Novel approaches to the automated assay of β-glucanase and lichenase activity.
Mangan, D., Liadova, A., Ivory, R. & McCleary, B. V. (2016). Carbohydrate Research, 435, 162-172.
We report herein the development of a novel assay procedure for the measurement of β-glucanase and lichenase (EC 18.104.22.168) in crude enzyme extracts. Two assay formats based on a) a direct cleavage or b) an enzyme coupled substrate were initially investigated. The ‘direct cleavage’ substrate, namely 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-31-cellotriosyl-β-glucopyranoside (MBG4), was found to be the more generally applicable reagent. This substrate was fully characterised using a crude malt β-glucanase extract, a bacterial lichenase (Bacillus sp.) and a non-specific endo-1,3(4)-β-glucanase from Clostridium thermocellum (EC 22.214.171.124). Standard curves were derived that allow the assay absorbance response to be directly converted to β-glucanase/lichenase activity on barley β-glucan. The specificity of MBG4 was confirmed by analysing the action of competing glycosyl hydrolases that are typically found in malt on the substrate. Manual and automated assay formats were developed for the analysis of a) β-glucanase in malt flour and b) lichenase enzyme extracts and the repeatability of these assays was fully investigated.
Measurement of (1→3)(1→4)-β-D-glucan in malt, wort and beer.
McCleary, B. V. & Nurthen, E. (1986). Journal of the Institute of Brewing, 92(2), 168-173.
A method developed for the quantification of (1→3)(1→4)-β-D-glucan in barley flour has been modified to allow its use in the measurement of this component in malt, wort, beer and spent grain. For malt samples, free D-glucose was first removed with aqueous ethanol. Quantification of the polymer in wort and beer samples involved precipitation of the β-glucan with ammonium sulphate followed by washing with aqueous ethanol to remove free D-glucose. Spent grain was lyophilised and milled and then analysed by the method developed for malt. In all cases, the β-glucan was depolymerised with lichenase and the resultant β-gluco-oligosaccharides hydrolysed to D-glucose with β-D-glucosidase. The released D-glucose was then specifically determined using glucose oxidase-peroxidase reagent.
Enzymic quantification of (1→3) (1→4)-β-D-glucan in barley and malt.
McCleary, B. V. & Glennie-Holmes, M. (1985). Journal of the Institute of Brewing, 91(5), 285-295.
A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in malt samples. α-Amylase does not interfere with the assay. The method is suitable for the routine analysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0–1 for barley flour samples containing 3–8 and 4–6% (w/w) β-glucan content.
Measurement of (1→3),(1→4)-β-D-glucan in barley and oats: A streamlined enzymic procedure.
McCleary, B. V. & Codd, R. (1991). Journal of the Science of Food and Agriculture, 55(2), 303-312.
A commercially available enzymic method for the quantitative measurement of (1→3),(1→4)-β-glucan has been simplified to allow analysis of up to 10 grain samples in 70 min or of 100–200 samples by a single operator in a day. These improvements have been achieved with no loss in accuracy or precision and with an increase in reliability. The glucose oxidase/peroxidase reagent has been significantly improved to ensure colour stability for periods of up to 1 h after development. Some problems experienced with the original method have been addressed and resolved, and further experiments to demonstrate the quantitative nature of the assay have been designed and performed.
Dietary fibers from mushroom sclerotia: 3. In vitro fermentability using human fecal microflora.
Wong, K. H., Wong, K. Y., Kwan, H. S. & Cheung, P. C. K. (2005). Journal of Agricultural and Food Chemistry, 53(24), 9407-9412.
The in vitro fermentability of three novel dietary fibers (DFs) prepared from mushroom sclerotia, namely, Pleurotus tuber-regium, Polyporous rhinocerus, and Wolfiporia cocos, was investigated and compared with that of the cellulose control. All DF samples (0.5 g each) were fermented in vitro with a human fecal homogenate (10 mL) in a batch system (total volume, 50 mL) under strictly anaerobic conditions (using oxygen reducing enzyme and under argon atmosphere) at 37°C for 24 h. All three novel sclerotial DFs exhibited notably higher dry matter disappearance (P. tuber-regium, 8.56%; P. rhinocerus, 13.5%; and W. cocos, 53.4%) and organic matter disappearance (P. tuber-regium, 9.82%; P. rhinocerus, 14.6%; and W. cocos, 57.4%) when compared with those of the cellulose control. Nevertheless, only the W. cocos DF was remarkably degraded to produce considerable amounts of total short chain fatty acids (SCFAs) (5.23 mmol/g DF on organic matter basis, with a relatively higher molar ratio of propionate) that lowered the pH of its nonfermented residue to a slightly acidic level (5.89). Variations on the in vitro fermentability among the three sclerotial DFs might mainly be attributed to their different amounts of interwoven hyphae present (different amounts of enzyme inaccessible cell wall components) as well as the possible different structural arrangement (linkage and degree of branching) of their β-glucans.
