Cellodextrin Phosphorylase Research Articles

Glycoside Phosphorylase Catalyzed Cellulose and β-1,3-Glucan Synthesis Using Chromophoric Glycosyl Acceptors.

Glycoside phosphorylases are enzymes that are frequently used for polysaccharide synthesis. Some of these enzymes have broad substrate specificity, enabling the synthesis of reducing-end-functionalized glucan chains. Here, we explore the potential of glycoside phosphorylases in synthesizing chromophore-conjugated polysaccharides using commercially available chromophoric model compounds as glycosyl acceptors. Specifically, we report cellulose and β-1,3-glucan synthesis using 2-nitrophenyl β-d-glucopyranoside, 4-nitrophenyl β-d-glucopyranoside, and 2-methoxy-4-(2-nitrovinyl)phenyl β-d-glucopyranoside with Clostridium thermocellum cellodextrin phosphorylase and Thermosipho africanus β-1,3-glucan phosphorylase as catalysts. We demonstrate activity for both enzymes with all assayed chromophoric acceptors and report the crystallization-driven precipitation and detailed structural characterization of the synthesized polysaccharides, i.e., their molar mass distributions and various structural parameters, such as morphology, fibril diameter, lamellar thickness, and crystal form. Our results provide insights for the studies of chromophore-conjugated low molecular weight polysaccharides, glycoside phosphorylases, and the hierarchical assembly of crystalline cellulose and β-1,3-glucan.

Effect of Free Cysteine Residues to Serine Mutation on Cellodextrin Phosphorylase.

Cellodextrin phosphorylase (CDP) plays a key role in energy-efficient cellulose metabolism of anaerobic bacteria by catalyzing phosphorolysis of cellodextrin to produce cellobiose and glucose 1-phosphate, which can be utilized for glycolysis without consumption of additional ATP. As the enzymatic phosphorolysis reaction is reversible, CDP is also employed to produce cellulosic materials in vitro. However, the enzyme is rapidly inactivated by oxidation, which hinders in vitro utilization in aerobic environments. It has been suggested that the cysteine residues of CDP, which do not form disulfide bonds, are responsible for the loss of activity, and the aim of the present work was to test this idea. For this purpose, we replaced all 11 free cysteine residues of CDP from Acetivibrio thermocellus (formerly known as Clostridium thermocellum) with serine, which structurally resembles cysteine in our previous work. Herein, we show that the resulting CDP variant, named CDP-CS, has comparable activity to the wild-type enzyme, but shows increased stability to oxidation during long-term storage. X-Ray crystallography indicated that the mutations did not markedly alter the overall structure of the enzyme. Ensemble refinement of the crystal structures of CDP and CDP-CS indicated that the C372S and C625S mutations reduce structural fluctuations in the protein main chain, which may contribute to the increased stability of CDP-CS to oxidation.

Production of cello-oligosaccharides from corncob residue by degradation-synthesis reactions.

The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. KEY POINTS: • Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. • Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. • Spraying cello-oligosaccharides promoted tomato growth.

Xylulose 5-phosphate production using enzymes immobilized on silica nanoparticles and its application for biohydrogen generation

Development of green hydrogen production is one of sustainable approaches to replace fossil fuels. An in vitro cell-free synthetic enzymatic pathway (SyPaB) for hydrogen production is an alternative to less effective microbial fermentation. To increase the production of xylulose 5-phosphate (Xu5-P); the rate-limiting step's product in the ATP-free SyPaB for biohydrogen production powered by xylooligomers, immobilization of cellodextrin phosphorylase, phosphopentomutase, and ribose-5-phosphate isomerase, on silica nano-particles (SiNPs) was evaluated. Immobilization of 1 Unit/mL of each enzyme on 15 to 20 mg of amorphous SiNPs for 120 min, at 4 °C gave the highest immobilization yield. Immobilized enzymes along with other 10 free enzymes gave maximum hydrogen generation rate of 3.618 mmole/L.hr after 0.8 h which 1.3 fold higher than using 13 free enzymes at the same conditions. The study reveals that immobilized enzymes efficiently produce Xylulose 5-phosphate, and its application in effective biohydrogen production, indicating potential for future enzyme immobilization.

Broadening the Substrate Specificity of Cellobiose Phosphorylase from Clostridium thermocellum for Improved Transformation of Cellodextrin to Starch.

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (PNAS, 110: 7182-7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from Clostridium thermocellum (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin's phosphorolysis.

