Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli

et al. 2013

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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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

et al. 2013

“…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).…”

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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

Deng

et al. 2014

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.

“…Alkaline ␣-amylases have high catalytic efficiency and stability at alkaline pHs from 9 to 11 (5,9,11,17) and have applications in the starch and textile industries, where starch is hydrolyzed under alkaline conditions (5,10,16,21,22).…”

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confidence: 99%

“…␣ -Amylases hydrolyze starch by cleaving ␣-1,4-glucosidic linkages and have been used widely in the food, textile, and pharmaceutical industries (10,16,(20)(21)(22)25). Alkaline ␣-amylases have high catalytic efficiency and stability at alkaline pHs from 9 to 11 (5, 9, 11, 17) and have applications in the starch and textile industries, where starch is hydrolyzed under alkaline conditions (5,10,16,21,22).Oxidative stability is one of the most important quality parameters for alkaline amylase, especially in detergents where the washing environment is oxidizing (13). Methionine (Met) residues in proteins are especially oxidation prone (4, 27).…”

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confidence: 99%

Structure-Based Engineering of Methionine Residues in the Catalytic Cores of Alkaline Amylase from Alkalimonas amylolytica for Improved Oxidative Stability

Wang

et al. 2012

dThis work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H 2 O 2 at 35°C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, the k cat values of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 ؋ 10 4 to 5.6 ؋ 10 4 min ؊1 . The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries. ␣ -Amylases hydrolyze starch by cleaving ␣-1,4-glucosidic linkages and have been used widely in the food, textile, and pharmaceutical industries (10,16,(20)(21)(22)25). Alkaline ␣-amylases have high catalytic efficiency and stability at alkaline pHs from 9 to 11 (5, 9, 11, 17) and have applications in the starch and textile industries, where starch is hydrolyzed under alkaline conditions (5,10,16,21,22).Oxidative stability is one of the most important quality parameters for alkaline amylase, especially in detergents where the washing environment is oxidizing (13). Methionine (Met) residues in proteins are especially oxidation prone (4, 27). The oxidation of this residue has been shown to cause a decrease in activity or an outright inactivation of amylases (12, 23). To reduce inactivation caused by oxidation, the replacement of methionine by an oxidative-resistant amino acid may be effective. The nonoxidizable amino acid residues include leucine (Leu), serine (Ser), isoleucine (Ile), threonine (Thr), and alanine (Ala). For example, Met 197 of the ␣-amylase from Geobacillus stearothermophilus US110 was replaced with Ala, and the mutant retained 70% of the activity in the pre...