Purification of β-D-glucosidase from Aspergillus niger.
McCleary, B. V. & Harrington, J. (1988). “Methods in Enzymology”, Volume 160, (H. Gilbert, Ed.), Elsevier Inc., pp. 575-583.
β-D-Glucosidases have been isolated and purified from a number of fungal culture filtrates. A number of β-glucosidases, including those from almond emulsin and from Aspergillus niger 15, do not have a strict requirement for a D-gluco configuration. The enzyme from almond emulsin catalyzes hydrolysis of both β-D-glucopyranosides and β-D-galactopyranosides and evidence has been obtained for the involvement of different enzyme active sites. An enzyme purified to homogeneity from culture filtrates of Aspergillus niger 15 has a very broad specificity with activity on β-D-glucosides, β-D-xylosides, β-D-galactosides, and β-L-arabinosides. β-glucosidase in combination with a specific endo-β-glucanase could find widespread application in the quantification of a range of β-D-glucans such as (1→4)-β-D-glucan, (1→3)-β-D-glucan, (1→3),(1→4)-β-D-glucan, and (1→3),1→6)-β-D-glucan. Together with endo-1,4-β-D-mannanase and β-D-mannosidase it may also prove useful in the measurement of β-D-glucomannans. A method for the assay of (1→3),(1→4)-β-D-glucan has already been developed using a highly purified β-D-glucosidase from a commercially available Aspergillus niger enzyme preparation. This chapter describes the purification of this enzyme and report on some of its properties.
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 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 hydrolysis and industrial importance of barley β-glucans and wheat flour pentosans.
McCleary, B. V., Gibson, T. S., Allen, H. & Gams, T. C. (1986). Starch-Stärke, 38(12), 433-437.
Mixed linkage β-glucane and pentosanes (mainly arabinoxylanes) are the major endosperm cell-wall polysaccharides of barley and wheat respectively. These polysaccharides, although minor components of the whole grain, significantly affect the industrial utilization of these cereals. The modification of barley corns during malting requires the dissolution of the β-glucane in the cell-wall of the starch endosperm. High β-glucane concentration in wort and beer effect the rate of filtration and can also lead to precipitate or gel formation in the final product. In a similar manner, pentosane is thought to cause filtration problems with wheat starch hydrolysates by increasing viscosity and by producing gelatinous precipitate which blocks filters. Ironically, it is this same viscosity building and water binding capacity which is considered to render pentosanes of considerable value in dough development and bread storage (anti-staling functions). In the current paper, some aspects of the beneficial and detrimental effects of pentosanes and β-glucane in the industrial utilization of wheat and barley are discussed. More specifically, enzymic methods for the preparation, analysis and identification of these polysaccharides and for the removal of their functional properties, are described in detail.
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.
Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition.
Strobel, K. L., Pfeiffer, K. A., Blanch, H. W. & Clark, D. S. (2016).
Biotechnology and Bioengineering, 133(6), 1369-1374.
Non-productive binding of cellulases to lignin inhibits enzymatic hydrolysis of biomass, increasing enzyme requirements and the cost of biofuels. This study used site-directed mutagenesis of the Trichoderma Cel7A carbohydrate binding module (CBM) and linker to investigate the mechanisms of adsorption to lignin and engineer a cellulase with increased binding specificity for cellulose. CBM mutations that added hydrophobic or positively charged residues decreased the specificity for cellulose, while mutations that added negatively charged residues increased the specificity. Linker mutations that altered predicted glycosylation patterns selectively impacted lignin affinity. Beneficial mutations were combined to generate a mutant with 2.5-fold less lignin affinity while fully retaining cellulose affinity. This mutant was uninhibited by added lignin during hydrolysis of Avicel and generated 40% more glucose than the wild-type enzyme from dilute acid-pretreated Miscanthus.
Enzymatic preparation of mushroom dietary fibre: A comparison between analytical and industrial enzymes.
Wong, K. H. & Cheung, P. C. K. (2009). Food Chemistry, 115(3), 795-800.
