title: 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.
abstract: 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 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.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Zhuangmei
authors.initials: Z
authors.lastname: Deng
authors.forename: Haiquan
authors.initials: H
authors.lastname: Yang
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Hyun-dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Chen
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D020134:Catalytic Domain; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D000516:alpha-Amylases; D003545:Cysteine