Mn2+ further stimulated the activity of the amylase to 136.7%. Compared to the metal ions tested, Mn2+ had the most stabilizing effect on the amylase; the amylase exhibited 148.2% and 136.5% activity at 70ºC and 80ºC respectively in the presence of 5 mM Mn2+. Ca2+ inhibited the amylase activity and inhibition rate increased with increasing concentration of Ca2+ concentrations. Km of the reactivated amylase was 0.18 mg/ml.

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Functional and cooperative stabilization of a two-metal (Ca, Zn) center in α-amylase derived from Flavobacteriaceae species

Cited by 17 publications

References 37 publications

An assessment of the interaction for three Chrysanthemum indicum flavonoids and α‐amylase by surface plasmon resonance

An assessment of the interaction for three Chrysanthemum indicum flavonoids and α‐amylase by surface plasmon resonance

Native to designed: microbial α-amylases for industrial applications

Impact of Metal Ion Substitution on the Activity and Stability of Saccharifying Raw Starch Digesting Amylase from Aspergillus carbonarius

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