当前位置： SCI文献检索 > INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES期刊下所有文献 > Selection and mutational analyses of the substrate interacting residues of a chitinase from Enterobacter cloacae subsp. cloacae (EcChi2) to improve transglycosylation.

Selection and mutational analyses of the substrate interacting residues of a chitinase from Enterobacter cloacae subsp. cloacae (EcChi2) to improve transglycosylation.

Abstract：

:Transglycosylation (TG) by Enterobacter cloacae subsp. cloacae chitinase 2 (EcChi2) has been deciphered by site-directed mutagenesis. EcChi2 originally displayed feeble TG with chitin oligomer with a degree of polymerization (DP4), for a short duration. Based on the 3D modelling and molecular docking analyses, we altered the substrate interactions at the substrate-binding cleft, catalytic center, and catalytic groove of EcChi2 by mutational approach to improve TG. The mutation of W166A and T277A increased TG by EcChi2 and also affected its catalytic efficiency on the polymeric substrates. Whereas, R171A had a drastically decreased hydrolytic activity but, retained TG activity. In the increased hydrolytic activity of the T277A, altered interactions with the substrates played an indirect role in the catalysis. Mutation of the central Asp, in the conserved DxDxE motif, to Ala (D314A) and Asn (D314N) conversion yielded DP5-DP8 TG products. The quantifiable TG products (DP5 and DP6) increased to 8% (D314A) and 7% (D314N), resulting in a hyper-transglycosylating mutant. Mutation of W276A and W398A resulted in the loss of TG activity, indicating that the aromatic residues (W276 and W398) at +1 and +2 subsites are essential for the TG activity of EcChi2.

journal_name

Int J Biol Macromol

journal_title

International journal of biological macromolecules

authors

Mallakuntla MK,Vaikuntapu PR,Bhuvanachandra B,Podile AR

doi

10.1016/j.ijbiomac.2020.10.125

subject

Has Abstract

pub_date

2020-12-15 00:00:00

pages

2432-2441

issue

Pt B

eissn

0141-8130

issn

1879-0003

pii

S0141-8130(20)34755-3

journal_volume

165

pub_type

杂志文章

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