High-level expression and characterization of GH61 glycoside hydrolase from Trichoderma reesei

Abstract:

[Objective] The GH61 family glycoside hydrolases exhibit glucan oxidative activity; they randomly oxidize glucan and thus partially destroy the crystallized structure of lignin cellulose, overcoming the major hurdle for the activity of cellulase upon lignin cellulose. In this work, a GH61 family glycoside hydrolase in Trichoderma reesei (TrGH61, previous name is endoglucanase IV) was obtained through homologous expression and purification, and its function in enzymatic degradation of lignin cellulose was studied. [Methods] The promoter of T. reesei pyvurate decarboxylase gene, the signal peptide of cellubiohydrolase, the endoglucanase IV gene, and the terminal region of the PDC gene were sequentially ligated through overlap PCR, forming the expression cassette of TrGH61. The expression cassette was then transformed into T. reesei QM9414 with the aid of plas-mid pAN7.1, and TrGH61 was homologously expressed and purified to homogeniety. The hydro-lytic activity towards cellulosic materials, the synergistic effect with cellulase and the oxidase ac-tivity were studied. [Results] Highly efficient production of the TrGH61 was achieved under the control of the PDC promoter, and the expressing level was 2.33 g/L in shake flask cultivation. The expressed TrGH61 exhibited feint endoglucanase activity, with a specific activity of 0.02 IU/mg. Moreover, it enhanced the activity of cellulase towards straw mill when it was added into the reac-tion mixture, with a synergistic degree of 1.998. Metal ions Cu2+ and Co2+, and electron donor re-duced glutathione, L-ascorbic acid, and pyrogallic acid, enhanced the activity of TrGH61 effectively. The cellulose crystallinity and degree of polymerization of rice straw were decreased by TrGH61. [Conclusion] TrGH61 can be effectively expressed with the T. reesei PDC promoter, and the puri-fied TrGH61 can act as a cellulase activity enhancer, improving the action of cellulose by destroy the crystal structure of cellulose on lignin cellulose.