Nattokinase (NK, EC3.4.21.62) is a bacterial serine protease derived from the traditional Japanese food natto with strong fibinolytic activity. However, the thermal stability of NK is too low to ensure high enzyme activity in the production process, and thus limits its production and application. Proteins deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartate (Asp) and glutamic acid (Glu),which may change the local structure of protein and affect the enzyme activity, pH optimum, and stability. Therefore, simulating this process can efficiently modify the target enzyme. In order to improve the activity and stability of NK, Asn and Gln located on the surface were mutated to Asp and Glu, respectively. The mutant Q59E with increased activity (about 1.54 times of the wild type) and mutant N218D with increased thermal stability were obtained. The thermal stability of the double mutant Q59E-N218D was further improved, and its half-life (t1/2, 33 min) was 2.75 times of that of the wild type NK (t1/2, 12 min). It provides a method for enzyme engineering and a new enzyme material for the industrial application of NK.
ZHAO Han
,
ZHOU Li
,
ZHOU Zhe-min
. Enhancing the thermostability and activity of nattokinase by site-directed mutagenesis[J]. Food and Fermentation Industries, 2018
, 44(9)
: 36
-40
.
DOI: 10.13995/j.cnki.11-1802/ts.016991
[1] BRYAN P N.Protein engineering of subtilisin[J].Biochimica Et Biophysica Acta,2000,1543(2):203-222.
[2] MARTINEZ R,JAKOB F,TU R,et al.Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution[J].Biotechnology & Bioengineering,2013,110(3):711-720.
[3] WENG M,DENG X,BAO W,et al.Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation[J].Biochemical & Biophysical Research Communications,2015,465(3):580-586.
[4] RUAN B,LONDON V,FISHER K E,et al.Engineering substrate preference in subtilisin:structural and kinetic analysis of a specificity mutant[J].Biochemistry,2008,47(25):6 628.
[5] DOHNALEK J,MCAULEY K,BRZOZOWSKI A,et al.Understanding enzymes:Function,design,engineering,and analysis[M].Singapore:Pan Stanford Publishing Pte. Ltd,2016:203-265.
[6] NAKAMURA T,YAMAGATA Y,ICHISHIMA E.Nucleotide sequence of the subtilisin NAT gene,aprN,of Bacillus subtilis (natto)[J].Bioscience Biotechnology & Biochemistry,1992,56(11):1 869-1 871.
[7] URANO T,IHARA H,UMEMURA K,et al.The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1[J].Journal of Biological Chemistry,2001,276(27):24 690.
[8] FUJITA M, ITO Y, HONG K,et al.Characterization of nattokinase-degraded products from human fibrinogen or cross-linked fibrin[J].Fibrinolysis,1995,9(3):157-164.
[9] DABBAGH F,NEGAHDARIPOUR M,BERENJIAN A,et al.Nattokinase:production and application[J].Applied Microbiology & Biotechnology,2014,98(22):9 199.
[10] 何孝天.Bacillus natto纳豆激酶的重组表达及热稳定性改造[D].无锡:江南大学,2014.
[11] BISCHOFF R,SCHLUTER.Amino acids:Chemistry,functionality and selected non-enzymatic post-translational modifications[J].Journal of Proteomics,2012,75(8):2 275.
[12] KATO A,TANIMOTO S,MURAKI Y,et al.Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering[J].Journal of the Agricultural Chemical Society of Japan,1992,56(9):1 424.
[13] JAKOB F,MARTINEZ R,MANDAWE J,et al.Surface charge engineering of a Bacillus gibsonii subtilisin protease[J].Applied Microbiology & Biotechnology,2013,97(15):6 793-6 802.
[14] KHURANA J,SINGH R,KAUR J.Engineering of Bacillus lipase by directed evolution for enhanced thermal stability:effect of isoleucine to threonine mutation at protein surface[J].Molecular Biology Reports,2011,38(5):2 919.
[15] PANTOLIANO M W,WHITLOW M,WOOD J F,et al.Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding[J].Biochemistry,1989,28(18):7 205-7 213.
[16] DE K A,VAN D B B,VENEMA G,et al.The effects of modifying the surface charge on the catalytic activity of a thermolysin-like protease[J].Journal of Biological Chemistry,2002,277(18):15 432.
[17] JAOUADI B,AGHAJARI N,HASER R,et al.Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis[J].Biochimie,2010,92(4):360.
[18] MIYAZAKI K,WINTRODE,GRAYLING R,et al.Directed evolution study of temperature adaptation in a psychrophilic enzyme 1[J].Journal of Molecular Biology,2000,297(4):1 015-1 026.
[19] STUDIER F W.Protein production by auto-induction in high density shaking cultures[J].Protein Expression & Purification,2005,41(1):207-234.