A comparative study on preparing dietary fibres (DFs) from three mushroom sclerotia, namely, Pleurotus tuber-regium (PTR), Polyporus rhinocerus (PR) and Wolfiporia cocos (WC), using analytical or industrial enzymes (including α-amylase, protease and amyloglucosidase), was conducted. Apart from enzyme activity and purity, their effects on the yield of sclerotial DF as well as its major components, such as β-glucans, chitin and resistant glycogen (RG), were investigated and compared. The activities of all industrial enzymes were significantly lower than those of their corresponding analytical ones, except for the Fungamyl® Super MA, which had the highest α-amylase activity (6395 U/g). However, this fungal α-amylase was less able to digest the three sclerotial glycogens when compared with the bacterial alternatives. Amongst all tested enzymes, only analytical and industrial amyloglucosidases were found to have significant amount of contaminating cellulase (7.05–7.07 U/ml) and lichenase (4.62–4.67 U/ml) activities, which would cause endo-depolymerization of the β-glucan-type cell wall components (3.39% reduction in glucose residue after RG correction) of the PTR, leading to a marked α-amylase hydrolysis of its otherwise physically-inaccessible cytoplasmic glycogen (20.3% reduction in RG content). Commercial production of the three novel sclerotial DFs, using the industrial enzymes, would be feasible since, in addition to their economic advantage, both the yield (PTR: 81.2%; PR: 86.5%; WC: 96.2% of sample DM) and total non-starch polysaccharide contents (PTR: 88.0%; PR: 92.5%; WC: 91.1% DF-rich materials of DM) of their resulting sclerotial DFs were comparable to the levels of those prepared using analytical enzymes.
Enzymatic hydrolysis of steryl ferulates and steryl glycosides.
Nyström, L., Moreau, R. A., Lampi, A. M., Hicks, K. B. & Piironen, V. (2008). European Food Research and Technology, 227(3), 727-733.
Steryl ferulates (SF) and steryl glycosides (SG) are phytosterol conjugates found characteristically in cereals. Currently, little is known about their properties with respect to enzymatic hydrolysis. SF and SG were extracted and purified from rye and wheat bran. Their percentages of hydrolysis with different enzymes were studied using normal phase HPLC with UV detection for steryl ferulates and evaporative light scattering detection for steryl glycosides. Steryl ferulates were hydrolysed by mammalian digestive steryl esterases. It was further demonstrated that a mixture of steryl ferulates from rye and wheat was hydrolysed much more effectively than a steryl ferulate mixture from rice (commonly known as γ-oryzanol), suggesting greater bioavailability in non-rice steryl ferulates. Steryl glycosides were hydrolysed by a commercial microbial β-glucosidase preparation (cellobiase), but were not effectively hydrolysed by two other highly purified β-glucosidases. These results demonstrate for the first time the potential use of enzymes as a replacement for acid hydrolysis in analytical procedures for SG and also provide insights about the potential bioavailability of these sterol derivatives in human digestive systems.
Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases.
Badhan, A., Wang, Y., Gruninger, R., Patton, D., Powlowski, J., Tsang, A. & McAllister, T. (2014). BMC Biotechnology, 14(1), 31.
Background: Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Results: Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. Conclusion: The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw.
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.
Biomass hydrolyzing enzymes from plant pathogen Xanthomonas axonopodis pv. punicae: optimizing production and characterization.
Amat, D., Arora, A., Nain, L. & Saxena, A. K. (2014). Annals of Microbiology, 64(1), 267-274.
Xanthomonas axonopodis pv. Punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28°C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55°C while xylanase had highest activity at 45°C. Heat treatment of enzyme extract at 75°C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.
Droplet-based microfluidic platform for heterogeneous enzymatic assays.
Chang, C., Sustarich, J., Bharadwaj, R., Chandrasekaran, A., Adams, P. D. & Singh, A. K. (2013). Lab Chip, 13(9), 1817-1822.
Heterogeneous enzymatic reactions are used in many industrial processes including pulp and paper, food, and biofuel production. Industrially-relevant optimization of the enzymes used in these processes requires assaying them with insoluble substrates. However, platforms for high throughput heterogeneous assays do not exist thereby severely increasing the cost and time of enzyme optimization, or leading to the use of assays with soluble substrates for convenient, but non-ideal, optimization. We present an innovative approach to perform heterogeneous reactions in a high throughput fashion using droplet microfluidics. Droplets provide a facile platform for heterogeneous reactions as internal recirculation allows rapid mixing of insoluble substrates with soluble enzymes. Moreover, it is easy to generate hundreds or thousands of picoliter droplets in a small footprint chip allowing many parallel reactions. We validate our approach by screening combinations of cellulases with real-world insoluble substrates, and demonstrate that the chip-based screening is in excellent agreement with the conventional screening methods, while offering advantages of throughput, speed and lower reagent consumption. We believe that our approach, while demonstrated for a biofuel application, provides a generic platform for high throughput monitoring of heterogeneous reactions.
