Recombinant exochitinase of the thermophilic mould Myceliopthora thermophila BJA: Characteristics and utility in generating N-acetyl glucosamine and in biocontrol of phytopathogenic fungi.
Chitinase from the thermophilic mould Myceliopthora thermophila BJA (MtChit) is an acid tolerant, thermostable and organic solvent stable biocatalyst which does not require any metal ions for its activity. To produce high enzyme titres, reduce fermentation time and overcome the need for induction, this enzyme has been heterologously expressed under GAP promoter in the GRAS yeast, Pichia pastoris. The production medium supplemented with the permeabilizing agent Tween-20 supported two-fold higher rMtChit production (5.5 × 103U L-1). The consensus sequences S(132)xG(133)G(134) and D(168)xxD(171)xD(173)xE(175) in the enzyme have been found to represent the substrate binding and catalytic sites, respectively. The rMtChit, purified to homogeneity by a two-step purification strategy, is a monomeric glycoprotein of ∼48 kDa, which is optimally active at 55°C and pH 5.0. The enzyme is thermostable with t1/2values of 113 and 48 min at 65 and 75°C, respectively. Kinetic parameters Km, Vmax, kcat, and kcat/Kmof the enzyme are 4.655 mg mL-1, 34.246 nmol mg-1s-1, 3.425 × 106min-1, and 1.36 × 10-6mg mL-1min-1, respectively. rMtChit is an unique exochitinase, since its action on chitin liberates N-acetylglucosamine NAG. The enzyme inhibits the growth of phytopathogenic fungi like Fusarium oxysporum and Curvularia lunata, therefore, this finds application as biofungicide at high temperatures during summer in tropics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:70-80, 2017.
Dept. of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India.
Ashima
A
Dua
Dept. of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India.
Swati
S
Joshi
Dept. of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India.
T
T
Satyanarayana
D000117:Acetylglucosamine / Q000096:biosynthesis*; D002686:Chitin / Q000096:biosynthesis / Q000378:metabolism; D005285:Fermentation; D005658:Fungi / Q000254:growth & development* / Q000472:pathogenicity; D006596:Hexosaminidases / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D020037:Sordariales / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002686:Chitin; C079639:1,4-beta-chitobiosidase; D006596:Hexosaminidases; D000117:Acetylglucosamine
Myceliopthora thermophila BJA; Pichia pastoris; biocontrol; constitutive expression; recombinant exochitinase