Rational introduction of disulfide bond to enhance optimal temperature of Lipomyces starkeyi alpha-dextranase expressed in Pichia pastoris.
Alpha-dextranase, which can hydrolyze dextran, is largely used in the sugar industry. However, a thermostable alpha- dextranase is needed to alleviate the viscosity of syrups and clean blocked machines. Thus, to improve the optimal temperature of Lipomyces starkeyi alpha-dextranase expressed by Pichia pastoris, the rational introduction of a de novo designed disulfide bond was investigated. Based on the known structure of Penicillium minioluteum dextranase, L. starkeyi alpha-dextranase was constructed using homology modeling. Four amino acids residues were then selected for site-directed mutagenesis to cysteine. When compared with the wildtype dextranase, the mutant DexM2 (D279C/S289C) showed a more than 13oC improvement on its optimal temperature. DexM2 and DexM12 (T245C/N248C, D279C/S289C) also showed a better thermal stability than the wild-type dextranase. After the introduction of two disulfide bonds, the specific activity of DexM12 was evaluated and found to be two times higher than that of the wild-type. Moreover, DexM12 also showed the highest Vmax.
Chen
Lin
L
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Yu
Chao
C
Zhou
Xiangshan
X
Zhang
Yuanxing
Y
D019943:Amino Acid Substitution; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003910:Dextranase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D007218:Industrial Microbiology; D055160:Lipomyces / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D004220:Disulfides; D011994:Recombinant Proteins; D003910:Dextranase; D003545:Cysteine