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Integrating terminal truncation and oligopeptide fusion for a novel protein engineering strategy to improve specific activity and catalytic efficiency: alkaline ?-amylase as a case study.

ABSTRACT: In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline ?-amylase (AmyK) from Alkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyK?C500-587::OP and AmyK?C492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. The kcat/Km was increased from 1.0 × 10(5) liters · mol(-1) · s(-1) for AmyK to 30.6 × and 23.2 × 10(5) liters · mol(-1) · s(-1) for AmyK?C500-587::OP and AmyK?C492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.

SUBMITTER: Yang H

PROVIDER: S-EPMC3811186 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Integrating terminal truncation and oligopeptide fusion for a novel protein engineering strategy to improve specific activity and catalytic efficiency: alkaline α-amylase as a case study.

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

Applied and environmental microbiology 20130816 20

In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) from Alkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activitie ...[more]

PMID: 23956385

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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.

Dataset Information

Integrating terminal truncation and oligopeptide fusion for a novel protein engineering strategy to improve specific activity and catalytic efficiency: alkaline ?-amylase as a case study.

Publications

Integrating terminal truncation and oligopeptide fusion for a novel protein engineering strategy to improve specific activity and catalytic efficiency: alkaline α-amylase as a case study.

Similar Datasets

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