Synthesis of cello-oligosaccharides which promotes the growth of intestinal probiotics by multi-enzyme cascade reaction

Soluble cello-oligosaccharide with 2-6 oligosaccharide units is a kind of oligosaccharide with various biological functions, which can promote the proliferation of intestinal probiotics such as Bifidobacteria and Lactobacillus paracei. Therefore, it has a regulatory effect on human intestinal microbiota. In this study, a Cc 01 strain was constructed by expressing cellodextrin phosphorylase (CDP) in Escherichia coli. By combining with a previously constructed COS 01 strain, a three-enzyme cascade reaction system based on strains COS 01 and Cc 01 was developed, which can convert glucose and sucrose into cello-oligosaccharide. After optimization, the final titer of soluble cello-oligosaccharides with 2-6 oligosaccharide units reached 97 g/L, with a purity of about 97%. It contained cellobiose (16.8 wt%), cellotriose (49.8 wt%), cellotetrose (16.4 wt%), cellopentaose (11.5 wt%) and cellohexose (5.5 wt%). When using inulin, xylo-oligosaccharide and fructooligosaccharide as the control substrate, the biomass (OD600) of Lactobacillus casei (WSH 004), Lactobacillus paracei (WSH 005) and Lactobacillus acidophilus (WSH 006) on cello-oligosaccharides was about 2 folds higher than that of the control. This study demonstrated the efficient synthesis of cello-oligosaccharides by a three-enzyme cascade reaction and demonstrated that the synthesized cello-oligosaccharides was capable of promoting intestinal microbial proliferation.

Key roles of β-glucosidase BglA for the catabolism of both laminaribiose and cellobiose in the lignocellulolytic bacterium Clostridium thermocellum

The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of β-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the β-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the β-1,3-disaccharide laminaribiose with much higher activity than that against the β-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing β-1,3-linked glycosidic bonds in lignocellulose biorefinery.

Insights to improve the activity of glycosyl phosphorylases from Ruminococcus albus 8 with cello-oligosaccharides.

The phosphorolysis of cello-oligosaccharides is a critical process played in the rumen by Ruminococcus albus to degrade cellulose. Cellodextrins, made up of a few glucosyl units, have gained lots of interest by their potential applications. Here, we characterized a cellobiose phosphorylase (RalCBP) and a cellodextrin phosphorylase (RalCDP) from R. albus 8. This latter was further analyzed in detail by constructing a truncated mutant (Ral∆N63CDP) lacking the N-terminal domain and a chimeric protein by fusing a CBM (RalCDP-CBM37). RalCBP showed a typical behavior with high activity on cellobiose. Instead, RalCDP extended its activity to longer soluble or insoluble cello-oligosaccharides. The catalytic efficiency of RalCDP was higher with cellotetraose and cellopentaose as substrates for both reaction directions. Concerning properties of Ral∆N63CDP, results support roles for the N-terminal domain in the conformation of the homo-dimer and conferring the enzyme the capacity to catalyze the phosphorolytic reaction. This mutant exhibited reduced affinity toward phosphate and increased to glucose-1-phosphate. Further, the CBM37 module showed functionality when fused to RalCDP, as RalCDP-CBM37 exhibited an enhanced ability to use insoluble cellulosic substrates. Data obtained from this enzyme's binding parameters to cellulosic polysaccharides agree with the kinetic results. Besides, studies of synthesis and phosphorolysis of cello-saccharides at long-time reactions served to identify the utility of these enzymes. While RalCDP produces a mixture of cello-oligosaccharides (from cellotriose to longer oligosaccharides), the impaired phosphorolytic activity makes Ral∆N63CDP lead mainly toward the synthesis of cellotetraose. On the other hand, RalCDP-CBM37 remarks on the utility of obtaining glucose-1-phosphate from cellulosic compounds.

Continuous process technology for bottom-up synthesis of soluble cello-oligosaccharides by immobilized cells co-expressing three saccharide phosphorylases

