Improved production of recombinant β-mannanase (TaMan5) in Pichia pastoris and its synergistic degradation of lignocellulosic biomass

Abstract

Mannan, a highly abundant and cost-effective natural resource, holds great potential for the generation of high-value compounds such as bioactive polysaccharides and biofuels. In this study, we successfully enhanced the expression of constructed GH5 β-mannanase (TaMan5) from Trichoderma asperellum ND-1 by employing propeptide in Pichia pastoris. By replacing the α-factor with propeptide (MGNRALNSMKFFKSQALALLAATSAVA), TaMan5 activity was significantly increased from 67.5 to 91.7 U/mL. It retained higher activity in the presence of 20% ethanol and 15% NaCl. When incubated with a high concentration of mannotriose or mannotetraose, the transglycosylation action of TaMan5 can be detected, yielding the corresponding production of mannotetraose or mannooligosaccharides. Moreover, the unique mechanism whereby TaMan5 catalyzes the degradation of mannan into mannobiose involves the transglycosylation of mannose to mannotriose or mannotetraose as a substrate to produce a mannotetraose or mannopentose intermediate, respectively. Additionally, the production of soluble sugars from lignocellulose is a crucial step in bioethanol development, and it is noteworthy that TaMan5 could synergistically yield fermentable sugars from corn stover and bagasse. These findings offered valuable insights and strategies for enhancing β-mannanase expression and efficient conversion of lignocellulosic biomass, providing cost-effective and sustainable approaches for high-value biomolecule and biofuel production.

Keywords: Trichoderma asperellum; catalysis mechanism; improved production; synergism; transglycosylation; β-mannanase.

FIGURE 1

Schematic representation of recombinant strains α-oTaMan5 and p-oTaMan5.

FIGURE 2

High-efficiency expression of TaMan5 in Pichia pastoris. (A) Extracellular enzyme activity of recombinant strains α-oTaMan5 and p-oTaMan5. (B) SDS-PAGE analysis of proteins expressed by p-oTaMan5. 1, 24 h; 2, 48 h; 3, 72 h; 4, 96 h; 5, 120 h; and 6, 144 h.

FIGURE 3

Effect of varying ethanol (A) and NaCl (B) concentrations on TaMan5 activity.

FIGURE 4

Transglycosylation reaction of TaMan5 by TLC analysis. (A, B) Time-course hydrolysis of mannotetraose and mannotriose by TaMan5. Mannohexaose (M6), mannopentaose (M5), mannotetraose (M4), mannotriose (M3), mannobiose (M2), and mannose (M1). Substrates containing inactive enzymes were used as blank controls (0 min).

FIGURE 5

Catalytic mechanism of TaMan5. (A–H) Molecular docking studies of TaMan5 showing substrates and intermediate products in predicted active sites during the hydrolysis of mannotetraose (A–D), mannotriose (E–H), and mannan (J). (I) Schematic model of the hydrolysis mechanism of mannotriose and mannotetraose by TaMan5. (K) Enzyme structure of the catalytic region showing distances (Å) among catalytic residues.

FIGURE 6

Synergistic action of TaMan5 with the addition of commercial cellulase (CEL) and β-xylanase (XYL) on the degradation of corn stover (A) and bagasse (B). Data are presented as the mean ± SD (n = 3).