Engineering of a truncated α-amylase of Bacillus sp. strain TS-23 for the simultaneous improvement of thermal and oxidative stabilities

Chi, Meng-Chun; Chen, Yan-Hung; Wu, Tai-Jung; Lo, Huei-Fen; Lin, Long-Liu

doi:10.1016/j.jbiosc.2009.11.012

Cited by 40 publications

(21 citation statements)

References 54 publications

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“…These data may indicate that single mutagenesis is not sufficient to drastically modify the catalytic parameters of the cold-adapted enzyme to convert it to a true mesophilic α-amylase. Similar phenomena were also observed with a number of other microbial α-amylases (47–49). When the mutations were combined, the generated variant enzymes (E166P/S185P/T350P and E166P/S185P/T350P/V212T/V232T) displayed a shift in optimal temperature from 25 to 30°C that is typical of the mesophilic counterpart, Ec Amy (Fig.…”

Section: Resultssupporting

confidence: 83%

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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Section: Resultssupporting

confidence: 83%

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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“…Oxidation of the methionine residue situated in the cavity of the AmyK active site has been shown to decrease its activity or even inactivate the enzyme (11,33). The model structure of AmyK from Bacillus sp.…”

Section: Resultsmentioning

confidence: 99%

Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

Yang

Liu

et al. 2013

Appl Environ Microbiol

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In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline ␣-amylase (AmyK) from Alkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (k cat ) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK: k cat /K m increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ϳ2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H 2 O 2 , compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes. Most industrial enzymes are obtained from the natural environment. However, these enzymes are often used under conditions drastically different from their natural environment, in which the catalytic performance of the enzymes decreases, restricting their applications. Consequently, increasing attention is being paid to improving the catalytic performance of enzymes under extreme but application-relevant conditions such as high temperature, strong acid and alkali, and oxidative stress (1).Protein engineering has emerged as an important tool to overcome the limitations of natural enzymes (2). Common strategies to alter proteins include site-directed mutagenesis and directed evolution (e.g., error-prone PCR). However, site-directed mutagenesis requires a clear understanding of the relationship between enzyme structure and function, and directed evolution requires a straightforward and efficient high-throughput screening method (3-5).Alkaline ␣-amylases have high catalytic efficiency and stability at an alkaline pH range between 9 and 11 (6, 7) and are widely used in the detergent and textile industries for starch hydrolysis under alkaline conditions (8-10). This application requires high catalytic efficiency, high alkaline stability, and high oxidative stability (11). Although site-directed mutagenesis has been used to improve the oxidative stability of ␣-amylases, this was accompanied by decreased catalytic efficiency, alkaline stability,...

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“…Therefore, we selected those asparagines that are exposed to solvent and/or have relatively high B-factors (Table 2). It had been shown that the structural determinants contributing to the overall thermostability of ␣-amylases concentrate in domain B and at its interface with the central A domain [25][26][27][28]. Three out of four selected Asn residues are located in domain B (Asn129, Asn112, and Asn119).…”

Section: Choosing Targetsmentioning

confidence: 99%

Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase

Rahimzadeh

Khajeh

Mirshahi

et al. 2012

International Journal of Biological Macromolecules

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Engineering of a truncated α-amylase of Bacillus sp. strain TS-23 for the simultaneous improvement of thermal and oxidative stabilities

Cited by 40 publications

References 54 publications

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

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

Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase

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