BackgroundContinuous processing with enzyme reuse is a well-known engineering strategy to enhance the efficiency of biocatalytic transformations for chemical synthesis. In one-pot multistep reactions, continuous processing offers the additional benefit of ensuring constant product quality via control of the product composition. Bottom-up production of cello-oligosaccharides (COS) involves multistep iterative β-1,4-glycosylation of glucose from sucrose catalyzed by sucrose phosphorylase from Bifidobacterium adeloscentis (BaScP), cellobiose phosphorylase from Cellulomonas uda (CuCbP) and cellodextrin phosphorylase from Clostridium cellulosi (CcCdP). Degree of polymerization (DP) control in the COS product is essential for soluble production and is implemented through balance of the oligosaccharide priming and elongation rates. A whole-cell E. coli catalyst co-expressing the phosphorylases in high yield and in the desired activity ratio, with CdP as the rate-limiting enzyme, was reported previously.ResultsFreeze-thaw permeabilized E. coli cells were immobilized in polyacrylamide (PAM) at 37–111 mg dry cells/g material. PAM particles (0.25–2.00 mm size) were characterized for COS production (~ 70 g/L) in mixed vessel with catalyst recycle and packed-bed reactor set-ups. The catalyst exhibited a dry mass-based overall activity (270 U/g; 37 mg cells/g material) lowered by ~ 40% compared to the corresponding free cells due to individual enzyme activity loss, CbP in particular, caused by the immobilization. Temperature studies revealed an operational optimum at 30 °C for stable continuous reaction (~ 1 month) in the packed bed (volume: 40 mL; height: 7.5 cm). The optimum reflects the limits of PAM catalyst structural and biological stability in combination with the requirement to control COS product solubility in order to prevent clogging of the packed bed. Using an axial flow rate of 0.75 cm− 1, the COS were produced at ~ 5.7 g/day and ≥ 95% substrate conversion (sucrose 300 mM). The product stream showed a stable composition of individual oligosaccharides up to cellohexaose, with cellobiose (48 mol%) and cellotriose (31 mol%) as the major components.ConclusionsContinuous process technology for bottom-up biocatalytic production of soluble COS is demonstrated based on PAM immobilized E. coli cells that co-express BaScP, CuCbP and CcCdP in suitable absolute and relative activities.

Engineering cascade biocatalysis in whole cells for bottom-up synthesis of cello-oligosaccharides: flux control over three enzymatic steps enables soluble production

BackgroundSoluble cello-oligosaccharides (COS, β‐1,4‐D‐gluco‐oligosaccharides with degree of polymerization DP 2–6) have been receiving increased attention in different industrial sectors, from food and feed to cosmetics. Development of large-scale COS applications requires cost-effective technologies for their production. Cascade biocatalysis by the three enzymes sucrose-, cellobiose- and cellodextrin phosphorylase is promising because it enables bottom-up synthesis of COS from expedient substrates such as sucrose and glucose. A whole-cell-derived catalyst that incorporates the required enzyme activities from suitable co-expression would represent an important step towards making the cascade reaction fit for production. Multi-enzyme co-expression to reach distinct activity ratios is challenging in general, but it requires special emphasis for the synthesis of COS. Only a finely tuned balance between formation and elongation of the oligosaccharide precursor cellobiose results in the desired COS.ResultsHere, we show the integration of cellodextrin phosphorylase into a cellobiose-producing whole-cell catalyst. We arranged the co-expression cassettes such that their expression levels were upregulated. The most effective strategy involved a custom vector design that placed the coding sequences for cellobiose phosphorylase (CbP), cellodextrin phosphorylase (CdP) and sucrose phosphorylase (ScP) in a tricistron in the given order. The expression of the tricistron was controlled by the strong T7lacO promoter and strong ribosome binding sites (RBS) for each open reading frame. The resulting whole-cell catalyst achieved a recombinant protein yield of 46% of total intracellular protein in an optimal ScP:CbP:CdP activity ratio of 10:2.9:0.6, yielding an overall activity of 315 U/g dry cell mass. We demonstrated that bioconversion catalyzed by a semi-permeabilized whole-cell catalyst achieved an industrial relevant COS product titer of 125 g/L and a space–time yield of 20 g/L/h. With CbP as the cellobiose providing enzyme, flux into higher oligosaccharides (DP ≥ 6) was prevented and no insoluble products were formed after 6 h of conversion.ConclusionsA whole-cell catalyst for COS biosynthesis was developed. The coordinated co-expression of the three biosynthesis enzymes balanced the activities of the individual enzymes such that COS production was maximized. With the flux control set to minimize the share of insolubles in the product, the whole-cell synthesis shows a performance with respect to yield, productivity, product concentration and quality that is promising for industrial production.

