Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of <i>Bacillus stearothermophilus</i> US100 strain

Ali, Mamdouh Ben; Khemakhem, Bassem; Robert, Xavier; Haser, R.; Béjar, Samír

doi:10.1042/bj20050726

Cited by 63 publications

(25 citation statements)

References 33 publications

(45 reference statements)

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“…The results demonstrated that the maximal thermostability was increased by ten degrees compared with the wild type BAA [92]. Similar studies by Mamdouh et al [94] reported that the amylase of Bacillus stearothermophilus US100 has an additional loop compared with the model of BLA. The deletion of two residues (Ile214 and Gly215) increased thermostability and reduced calcium requirements.…”

Section: Amylasesupporting

confidence: 53%

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

2016

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Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes.

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Section: Amylasesupporting

confidence: 53%

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

2016

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“…Analysis of refined modeled structures of WT and mutated enzymes, which were simulated to mimic experimental conditions through atomistic molecular dynamic simulations at 27 and 80°C beside the biochemical analyses achievements, revealed that among manipulated phytases, the P9 and P12 mutants reached the desired thermostability and higher melting temperature simultaneously with catalytic efficiency improvement. Several recent studies have succeeded in combining thermostability with other enzyme properties such as hydrolysis profile [35] and substrate specificity [36]. In this study, we herein achieved improvement in both the thermostability and catalytic efficiency of mutants P9 and P12, suggesting that thermostability and catalytic efficiency may be fairly independent in the case of PhyA phytases, and both of them can be optimized at the same time.…”

Section: Discussionmentioning

confidence: 69%

Enhancement of Thermostability and Kinetic Efficiency of Aspergillus niger PhyA Phytase by Site-Directed Mutagenesis

Hesampour

Siadat

Malboobi

et al. 2014

Appl Biochem Biotechnol

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Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P < 0.05) with 24 and 22.6 % greater retention, respectively, compared with the PhyA of the wild type at 80 °C. The K m value of the improved thermostable P9 and P12 mutant enzymes for sodium phytate were 35 and 20 % lower (P < 0.05) with respect to the wild-type enzyme. In conclusion, it is feasible to simultaneously improve the thermostability and the catalytic efficiency of phytase to be used as an animal feed supplement.

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“…Among the ␣-amylases, alkaline ␣-amylases are of special interest because they work under alkaline conditions encountered in the starch, detergent, and textile industries (2,3).…”

mentioning

confidence: 99%

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Liu

Deng

Yang

et al. 2014

Appl Environ Microbiol

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High thermostability is required for alkaline ␣-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline ␣-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants-P35C-G426C, G116C-Q120C, and R436C-M480C-of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (k cat /K m ) increased from 1.8 ؋ 10 4 to 2.4 ؋ 10 4 liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.

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Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain

Cited by 63 publications

References 33 publications

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

Enhancement of Thermostability and Kinetic Efficiency of Aspergillus niger PhyA Phytase by Site-Directed Mutagenesis

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

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