Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions

Mabrouk, Sameh; Aghajari, Nushin; Ali, Mamdouh Ben; Messaoud, Ezzedine Ben; Juy, Michel; Haser, R.; Béjar, Samír

doi:10.1016/j.biortech.2010.08.082

Cited by 38 publications

(12 citation statements)

References 40 publications

(43 reference statements)

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“…It can be a limiting factor in the selection of enzymes for industrial applications due to the elevated temperatures or the extreme pH of many biotechnological processes. The stability of an enzyme can be improved by site-directed mutagenesis (Ben Mabrouk et al, 2011;Ghollasi et al, 2010;Leemhuis et al, 2004;Liu et al, 2008;Yin et al, 2011). One of the most exhaustive efforts was done by Palackal and coworkers (Palackal et al, 2004), who used saturation mutagenesis for each of the 189 amino acid residues of a xylanase.…”

Section: Industrial Uses Of Glycosyl Hydrolasesmentioning

confidence: 99%

Site-Directed Mutagenesis as Applied to Biocatalysts

Damián-Almazo¹,

Saab‐Rincón²

2013

Genetic Manipulation of DNA and Protein - Examples From Current Research

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Section: Industrial Uses Of Glycosyl Hydrolasesmentioning

confidence: 99%

Site-Directed Mutagenesis as Applied to Biocatalysts

Damián-Almazo¹,

Saab‐Rincón²

2013

Genetic Manipulation of DNA and Protein - Examples From Current Research

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“…One way to solve this problem is to modify existing cold-adapted enzymes by site-directed mutagenesis combined with a rational design approach (28), which generally is based on structure-guided consensus sequence alignments with sequences that have moderate or high amino acid identity to reduce the number of possible target residues to be mutated. Previously, rational design principles were successfully utilized to enhance the stability and/or catalytic efficiency of enzymes such as maltogenic amylase and patatin-like phospholipase (29–31). These changes reduced the molecular flexibility of the polypeptide by (i) introducing hydrophobic residues in the protein core, (ii) introducing disulfide and salt bridges to increase electrostatic interactions in the polypeptide, (iii) stabilizing α-helix dipoles, (iv) introducing Pro residues, and (v) loop shortening to decrease loop entropy (28, 32).…”

Section: Introductionmentioning

confidence: 99%

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

Yang

Mozzicafreddo

et al. 2017

Appl Environ Microbiol

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The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

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“…Maltogenic amylases are widely distributed in microorganisms, plants, and higher organisms and constitute a subfamily of amylolytic enzymes [ 1 , 2 ]. Through their transglycosylation activity, they were responsible for the solubility increase, the oxidative stability, the sweetness, and the carcinogenicity decrease [ 3 , 4 ].…”

Section: Introductionmentioning

confidence: 99%

Optimization, Purification, and Starch Stain Wash Application of Two Newα-Amylases Extracted from Leaves and Stems ofPergularia tomentosa

Lahmar

Abed

Khemakhem

et al. 2017

BioMed Research International

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A continuous research is attempted to fulfil the highest industrial demands of natural amylases presenting special properties. New α-amylases extracted from stems and leaves of Pergularia tomentosa, which is widespread and growing spontaneously in Tunisia, were studied by the means of their activities optimization and purification. Some similarities were recorded for the two identified enzymes: (i) the highest amylase activity showed a promoted thermal stability at 50°C; (ii) the starch substrate at 1% enhanced the enzyme activity; (iii) the two α-amylases seem to be calcium-independent; (iv) Zn2+, Cu2+, and Ag2+ were considered as important inhibitors of the enzyme activity. Following the increased gradient of elution on Mono Q-Sepharose column, an increase in the specific activity of 11.82-fold and 10.92-fold was recorded, respectively, for leaves and stems with the presence of different peaks on the purification profiles. Pergularia amylases activities were stable and compatible with the tested commercial detergents. The combination of plant amylase and detergent allowed us to enhance the wash performance with an increase of 35.24 and 42.56%, respectively, for stems and leaves amylases. Characterized amylases were reported to have a promoted potential for their implication notably in detergent industry as well as biotechnological sector.

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Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions

Cited by 38 publications

References 40 publications

Site-Directed Mutagenesis as Applied to Biocatalysts

Site-Directed Mutagenesis as Applied to Biocatalysts

Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

Optimization, Purification, and Starch Stain Wash Application of Two Newα-Amylases Extracted from Leaves and Stems ofPergularia tomentosa

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