Driving an in vitro multienzymatic cascade of laminaribiose biosynthesis from non-food cellulose with balancing the precursor supply

High-valued disaccharide laminaribiose, derived from two glucose units, is widely used in the pharmaceutical, cosmetic and food industries. The technology for laminaribiose synthesis from non-food feedstock cellulose instead of sucrose, glucose and starch was developed. First, we investigated a two-enzyme cascade converting cellobiose to laminaribiose by cellobiose phosphorylase and laminaribiose phosphorylase and 0.47 mol/mol cellobiose yield was obtained with cellobiose. An in vitro enzymatic cascade composed of cellobiohydrolase, cellodextrin phosphorylase, cellobiose phosphorylase and laminaribiose phosphorylase was designed to synthesize laminaribiose from cellulose. Laminaribiose production reached 2.35 and 1.23 g/L in pathways I & II, respectively. Furthermore, another multienzyme cascade was designed to eliminate the imbalance of glucose and glucose-1-p supply and laminaribiose yield finally reached 72% of that yield from cellobiose. This in vitro enzymatic cascades for laminaribiose biosynthesis not only widen the utilization of the non-food cellulose, but also provide a model that improves the economy of lignocellulosic biorefineries.

Enzymatic synthesis of cellulose in space: gravity is a crucial factor for building cellulose II gel structure

We previously reported in vitro synthesis of highly ordered crystalline cellulose II by reverse reaction of cellodextrin phosphorylase from the cellulolytic bacterium Clostridium (Hungateiclostridium) thermocellum (CtCDP), but the formation mechanism of the cellulose crystals and highly ordered structure has long been unclear. Considering the specific density of cellulose versus water, the formation of crystalline and highly ordered structure in an aqueous solution should be affected by gravity. Thus, we synthesized cellulose with CtCDP stable variant at the International Space Station, where sedimentation and convection due to gravity are negligible. Optical microscopic observation suggested that cellulose in space has a gel-like appearance without apparent aggregation, in contrast to cellulose synthesized on the ground. Small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) indicated that cellulose synthesized in space has a more uniform particle distribution in the ~ 100 nm scale region than cellulose synthesized on the ground. Scanning electron microscopy (SEM) showed that both celluloses have a micrometer scale network structure, whereas a fine fiber network was constructed only under microgravity. These results indicate that gravity plays a role in cellulose II crystal sedimentation and the building of network structure, and synthesis in space could play a role in designing unique materials.Graphical abstract

Precision synthesis of reducing-end thiol-modified cellulose enabled by enzyme selection

Enzyme-catalyzed iterative β-1,4-glycosylation of β-glycosides is promising for bottom-up polymerization of reducing-end-modified cello-oligosaccharide chains. Self-assembly of the chains from solution yields crystalline nanocellulose materials with properties that are tunable by the glycoside group used. Cellulose chains with a reducing-end thiol group are of interest to install a controllable pattern of site-selective modifications into the nanocellulose material. Selection of the polymerizing enzyme (cellodextrin phosphorylase; CdP) was pursued here to enhance the synthetic precision of β-1-thio-glucose conversion to generate pure “1-thio-cellulose” (≥95%) unencumbered by plain (unlabeled) cellulose resulting from enzymatic side reactions. The CdP from Clostridium stercorarium (CsCdP) was 21 times more active on β-1-thio-glucose (0.17 U/mg; 45 °C) than the CdP from Clostridium cellulosi (CcCdP), and it lacked hydrolase activity, which is substantial in CcCdP, against the α-d-glucose 1-phosphate donor substrate. The combination of these enzyme properties indicated that CsCdP is a practical catalyst for 1-thio-cellulose synthesis directly from β-1-thio-glucose (8 h; 25 mol% yield) that does not require a second enzyme (cellobiose phosphorylase), which was essential when using the less selective CcCdP. The 1-thio-cellulose chains had an average degree of polymerization of ∼10 and were assembled into highly crystalline cellulose II crystallinity material.

Molecular Recognition of Natural and Non-Natural Substrates by Cellodextrin Phosphorylase from Ruminiclostridium Thermocellum Investigated by NMR Spectroscopy.

β‐1→4‐Glucan polysaccharides like cellulose, derivatives and analogues, are attracting attention due to their unique physicochemical properties, as ideal candidates for many different applications in biotechnology. Access to these polysaccharides with a high level of purity at scale is still challenging, and eco‐friendly alternatives by using enzymes in vitro are highly desirable. One prominent candidate enzyme is cellodextrin phosphorylase (CDP) from Ruminiclostridium thermocellum, which is able to yield cellulose oligomers from short cellodextrins and α‐d‐glucose 1‐phosphate (Glc‐1‐P) as substrates. Remarkably, its broad specificity towards donors and acceptors allows the generation of highly diverse cellulose‐based structures to produce novel materials. However, to fully exploit this CDP broad specificity, a detailed understanding of the molecular recognition of substrates by this enzyme in solution is needed. Herein, we provide a detailed investigation of the molecular recognition of ligands by CDP in solution by saturation transfer difference (STD) NMR spectroscopy, tr‐NOESY and protein‐ligand docking. Our results, discussed in the context of previous reaction kinetics data in the literature, allow a better understanding of the structural basis of the broad binding specificity of this biotechnologically relevant enzyme.

