High efficient degradation of glucan/glucomannan to cello-/mannan-oligosaccharide by endoglucanase via tetrasaccharide as intermediate.

Affiliation

Miao T(1), Basit A(2), Wen J(1), Liu J(1), Zheng F(1), Cao Y(3), Jiang W(4).
Author information:
(1)State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
(2)State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; Department of Microbiology and Molecular Genetics, Faculty of Life Sciences, University of Okara, Punjab 56300, Pakistan.
(3)State Key Laboratory of Animal Nutrition, China Agricultural, University, Beijing, China. Electronic address: [Email]
(4)State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China. Electronic address: [Email]

Abstract

Here, we report an efficient endoglucanase from Aureobasidium pullulans (termed ApCel5A) was expressed in Pichia pastoris. ApCel5A shows two different enzyme activities of endoglucanase (1270 U/mg) and mannanase (31.2 U/mg). Through engineering the signal peptide and fed-batch fermentation, the enzyme activity of endoglucanase was improved to 6.63-folds, totally. Its efficient synergism with Celluclast 1.5 L, excellent tolerance to low pH (2.5), cholate and protease suggests potential application in bioresources, food and feed industries. Site-directed mutagenesis experiments present that ApCel5A residues Glu245 and Glu358 are key catalytic sites, while Asp118, Asp122, Asp198 and Asp314 play an auxiliary role. More importantly, ApCel5A display high degradation efficiency of glucan and glucomannan substrates by using tetrasaccharide contained reducing end of glucose residue as an intermediate. This study elucidated the effective methods to improve an endoglucanase expression and detailed catalytic mechanism for degradation of various substrates, which provides a new insight for endoglucanase application.