Expression and characterization of a cellobiohydrolase (CBH7B) from the thermophilic fungus Thielavia terrestris in Pichia pastoris

Abstract

The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.

Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus

“…CBHs are exo-acting enzymes that cleave from the opposite ends of cellulose chains [12][13][14][15] and are only found in the hindgut of lower termites [7,14,15]. EGLs are endo-acting enzymes that initiate random cuts in the central region of cellulose chains.…”

“…BGLs are cellobiases that cleave cellobioses into glucose subunits [12]. Both EGLs and BGLs are found in the midgut and hindgut of lower termites [6].…”

Peer Review #2 of "Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches (v0.1)"

“…Other reports of CBHs derived from P. pastoris mentioned decreased activity towards insoluble crystalline substrates such as Avicel ® or bacterial microcrystalline cellulose (BMCC) (Boer, Teeri & Koivula, 2000;Bronnenmeier, Rücknagel & Staudenbauer, 1991;Kanokratana et al, 2008;Woon et al, 2017). Most probably, this decreased activity is linked to the high glycan content (both N-and O-linked) of CBHs that potentially perturbs folding of the enzyme, substrate binding and enzyme activity (Gao et al, 2012;Woon et al, 2016b). The K m and V max of purified CBHB were calculated to be 0.25 mM and 1.41 U mg -1 , respectively using MUC as the substrate (Fig.6).…”

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“…Other reports of CBHs derived from P. pastoris mention decreased activity towards insoluble crystalline substrates such as Avicel ® or bacterial microcrystalline cellulose (BMCC) (Boer, Teeri & Koivula, 2000;Bronnenmeier, Rücknagel & Staudenbauer, 1991;Kanokratana et al, 2008;Woon et al2017). Most probably, this decreased activity is linked to the high glycan content (both N-and O-linked) of CBHs that potentially perturbs folding of the enzyme, substrate binding and enzyme activity (Gao et al, 2012;Woon et al, 2016b). K m and V max of purified CbhB were calculated to be 0.25 mM and 1.41 U mg -1 , respectively using MUC as the substrate (Fig.6).…”

“…According to the glycoside hydrolase classification system, fungi generally produce two classes of CBHs (Henrissat & Bairoch, 1996). CBHs that belong to glycoside hydrolase (GH) family 6 (EC 3.2.1.176) are inverting enzymes that cleave from the reducing ends of cellulose chains whilst CBHs of GH family 7 (EC 3.2.1.91) are retaining enzymes that cleave from the non-reducing termini of cellulose chains (Woon et al, 2016b). Although heterologous expression of CBHs has been widely reported in yeasts (Den Haan et al, 2013) and fungi (Zoglowek et al, 2015), the use of recombinant cellobiohydrolases in the biomass conversion industry has been limited by their expression levels and unpredictable activity profiles caused by non-native glycosylation patterns of the expression hosts (Gao et al, 2012;Jeoh et al, 2008).…”