Facile biosynthesis of synthetic crystalline cellulose nanoribbon from maltodextrin through a minimized two-enzyme phosphorylase cascade and its application in emulsion

Nanocellulose has many promising applications such as a green ingredient for Pickering emulsion. Traditional strategies to produce nanocellulose, which are acid or enzymatic hydrolysis and mechanical methods on natural complicated cellulose, are hard to control and can result in significant pollutants during the processes. Herein, we demonstrated a facile and sustainable method for the biocatalytic production of insoluble synthetic crystalline cellulose nanoribbon (CCNR) from cheap maltodextrin by coupling α-glucan phosphorylase (αGP) and cellodextrin phosphorylase (CDP) using cellobiose as a primer. And by optimizing the combination of different αGP and CDP, it turned out that the optimal enzyme combination is αGP from Thermotoga maritime and CDP from Clostridium thermocellum, in which CDP was attached to a family 9 cellulose-binding module. The product yield and degree of polymerization (DP) of insoluble synthetic CCNR was affected by the primer concentration at a fixed concentration of maltodextrin. After optimization of reaction conditions, the highest product yield of insoluble synthetic CCNR was 44.92 % and the highest DP of the insoluble synthetic CCNR was 24 from 50 g 1−1 maltodextrin. This insoluble synthetic CCNR can be used as a Pickering emulsions stabilizer, showing excellent emulsifiability. This study provides a promising alternative for cost-efficient production of insoluble synthetic CCNR which was used as a green emulsion stabilizer.

In Vitro Synthesis and Self-Assembly of Cellulose II Nanofibrils Catalyzed by the Reverse Reaction of Clostridium thermocellum Cellodextrin Phosphorylase.

In nature, various organisms produce cellulose as microfibrils, which are processed into their nano- and microfibrillar and/or crystalline components by humans in order to obtain desired material properties. Interestingly, the natural synthesis machinery can be circumvented by enzymatically synthesizing cellulose from precursor molecules in vitro. This approach is appealing for producing tailor-made cellulosic particles and materials because it enables optimization of the reaction conditions for cellulose synthesis in order to generate particles with a desired morphology in their pure form. Here, we present enzymatic cellulose synthesis catalyzed by the reverse reaction of Clostridium thermocellum cellodextrin phosphorylase in vitro. We were able to produce cellulose II nanofibril networks in all conditions tested, using varying concentrations of the glycosyl acceptors d-glucose or d-cellobiose (0.5, 5, and 50 mM). We show that shorter cellulose chains assemble into flat ribbon-like fibrils with greater diameter, while longer chains assemble into cylindrical fibrils with smaller diameter.

Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose

Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-d-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.Key points• Cellobiose phosphorylase is engineered for the production of cellotriose.• Cellotriose is synthesised with the highest purity and yield to date.• Both cellobiose and glucose can be used as acceptor for cellotriose production.

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.

Effect of C-6 Methylol Groups on Substrate Recognition of Glucose/Xylose Mixed Oligosaccharides by Cellobiose Dehydrogenase from the Basidiomycete Phanerochaete chrysosporium.

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar K m and k cat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher K m value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.

Preparative and Kinetic Analysis of β-1,4- and β-1,3-Glucan Phosphorylases Informs Access to Human Milk Oligosaccharide Fragments and Analogues Thereof.

The enzymatic synthesis of oligosaccharides depends on the availability of suitable enzymes, which remains a limitation. Without recourse to enzyme engineering or evolution approaches, herein we demonstrate the ability of wild‐type cellodextrin phosphorylase (CDP: β‐1,4‐glucan linkage‐dependent) and laminaridextrin phosphorylase (Pro_7066: β‐1,3‐glucan linkage‐dependent) to tolerate a number of sugar‐1‐ phosphate substrates, albeit with reduced kinetic efficiency. In spite of catalytic efficiencies of <1 % of the natural reactions, we demonstrate the utility of given phosphorylase–sugar phosphate pairs to access new‐to‐nature fragments of human milk oligosaccharides, or analogues thereof, in multi‐milligram quantities.