High-throughput enzymatic hydrolysis of lignocellulosic biomass via in-situ regeneration.
Bharadwaj, R., Wong, A., Knierim, B., Singh, S., Holmes, B. M., Auer, M., Simmons, B. A., Adams, P. D. & Singh, A. K. (2011). Bioresource Technology, 102(2), 1329-1337.
The high cost of lignocellulolytic enzymes is one of the main barriers towards the development of economically competitive biorefineries. Enzyme engineering can be used to significantly increase the production rate as well as specific activity of enzymes. However, the success of enzyme optimization efforts is currently limited by a lack of robust high-throughput (HTP) cellulase screening platforms for insoluble pretreated lignocellulosic substrates. We have developed a cost-effective microplate based HTP enzyme-screening platform for ionic liquid (IL) pretreated lignocellulose. By performing in-situ biomass regeneration in micro-volumes, we can volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for engineering novel IL-tolerant cellulases. Our platform only requires straightforward liquid-handling steps and allows the integration of biomass regeneration, washing, saccharification, and imaging steps in a single microtiter plate. The proposed method can be used to screen individual cellulases as well as to develop novel cellulase cocktails.
Pretreatment of paddy straw with Trametes hirsuta for improved enzymatic saccharification.
Saritha, M., Arora, A. & Nain, L. (2012). Bioresource Technology, 104, 459-465.
Delignification of paddy straw with the white-rot fungus, Trametes hirsuta under solid state fermentation, for enhanced sugar recovery by enzymatic saccharification was studied. T. hirsuta MTCC136 showed high ligninase and low cellulase activities. Solid state fermentation of paddy straw with T. hirsuta enhanced carbohydrate content by 11.1% within 10 days of incubation. Alkali extracts of Trametes pretreated paddy straw showed high absorbance at 205 nm indicating high lignin break down. The amount of value-added lignin recovered from the Trametes pretreated paddy straw was much higher than controls. Enzymatic hydrolysis of the Trametes pretreated paddy straw yielded much higher sugars than controls and yields increased till 120 h of incubation. Saccharification efficiency of the biologically pretreated paddy straw with Accelerase®1500 was 52.69% within 72 h and was higher than controls. Thus, the study brings out the delignification potential of T. hirsuta for pretreatment of lignocellulosic substrate and facilitating efficient enzymatic digestibility of cellulose.
Substrate specificities of glycosidases from Aspergillus species pectinase preparations on elderberry anthocyanins.
Pricelius, S., Murkovic, M., Souter, P. & Guebitz, G. M. (2009). Journal of Agricultural and Food Chemistry, 57(3), 1006-1012.
Attractive color is one of the most important sensory characteristics of fruit and berry products, and elderberry juice is widely used as natural colorant. When pectinase preparations were used in the production of elderberry juice for clarification, a concomitant decrease of anthocyanins and thus a color loss were observed. This paper demonstrates that this is due to side glycosidase activities contained in commercial pectinase preparations from Aspergillus sp. Using LC-MS, sequential deglycosylation of cyanidin-3-sambubioside, cy-3-glucoside, cy-3-sambubioside-5-glucoside, and cy-3,5-diglucoside was found to be catalyzed by specific glycosidases contained in the pectinase preparations. There was no big difference in the deglycosylation rate between monoglucosidic or diglucosidic anthocyanins. However, the degradation rate was decreased when rutinose was attached to cyanidin, whereas the structure of the aglycone itself had almost no influence. Pure β-glucosidases from Agrobacterium species and Aspergillus niger and the β-glucosidase N188 from A. niger did not show any conversion of anthocyanins, indicating the presence of specific glycosidases. Thus, an activity gel based assay was developed to detect anthocyanin-specific glycosidase activity in enzyme preparations, and according to LC-MS peptide mass mapping of digested bands, homologies to a β-glucosidase from Aspergillus kawachii were found.
New glycosidase substrates for droplet-based microfluidic screening.
Najah, M., Mayot, E., Mahendra-Wijaya, I. P., Griffiths, A. D., Ladame, S. & Drevelle, A. (2013). Analytical Chemistry, 85(20), 9807-9814.
Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30°C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems.