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In silico rational design and systems engineering of disulfide bridges in the catalytic domain of an alkaline ?-amylase from Alkalimonas amylolytica to improve thermostability.

ABSTRACT: 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 (kcat/Km) 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.

SUBMITTER: Liu L

PROVIDER: S-EPMC3911192 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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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 Long L Deng Zhuangmei Z Yang Haiquan H Li Jianghua J Shin Hyun-dong HD Chen Rachel R RR Du Guocheng G Chen Jian J

Applied and environmental microbiology 20131108 3

High thermostability is required for alkaline α-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 α-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 engineeri ...[more]

PMID: 24212581

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Project description:High oxidative stability and catalytic efficiency are required for the alkaline ?-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline ?-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317) were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I) were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants), the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline ?-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems engineering strategy may be useful for the protein engineering of the other microbial enzymes to fulfill industrial requirements.

Project description: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.

Dataset Information

In silico rational design and systems engineering of disulfide bridges in the catalytic domain of an alkaline ?-amylase from Alkalimonas amylolytica to improve thermostability.

Publications

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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