title: 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.
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 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 activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::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 AmyKΔC500-587::OP and AmyKΔC492-587::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.
authors: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
authors: Haiquan
authors: H
authors: Yang
authors: Long
authors: L
authors: Liu
authors: Hyun-dong
authors: HD
authors: Shin
authors: Rachel R
authors: RR
authors: Chen
authors: Jianghua
authors: J
authors: Li
authors: Guocheng
authors: G
authors: Du
authors: Jian
authors: J
authors: Chen
mesh_terms: D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases
references: Biochim Biophys Acta. 2006 Jun;1764(6):1080-6
references: 16765657
references: Int J Mol Sci. 2013 Jan 10;14(1):1232-77
references: 23306150
references: Curr Opin Chem Biol. 1999 Feb;3(1):54-9
references: 10021399
references: Eur J Biochem. 2001 Jul;268(14):3974-82
references: 11453991
references: Biotechnol Prog. 2005 Nov-Dec;21(6):1716-23
references: 16321056
references: PLoS One. 2013;8(3):e57403
references: 23554859
references: Biochem J. 2000 Sep 1;350 Pt 2:477-84
references: 10947962
references: FEBS Lett. 2006 Feb 6;580(3):763-8
references: 16413550
references: Appl Microbiol Biotechnol. 2011 Jul;91(2):329-39
references: 21479716
references: J Agric Food Chem. 2010 Oct 27;58(20):10899-905
references: 20873770
references: Curr Opin Struct Biol. 2007 Aug;17(4):454-9
references: 17884462
references: Appl Environ Microbiol. 2013 May;79(9):3049-58
references: 23455344
references: Bioinformatics. 2011 Feb 1;27(3):343-50
references: 21134891
references: Biotechnol Bioeng. 2009 Jul 1;103(4):676-86
references: 19288442
references: Electrophoresis. 1997 Dec;18(15):2714-23
references: 9504803
references: Biotechnol Bioeng. 2009 Feb 1;102(2):380-9
references: 18951544
references: Chem Biol. 2012 Mar 23;19(3):414-21
references: 22444596
references: Biochim Biophys Acta. 1993 Jan 18;1145(1):180-2
references: 8422410
references: Appl Microbiol Biotechnol. 2008 Feb;78(1):95-103
references: 18080120
references: Nat Struct Biol. 1999 May;6(5):432-6
references: 10331869
references: J Mol Biol. 1999 Jan 15;285(2):775-83
references: 9878443
references: J Mol Biol. 2005 Oct 14;353(1):26-37
references: 16168439
references: Curr Opin Microbiol. 2010 Jun;13(3):274-82
references: 20171138
references: Biotechnol J. 2006 Nov;1(11):1258-65
references: 17068753
references: BMC Biochem. 2011 May 31;12:30
references: 21624144
references: Biochemistry. 2005 Jun 28;44(25):9197-205
references: 15966744