Improved quantitative analysis of oligosaccharides from lichenase-hydrolyzed water-soluble barley β-glucans by high-performance anion-exchange chromatography.
Yoo, D. H., Lee, B. H., Chang, P. S., Lee, H. G. & Yoo, S. H. (2007). Journal of Agricultural and Food Chemistry, 55(5), 1656-1662.
Cereal β-glucan is a linear biopolymer linked by β-(1,3)/(1,4)-glycosidic bonds. More specifically, the β-(1,4)-linked glucose chain is interrupted with β-(1,3)-linkages in cereal β-glucan structure. Elucidation of the exact length and distribution of linear β-(1,4)-linked portion facilitates the understanding of the fine structure of cereal β-glucan. A HPAEC assisted by lichenase treatment has been used for the structural and quantitative analysis of cereal β-glucan. The absence of authentic standard oligosaccharides, putatively 3-O-β-cellobiosyl-D-glucose (DP3) and 3-O-β-cellotriosyl-D-glucose (DP4), was a potential problem to the characterization of β-glucan structure. In this study, two major lichenase-hydrolyzed products were generated from the barley β-glucan, and putative 3-O-β-cellobiosyl-D-glucose and 3-O-β-cellotriosyl-D-glucose were separated and highly purified by recycling preparative HPLC technology. Structural analysis of highly purified putative 3-O-β-cellobiosyl-D-glucose and 3-O-β-cellotriosyl-D-glucose was performed by TLC and LC-MS analysis. Two putative DP3 and DP4 displayed the nonreducing end/(1,4)/(1,3) linkage ratios of 1:0.96:0.90 and 1:2.18:1.16, respectively; the molecular masses (m/z) of their sodium adducts were 527.0 and 689.0, respectively. Using these structurally confirmed oligosaccharides, the exact amounts of β-glucan lichenase hydrolysates from domestic barley cultivars were quantified. The amount of two major DP3 and DP4 accounted for only 71.4−73.3% of water-extractable β-glucan fraction, and the (1,4)/(1,3) linkage ratios of the extracted β-glucans were almost identical in the range of 2.24−2.25 among the barley cultivars tested.
Enzymatic extraction of beta-glucan from oat bran cereals and oat crackers and optimization of viscosity measurement.
Gamel, T. H., Abdel-Aal, E. S. M., Ames, N. P., Duss, R., & Tosh, S. M. (2014). Journal of Cereal Science, 59(1), 33-40.
The viscosity of the soluble fibre, β-glucan, has been shown to influence its ability to lower serum cholesterol and postprandial blood glucose levels. The impact of various amylases, proteases and lipase on the solubility and resulting viscosity of β-glucan extracted from oat bran cereals with a range of β-glucan concentrations and molecular weights was investigated. Addition of enzymes increased the final viscosity of high molecular weight β-glucan in cereals by facilitating the release of β-glucan from the food matrix. For cereals with partially depolymerized β-glucan, the addition of digestive enzymes decreased the final viscosity by eliminating the contribution of starch and protein to viscosity. Final viscosity varied depending on enzyme combinations including pancreatin, salivary and microbial α-amylases, microbial protease, porcine protease, trypsin and α-chymotrypsin. Addition of lipase did not significantly affect viscosity or solubility of β-glucan extracted from oat crackers. Addition of lichenase showed that β-glucan was the major contributor of viscosity to the system, with negligible interference from other components. The viscosity of the optimized protocol was compared to physiological results previously obtained. The viscosity of β-glucan extracted with pancreatin plus microbial α-amylase (pH 6.9) was predictive of LDL-cholesterol reduction (R2 = 0.847) and glycemic response (R2 = 0.883).
Textural and Bile Acid-Binding Properties of Muffins Impacted by Oat β-Glucan with Different Molecular Weights.
Sayar, S., Jannink, J. L. & White, P. J. (2011). Cereal Chemistry, 88(6), 564-569.
