Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

Bacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions—multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

Background Efficient degradation of lignocellulosic biomass has become a major bottleneck in industrial processes which attempt to use biomass as a carbon source for the production of biofuels and materials. To make the most effective use of the source material, both the hemicellulosic as well as cellulosic parts of the biomass should be targeted, and as such both hemicellulases and cellulases are important enzymes in biorefinery processes. Using thermostable versions of these enzymes can also prove beneficial in biomass degradation, as they can be expected to act faster than mesophilic enzymes and the process can also be improved by lower viscosities at higher temperatures, as well as prevent the introduction of microbial contamination. Results This study presents the investigation of the thermostable, dual-function xylanase-glucuronoyl esterase enzyme CkXyn10C-GE15A from the hyperthermophilic bacterium Caldicellulosiruptor kristjanssonii. Biochemical characterization of the enzyme was performed, including assays for establishing the melting points for the different protein domains, activity assays for the two catalytic domains, as well as binding assays for the multiple carbohydrate-binding domains present in CkXyn10C-GE15A. Although the enzyme domains are naturally linked together, when added separately to biomass, the expected boosting of the xylanase action was not seen. This lack of intramolecular synergy might suggest, together with previous data, that increased xylose release is not the main beneficial trait given by glucuronoyl esterases. Conclusions Due to its thermostability, CkXyn10C-GE15A is a promising candidate for industrial processes, with both catalytic domains exhibiting melting temperatures over 70 °C. Of particular interest is the glucuronoyl esterase domain, as it represents the first studied thermostable enzyme displaying this activity.

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Objective: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg.L -1 ), and were secreted at moderate to high percentages of the total extracellular protein (51-94 %). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U.L -1 ) that could allow their direct application.

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Objective: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg.L -1 ), and were secreted at moderate to high percentages of the total extracellular protein (51-94 %). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U.L -1 ) that could allow their direct application.

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris

Objective Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. Results The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489–2780 mg L−1), and were secreted at moderate to high percentages of the total extracellular protein (51–94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5–6-fold, reaching enzyme activity levels (1020–202 U L−1) that could allow their direct application.

An integrative approach for high level production of glucuronoyl esterase in Pichia pastoris.

Objective: The commercial availability of lignin-modifying and accessory enzymes is limited, which delays the investigation of their functionality and development of industrial applications. Glucuronoyl esterase (GE) has been shown to improve fractionation of lignin-carbohydrate complexes, yet no pure commercial enzyme preparations are available. To improve accessibility to this enzyme for emerging research, this study reports a simple and effective heterologous expression strategy, coupled with a methanol-free production protocol in a bioreactor for high-level production of GE. This strategy was further validated by production of cellobiose dehydrogenase, a lignocellulose degrading enzyme which is scarcely available at high cost. Results: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg.L-1), and were secreted at moderate to high percentages of the total extracellular protein (51-94 %). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate, and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, in a two-step filtration process. This study describes a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research.