Expression and Characteristics of Two Glucose-Tolerant GH1 β-glucosidases From Actinomadura amylolytica YIM 77502T for Promoting Cellulose Degradation

Abstract

The bioconversion of lignocellulose in various industrial processes, such as biofuel production, requires the degradation of cellulose. Actinomadura amylolytica YIM 77502^T is an aerobic, Gram-positive actinomycete that can efficiently degrade crystalline cellulose by extracellular cellulases. Genomic analysis of A. amylolytica identified 9 cellulase and 11 β-glucosidase genes that could potentially encode proteins that digest cellulose. Extracellular proteome characterization of A. amylolytica cell-free culture supernatant by liquid chromatography tandem mass spectrometry analysis revealed that 4 of these cellulases and 2 of these β-glucosidases functioned during cellulose hydrolysis. Thin-layer chromatography analysis revealed extracellular β-glucosidases play a major role in carboxyl methyl cellulose (CMC) degradation of products in culture supernatants. In this study, 2 of the identified secreted β-glucosidases, AaBGL1 and AaBGL2, were functionally expressed in Escherichia coli and found to have β-glucosidase activity with wide substrate specificities, including for p-nitrophenyl β-D-glucopyranoside (pNPG), p-nitrophenyl-beta-D-cellobioside (pNPC), and cellobiose. Moreover, AaBGL1 and AaBGL2 had high tolerances for glucose. After adding these β-glucosidases to commercial cellulases, the degradation rates of CMC, Avicel, birch sawdust, and corncob powder increased by 37, 42, 33, and 9%, respectively. Overall, this work identifies an alternative potential source of β-glucosidases with potential applications in commercial cellulose utilization and the bioenergy industry.

Keywords: Actinomadura amylolytica; GH1; cellulose degradation; glucose tolerance; β-glucosidase.

FIGURE 1

Glycoside hydrolase (GH) families of Actinomadura amylolytica YIM 77502^T. (A) CAZyme analysis. (B) Number of GH genes. Histogram colors indicate different functions: yellow indicates predicted β-glucosidase, green indicates predicted cellulase, and blue indicates other predicted functions.

FIGURE 2

Cellulase activity of A. amylolytica YIM strain 77502^T. (A) Hydrolysis experiment using filter paper. (B) Thin-layer chromatography plate analysis of hydrolytic products of carboxyl methyl cellulose (CMC) in fermentation broth of A. amylolytica, which was cultured with microcrystalline cellulose. (C) CMCase activity of fermentation broth. (D) β-glucosidase activity of fermentation broth.

FIGURE 3

Glycoside hydrolases in the secretome of A. amylolytica.

FIGURE 4

Three-dimensional model and purification of β-glucosidases AaBGL1 and AaBGL2. Structural models of (A) AaBGL1 and (B) AaBGL2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of recombinant (C) AaBGL1 and (D) AaBGL2 produced by E. coli BL21. Lane 1, protein molecular weight marker, mass indicated on the left; lane 2, total protein in IPTG-induced E. coli BL21/pET28a-aabgl1 or pET28a-aabgl2; lane 3, purified AaBGL1 or AaBGL2.

FIGURE 5

Effects of temperature and pH on the activity and stability of the recombinant AaBGL1 and AaBGL2. (A,B) Effect of (A) temperature and (B) pH on the activity of the recombinant β-glucosidases. The primary activity was designated 100%. Each value in the figure represents the mean ± standard deviation (n = 3). AaBGL1: 100% = 6.2 U/mg. AaBGL2 100% = 5.6 U/mg.

FIGURE 6

Effect of glucose concentration on recombinant (A) AaBGL1 and (B) AaBGL2.

FIGURE 7

Cooperation of AaBGL1 and AaBGL2 with commercial cellulases in cellulosic material degradation.

FIGURE 8

Schematic diagram of hydrolytic cellulose from A. amylolytica YIM 77502^T.