Water-soluble β-glucan (BG) extracted from a high-BG oat line was treated with different amounts of lichenase (1→3)(1→4)-β-D-glucanase) enzyme to yield three different molecular weight (MW) BG extracts. Low (LMW-BG, 157,000), medium (MMW-BG, 277,000), and high (HMW-BG, 560,000) MW BG extracts were added to plain muffin formulations at a level of 0.52% (0.42% in the batter, 0.52% in the resultant muffins) to investigate the effect of MW of BG on textural and bile acid (BA) binding properties of the muffins. In addition, treatments were prepared containing LMW-BG, MMW-BG, and HMW-BG extracts in amounts providing equivalent batter firmness as determined on a texture analyzer. Resultant BG concentrations (and per serving amounts) of these muffins were 1.36% (0.81 g/60 g muffin), 1.05% (0.63 g/60 g muffin), and 0.52% (0.31 g/60 g muffin), respectively; thus, the LMW treatment complied with a U.S. Food and Drug Administration health claim requiring 0.75 g of BG per serving. The firmness, springiness, and BA-binding capacity of the muffins were unaffected by the MW of BG. However, when added at the maximum limit for equivalent batter firmness, the LMW treatment was more firm and less springy than the HMW treatment. Furthermore, BA-binding capacities of LMW and MMW fractions tended to be greater than that of the HMW fraction when added at the maximum limit. These results add further evidence to the importance of fine-tuning BG structure to provide maximum health benefits while maintaining high product quality.
Identification of quantitative trait loci affecting hemicellulose characteristics based on cell wall composition in a wild and cultivated rice species.
Zhang, S. J., Song, X. Q., Yu, B. S., Zhang, B. C., Sun, C. Q., Knox, J. P. & Zhou, Y. H. (2012). Molecular Plant, 5(1), 162-175.
Cell wall hemicellulosic polysaccharides are structurally complex and diverse. Knowledge about the synthesis of cell wall hemicelluloses and their biological roles is limited. Quantitative trait loci (QTL) mapping is a helpful tool for the dissection of complex phenotypes for gene identification. In this study, we exploited the natural variation in cell wall monosaccharide levels between a common wild rice, Yuanj, and an elite indica cultivar, Teqing, and performed QTL mapping with their introgression lines (ILs). Chemical analyses conducted on the culms of Yuanj and Teqing showed that the major alterations are found in glucose and xylose levels, which are correlated with specific hemicellulosic polymers. Glycosidic linkage examination revealed that, in Yuanj, an increase in glucose content results from a higher level of mixed linkage β-glucan (MLG), whereas a reduction in xylose content reflects a low level of xylan backbone and a varied arabinoxylan (AX) structure. Seventeen QTLs for monosaccharides have been identified through composition analysis of the culm residues of 95 core ILs. Four major QTLs affecting xylose and glucose levels are responsible for 19 and 21% of the phenotypic variance, respectively. This study provides a unique resource for the genetic dissection of rice cell wall formation and remodeling in the vegetative organs.
Generic tools to assess genuine carbohydrate specific effects on in vitro immune modulation exemplified by β-glucans.
Rieder, A., Grimmer, S., Aachmann, F. L., Westereng, B., Kolset, S. O. & Knutsen, S. H. (2013). Carbohydrate Polymers, 92(2), 2075-2083.
Even if carbohydrate preparations from plant/fungal sources have a high degree of purity, observed immune-stimulation may be caused by minute sample contaminations. Using the example of different β-glucans we present a range of analytical tools crucial for validation of possible immune-stimulatory effects. Two yeast (MacroGard and Zymosan) and one cereal β-glucan (CBG40) increased IL-8 secretion by HT-29 cells considerably. Degradation of the β-glucan samples with β-glucan specific enzymes did hardly influence the effect of Zymosan and CBG40 but significantly decreased the effect of MacroGard. Stimulation of IL-8 secretion by CBG40 and Zymosan was hence not due to their β-glucan content. Instead, the effect of the CBG40 sample was due to low levels of LPS despite the inability of the known LPS inhibitor Polymyxin B to supress its stimulatory effect. We conclude that targeted enzymatic degradation of samples is a powerful validation tool to investigate carbohydrate specific immune-modulation.
Mechanical and barrier properties of avenin, kafirin, and zein films.
Gillgren, T. & Stading, M. (2008). Food Biophysics, 3(3), 287-294.
Biodegradable and renewable materials can be manufactured from prolamins, which are the major storage protein fraction of cereals. This paper investigates the material properties of oat prolamin (avenin), corn prolamin (zein), and sorghum prolamin (kafirin). Glass transition temperature, dry solid content, stress at break, strain at break, oxygen permeability, and water vapor permeability were analyzed at different plasticizer contents. Avenin was plasticized with glycerol, and kafirin and zein were plasticized with a mixture of polyethylene glycol, glycerol, and lactic acid. Avenin displayed potential, although it did not exhibit the mechanical qualities of gluten, which resembles avenin at the molecular level. Compared to kafirin and zein, avenin was more extensible at low plasticizer contents, while kafirin and especially zein were more extensible at the highest plasticizer content. Avenin was far weaker than the other two at all plasticizer contents. Kafirin and zein displayed similar barrier properties, whereas avenin was notably more permeable.
