Molecular and biochemical characterization of recombinant cel12B, cel8C, and peh28 overexpressed in Escherichia coli and their potential in biofuel production

Abstract

BackgroundThe high crystallinity of cellulosic biomass myofibrils as well as the complexity of their intermolecular structure is a significant impediment for biofuel production. Cloning of celB-, celC-encoded cellulases (cel12B and cel8C) and peh-encoded polygalacturonase (peh28) from Pectobacterium carotovorum subsp. carotovorum (Pcc) was carried out in our previous study using Escherichia coli as a host vector. The current study partially characterizes the enzymes’ molecular structures as well as their catalytic performance on different substrates which can be used to improve their potential for lignocellulosic biomass conversion.Resultsβ-Jelly roll topology, (α/α)6 antiparallel helices and right-handed β-helices were the folds identified for cel12B, cel8C, and peh28, respectively, in their corresponding protein model structures. Purifications of 17.4-, 6.2-, and 6.0-fold, compared to crude extract, were achieved for cel12B and cel8C, and peh28, respectively, using specific membrane ultrafiltrations and size-exclusion chromatography. Avicel and carboxymethyl cellulose (CMC) were substrates for cel12B, whereas for cel8C catalytic activity was only shown on CMC. The enzymes displayed significant synergy on CMC but not on Avicel when tested for 3 h at 45 °C. No observed β-glucosidase activities were identified for cel8C and cel12B when tested on p-nitrophenyl-β-d-glucopyranoside. Activity stimulation of 130% was observed when a recombinant β-glucosidase from Pcc was added to cel8C and cel12B as tested for 3 h at 45 °C. Optimum temperature and pH of 45 °C and 5.4, respectively, were identified for all three enzymes using various substrates. Catalytic efficiencies (k cat/K m) were calculated for cel12B and cel8C on CMC as 0.141 and 2.45 ml/mg/s respectively, at 45 °C and pH 5.0 and for peh28 on polygalacturonic acid as 4.87 ml/mg/s, at 40 °C and pH 5.0. Glucose and cellobiose were the end-products identified for cel8C, cel12B, and β-glucosidase acting together on Avicel or CMC, while galacturonic acid and other minor co-products were identified for peh28 action on pectin.ConclusionsThis study provides some insight into which parameters should be optimized when application of cel8C, cel12B, and peh28 to biomass conversion is the goal.

Expression, characterization, and activity optimization of a novel cellulase from the thermophilic bacteria Cohnella sp. A01

“…CelC307 showed the highest cellulolytic activity against soluble CMC substrate (β-1 → 4 linkage) used to calculate kinetic parameters. Compared to some other studied cellulases 19 , 52 – 55 , CelC307 showed low K m and high k cat /K m , indicating a high preference of the enzyme for CMC that led the enzyme to hydrolyze β- 1 → 4 linkages more rapidly than other evaluated cellulases.…”

A Novel Neutral Endoglucanase from Streptomyces sp. H31 with Wide-pH-range Stability

“…However, most of the presently used endoglucanases, especially fungal endoglucanase, usually displayed their highest activity at acidic condition. Their activities are greatly reduced in case of the pH value above 7.0, limiting the application of those fungal enzymes under neutral and alkaline condition (Wang et al, 2014;Zeng et al, 2016;Ibrahim et al, 2017). Therefore, there has a large market and great demand for the endoglucanases which can be eff ective at broad pH with high activity.…”

“…GH12 endoglucanases usually presented low resistance to high pH, for example, cel12B (GH12) from Pectobacterium carotovorum subsp. carotovorum (Pcc) showed a pH optimum of 6.0 (Ibrahim et al, 2017), AcCel12B (GH12) from Acidothermus cellulolyticus 11B remained less than 10% of max enzyme activity at pH 7.0 (Wang et al, 2015). Especially, the neutral endoglucanase EgH31 showed outstanding stability in wide pH ranging from 3.0 to 11.0, and remained more than 70% of peak activity between pH 6.0 and 9.0, while most endoglucanases reported did not functionally work under the wide range of pH (Galante et al, 1998;Bhat, 2000;Trivedi et al, 2011;Yang et al, 2016;Zeng et al, 2016).…”

Recombinant E. coli Cellulases, β-Glucosidase, and Polygalacturonase Convert a Citrus Processing Waste into Biofuel Precursors

“…Recombinants cel12B , cel8C , β-glu , and peh28 encoding the respective cellulases, cel12B, cel8C, β-glucosidase, and polygalacturonase (peh28) expressed in E. coli were used for investigating their combined activities (cocktail formulation given below) on a grapefruit processing waste using their partially purified forms previously described …”

“…87128, C104450, and C0378, respectively) was added to all reactions each at 0.1 mg/g solid biomass. Hydrolysate samples (1.0 mL) were diluted with four volumes of ethanol and the filtrates containing mono- and disaccharide hydrolytic products were evaporated for derivatization and GC-MS quantification, as previously described . The GC (Model 6890, Agilent Technologies, Inc. Hewlett-Packard, Santa Clara, CA) operating conditions were as follows: 180 °C oven temperature for 2 min; increased by 230 °C for 15 min at 15 °C/min, 250 °C injector temperature, 1:10 split ratio, 1.0 μL injection sample/standard and 2 °C/min rate flow helium.…”