Characterization of a novel GH5 β-mannanase from Hahella sp. CR1 and its synergistic role in degradation of spent coffee grounds

Abstract

An efficient enzymatic degradation of plant biomass is essential for agricultural waste valorization and biofuel production. β-Mannanase is a key lignocellulolytic enzyme that targets mannan, a major component in plant cell walls. Hahella spp. are known to produce cellulolytic enzymes, but no β-mannanase was characterized from this genus up to date. In this study, a gene encoding for β-mannanase (HcrMan1) was identified and isolated from the genome of Hahella sp. CR1. HcrMan1 encodes a multidomain enzyme comprising a GH5 subfamily 8 catalytic domain, an immunoglobulin-like (Ig) linker domain, and a CBM2 substrate-binding domain. The presence of the rare Ig-like linker and a structurally distinct CBM2 makes its domain architecture unique compared to homologs. To investigate their contributions in the enzyme reaction, the wild-type HcrMan1 and two truncated variants (HcrMan1ΔCBM2 and HcrMan1ΔIgΔCBM2) were heterologously expressed and purified. Results revealed that the removal of Ig-like linker domain significantly affected protein solubility, whereas the deletion of CBM2 reduced the substrate-binding affinity but enhanced catalytic efficiency (K_cat/K_m) from 18.08 to 22.45. Truncated HcrMan1ΔCBM2 demonstrated a higher specific activity against locust bean gum (LBG) at optimal conditions (50 °C, pH 8.0) compared to its wild type. Furthermore, HcrMan1ΔCBM2 exhibited enhanced enzymatic performance when combined with the commercial cellulase CTec2 in saccharifying spent coffee grounds (SCG), achieving significant increase in reducing sugar release after alkali, ammonia and sonication pretreatment compared to CTec2 alone. These noteworthy findings highlight the compatibility and industrial relevance of engineered HcrMan1 in applications that involved with the utilization of lignocellulosic biomass.