Regulation of the cellulose synthase-like gene family by light in the maize mesocotyl.
Van Erp, H. & Walton, J. D. (2009). Planta, 229(4), 885-897.
The cellulose synthase-like (ZmCSL) gene family of maize was annotated and its expression studied in the maize mesocotyl. A total of 28 full-length CSL genes and another 13 partial sequences were annotated; four are predicted to be pseudogenes. Maize has all of the CSL subfamilies that are present in rice, but the CSLC subfamily is expanded from 6 in rice to 12 in maize, and the CSLH subfamily might be reduced from 3 to 1. Unlike rice, maize has a gene in the CSLG subfamily, based on its sequence similarity to two genes annotated as CSLG in poplar. Light regulation of glycan synthase enzyme activities and CSL gene expression were analyzed in the mesocotyl. A Golgi-localized glucan synthase activity is reduced by ~50% 12 h after exposure to light. Β-1,4-Mannan synthase activity is reduced even more strongly (>85%), whereas Β-1,4-xylan synthase, callose synthase, and latent IDPase activity respond only slightly, if at all, to light. At least 17 of the CSL genes (42%) are expressed in the mesocotyl, of which four are up-regulated at least twofold, seven are down-regulated at least twofold, and six are not affected by light. The results contribute to our understanding of the structure of the CSL gene family in an important food and biofuel plant, show that a large percentage of the CSL genes are expressed in the specialized tissues of the mesocotyl, and demonstrate that members of the CSL gene family are differentially subject to photobiological regulation.
Effects of barley and oat β-glucan structures on their rheological and thermal characteristics.
Ryu, J. H., Lee, S., You, S., Shim, J. H. & Yoo, S. H. (2012). Carbohydrate Polymers, 89(4), 1238-1243.
In order to understand the relationship between chemical structure and physical properties of cereal β-glucans, the β-glucans with identical Mw (98.4–99.2 kDa) and Rg (21.1–22.0 nm) were isolated from chal and gwangan barley, and ohl oat, and their linkage structure, flow behavior, and thermal properties were investigated. Previously, we established a purification method of 3-O-cellobiosyl-glucose (DP3) and 3-O-cellotriosyl-glucose (DP4) ( Yoo, Lee, Chang, Lee, & Yoo, 2007) and applied these authentic standards to quantify the ratio of β-(1,4)/(1,3) linkages in cereal β-glucans. β-Glucans isolated from two barley cultivars had greater proportion of DP3 than did the oat, and within barley cultivars chal barley β-glucan had significantly larger amount of DP3 over gwangan cultivar. Thus, chal barley β-glucan had the greatest molar ratio (2.53) of DP3 to DP4, and ohl oat had the lowest (1.51). While all the β-glucan solutions showed strong shear thinning behavior, ohl oat β-glucan with higher proportion of DP4 exhibited the highest viscosity among the β-glucan samples. After 3 freeze-thaw cycles of 3% (w/v) β-glucan samples, chal barley β-glucan had lower onset (To) and peak (Tp) temperatures (28.3 and 36.7°C, respectively) than those of gwangan barley (33.6 and 39.9°C) and ohl oat (37.9 and 46.9°C) did, and the heat scan profiles were thermoreversible. The To and Tp of inter-chain associations decreased as the DP3:DP4 ratio of the β-glucan increased. From this study, it was suggested that cellotetraosyl units and longer β-(1,4)-linked segments would be a major contributor for improving solution viscosity and gel formation of cereal β-glucans.
In vitro bile-acid binding and fermentation of high, medium, and low molecular weight β-glucan.
Kim, H. J. & White, P. J. (2010). Journal of Agricultural and Food Chemistry, 58(1), 628-634.
