Expression, homology modeling and enzymatic characterization of a new β-mannanase belonging to glycoside hydrolase family 1 from Enterobacter aerogenes B19

Abstract

Background: β-mannanase can hydrolyze β-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, β-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. β-mannanase is widespread in various organisms, however, microorganisms are the main source of β-mannanases. Microbial β-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial β-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce β-mannanase, which shows more advantages than traditional microbial fermentation in various aspects. Results: A β-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The β-mannanase activity was activated by low concentration of Co 2+ , Mn 2+ , Zn 2+ , Ba 2+ and Ca 2+. Man1E showed the highest affinity for Locust bean gum (LBG). The K m and V max values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 μmol/(mL min), respectively. Conclusions: A new type of β-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported β-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.

Computational design and characterization of a multiepitope vaccine against carbapenemase-producing Klebsiella pneumoniae strains, derived from antigens identified through reverse vaccinology

“…Putative TonB-dependent siderophore receptor, henceforth called Klebs#1, is an outer membrane protein which has an auxiliary role in iron sequestration, a process required for K. pneumoniae survival in the mammal host [2] , and has been identified as a potential antigen by previous in silico analyses studies [14] , [83] , [84] . β-1,4-mannanase, hereafter referred as Klebs#2, is a glycoside hydrolase which catalyzes hydrolysis of mannan (linear, galacto-, gluco - and galactogluco-), in the extracellular milieu of several microorganisms’ cell wall [85] . Type 3 fimbria adhesin subunit MrkD has been widely studied by its virulence potential, due to its role in biofilm formation in antibiotic-resistant K. pneumoniae strains [7] , [23] , [86] , [87] and it is widely disseminated in K. pneumoniae clinical isolates [7] , [88] , [89] , [90] .…”

Improvement of the Catalytic Ability of a Thermostable and Acidophilic β-Mannanase Using a Consensus Sequence Design Strategy

“…Nowadays, molecular simulation is widely used to infer the changes of non-covalent forces in the mutation site region before and after the alteration, which is helpful to explain the reasons for the changes in the thermostability and the catalytic efficiency of mutants. Molecular simulation can be used for most enzymes, including mannanases ( Chauhan et al, 2015 ; Chauhan and Jaiswar, 2017 ; Liu et al, 2020a ), xylanase ( Cheng et al, 2015 ), and β-glucosidases ( Dadheech et al, 2019 ). Therefore, in this study, the structure-function relationships of the three mutants were analyzed by docking analysis and a molecular simulation method.…”

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

“…However, low activities and high cost of β-mannanases are the crucial technological bottleneck increasing the conversion rate of mannan ( Katrolia et al, 2012 ). Nowadays, various genetic engineering technologies have been used to achieve the efficient expression of target proteins in plant, yeast, and bacterial expression hosts ( Liu S et al, 2020 ). Notably, recent advancements in protein engineering have facilitated the expression of β-mannanases in Pichia pastoris , paving the way for improved production and utilization of β-mannanases.…”