Identification of a thermostable cellobiose 2-epimerase from Caldicellulosiruptor sp. Rt8.B8 and production of epilactose using Bacillus subtilis.

BACKGROUND: Epilactose, a potential prebiotics, was derived from lactose through enzymatic catalysis. However, production and purification of epilactose are currently difficult due to powerless enzymes and inefficient downstream processing steps. RESULTS: The encoding gene of cellobiose 2-epimerase (CE) from Caldicellulosiruptor sp. Rt8.B8 was cloned and expressed in Escherichia coli BL21(DE3). The enzyme was purified and it was suitable for industrial production of epilactose from lactose without by-products, because of high kcat (197.6 s-1 ) and preferable thermostability. The Rt8-CE gene was further expressed in the Bacillus subtilis strain. We successfully produced epilactose from 700 g L-1 lactose in 30.4% yield by using the recombinant Bacillus subtilis whole cells. By screening of a ß-galactosidase from Bacillus stearothermophilus (BsGal), a process for separating epilactose and lactose was established, which showed a purity of over 95% in a total yield of 69.2%. In addition, a mixed rare sugar syrup composed of epilactose and d-tagatose was successfully produced from lactose through the co-expression of l-arabinose isomerase and ß-galactosidase. CONCLUSION: Our study shed light on the efficient production of epilactose using a food-grade host expressing a novel CE enzyme. Moreover, an efficient and low-cost process was attempted to obtain high purity epilactose. In order to improve the utilization of raw materials, the production process of mixed syrup containing epilactose and d-tagatose with prebiotic properties produced from lactose was also established for the first time. © 2021 Society of Chemical Industry.

Heterologous expression and characterization of thermostable chitinase and ß-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-d-glucosamine.

The bioconversion of chitin into N-acetyl-d-glucosamine (GlcNAc) using chitinolytic enzymes is one of the important avenues for chitin valorization. However, industrial applications of chitinolytic enzymes have been limited by their poor thermostability. Therefore, it is necessary to discover thermostable chitinolytic enzymes for GlcNAc production from chitin. In this study, two chitinolytic enzyme-encoding genes CaChiT and CaHex from Caldicellulosiruptor acetigenus were identified and heterologously expressed in Escherichia coli. The purified recombinant CaChiT and CaHex showed optimal activities at 70 °C and 90 °C respectively, and exhibited good thermostability over a range of temperature below 70 °C and broad pH stability at pH range of 3.0-8.0. CaChiT and CaHex were active on colloidal chitin, pNP-(GlcNAc)2, pNP-(GlcNAc)3, and pNP-GlcNAc, pNP-(GlcNAc)2, pNP-(GlcNAc)3, pNP-Glc respectively. Besides, the chitin oligosaccharides and colloidal chitin hydrolysis profiles revealed that CaChiT degraded chitin chains through exo-mode of action. Furthermore, CaChiT and CaHex exhibited a synergistic effect in the degradation of colloidal chitin, reaching 0.60 mg/mL of GlcNAc production after 1 h incubation. These results suggested that a combination of CaChiT and CaHex have great potential for industrial applications in the enzymatic production of GlcNAc from chitin-containing biowastes.

Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii.

The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1-CBM22.2-Xyn10C construct, indicating a compact arrangement at room temperature.

Perturbation of the peptidoglycan network and utilization of the signal recognition particle-dependent pathway enhances the extracellular production of a truncational mutant of CelA in Escherichia coli.

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.

Insight into the potential factors influencing the catalytic direction in cellobiose 2-epimerase by crystallization and mutagenesis.

Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Šresolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148-181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.

A Novel Glucose Isomerase from Caldicellulosiruptor bescii with Great Potentials in the Production of High-Fructose Corn Syrup.

Glucose isomerase (GI) that catalyzes the conversion of D-glucose to D-fructose is one of the most important industrial enzymes for the production of high-fructose corn syrup (HFCS). In this study, a novel GI (CbGI) was cloned from Caldicellulosiruptor bescii and expressed in Escherichia coli. The purified recombinant CbGI (rCbGI) showed neutral and thermophilic properties. It had optimal activities at pH 7.0 and 80°C and retained stability at 85°C. In comparison with other reported GIs, rCbGI exhibited higher substrate affinity (Km = 42.61 mM) and greater conversion efficiency (up to 57.3% with 3M D-glucose as the substrate). The high catalytic efficiency and affinity of this CbGI is much valuable for the cost-effective production of HFCS.