The impact of β-glucan molecular weight (MW) on in vitro bile-acid binding and in vitro fermentation with human fecal flora was evaluated. β-Glucan extracted from oat line ‘N979-5-4’ was treated with lichenase (1,3−1,4-β-D-glucanase) to yield high (6.87 × 105 g/mol), medium (3.71 × 105 g/mol), and low (1.56 × 105 g/mol) MW fractions. The low MW β-glucan bound more bile acid than did the high MW β-glucan (p < 0.05). If the positive control, cholestyramine, was considered to bind bile acid at 100%, the relative bile-acid binding of the original oat flour and the extracted β-glucan with high, medium, and low MW was 15, 27, 24, and 21%, respectively. Significant effects of high, medium, and low MW β-glucans on total SCFA were observed compared to the blank without substrate (p < 0.05). There were no differences in pH changes and total gas production among high, medium, and low MW β-glucans, and lactulose. The low MW β-glucan produced greater amounts of SCFA than the high MW after 24 h of fermentation. Among the major SCFA, more propionate was produced from all MW fractions of extracted β-glucans than from lactulose. In vitro fermentation of extracted β-glucan fractions with different MW lowered pH and produced SCFA, providing potential biological function.
How cell wall complexity influences saccharification efficiency in Miscanthus sinensis.
De Souza, A. P., Kamei, C. L. A., Torres, A. F., Pattathil, S., Hahn, M. G., Trindade, L. M. & Buckeridge, M. S. (2015). Journal of Experimental Botany, 66(14), 4351-4365.
The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis.
Starch structure in developing barley endosperm.
Källman, A., Bertoft, E., Koch, K., Sun, C., Åman, P. & Andersson, R. (2015). International Journal of Biological Macromolecules, 81, 730-735.
Barley spikes of the cultivars/breeding lines Gustav, Karmosé and SLU 7 were harvested at 9, 12 and 24 days after flowering in order to study starch structure in developing barley endosperm. Kernel dry weight, starch content and amylose content increased during development. Structural analysis was performed on whole starch and included the chain-length distribution of the whole starches and their β-limit dextrins. Karmosé, possessing the amo1 mutation, had higher amylose content and a lower proportion of long chains (DP ≥38) in the amylopectin component than SLU 7 and Gustav. Structural differences during endosperm development were seen as a decrease in molar proportion of chains of DP 22–37 in whole starch. In β-limit dextrins, the proportion of Bfp-chains (DP 4–7) increased and the proportion of BSmajor-chains (DP 15–27) decreased during development, suggesting more frequent activity of starch branching enzymes at later stages of maturation, resulting in amylopectin with denser structure.
Mixed‐Linkage Glucan Oligosaccharides Produced by Automated Glycan Assembly Serve as Tools to Determine the Substrate Specificity of Lichenase.
Dallabernardina, P., Schuhmacher, F., Seeberger, P. H. & Pfrengle, F. (2017). Chemistry-A European Journal, 23(13), 3191-3196.
The mixed-linkage (1→3),(1→4)-d-glucan (MLG) specific glycosyl hydrolase lichenase is an important biochemical tool for the structural characterization of MLGs. It holds potential for application in the brewery, animal feed, and biofuel industries. Several defined MLG oligosaccharides obtained by automated glycan assembly are used to analyze the substrate specificities of Bacillus subtilis lichenase. Two glucose building blocks (BBs), equipped with a temporary fluorenylmethyloxycarbonyl chloride (Fmoc) protecting group in the C-3 or C-4 position, served to assemble different oligosaccharides by using an automated oligosaccharide synthesizer. Light-induced cleavage of the glycan products from the solid support followed by global deprotection provided seven MLG oligosaccharides of different length and connectivity. After incubation of the MLG oligosaccharides with lichenase, the digestion products were analyzed by HPLC-MS. These digestion experiments provided insights into the enzyme's active site that is in line with other recent evidence suggesting that the substrate specificity of lichenases has to be reconsidered. These results demonstrate that synthetic MLG oligosaccharides are useful tools to analyze mixed-linkage β-glucanases.
Improvement of enzyme activity of β-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues.
Baek, S. C., Ho, T. H., Lee, H. W., Jung, W. K., Gang, H. S., Kang, L. W. & Kim, H. (2017). Applied Microbiology and Biotechnology, 101(10), 4073-4083.
β-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG2-6, BG2-10, and BG5-28 were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG5-28 was 2.11-fold higher than that of wild-type BGwt, whereas those of BG2-6 and BG2-10 were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BGwt (pH 5.0 and 40°C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (kcat/Km) of BG5-28 were 1.92- and 2.12-fold greater than those of BGwt at 40°C, respectively. The catalytic efficiency of BG5-28 increased to 3.09-fold that of BGwt at 60°C. These increases in the thermostability and catalytic efficiency of BG5-28 might be useful for the hydrolysis of β--glucans to produce fermentable sugars. Of the six mutated residues of BG5-28, five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α6/α6-double-barrel structure is essential for enzyme activity.