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 126.96.36.199) 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 188.8.131.52). 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 malt beta-glucanase.
McCleary, B. V. (1986). Proceedings of the 19th Convention of the Institute of Brewing (Aust. and N.Z. section), 181-187.
A Procedure has been developed for the assay of malt β-glucanase [a(1→3)(1→4)-β-D-glucanase] which employs as substrate, barley β-glucan dyed with Remazolbrilliant Blue and chemically modified with carboxymethyl groups to increase solubility. The described assay procedure together with a modified extraction format allows analysis of up to ten malt samples in less than 80 min. Also, the procedure is specific for enzymes active on barley β-glucan, is accurate and reliable, and can be readily applied to the analysis of β-glucanase in malt, green malt and wort.
A soluble chromogenic substrate for the assay of (1→3)(1→4)-β-D-glucanase (lichenase).
McCleary, B. V. (1986). Carbohydrate Polymers, 6(4), 307-318.
A simple procedure for the assay of (1→3)(1→4)-β-D-glucanase (lichenase) has been developed. This assay employs as substrate barley (1→3)(1→4)-β-D-glucan dyed with Remazolbrilliant Blue R and chemically modified with carboxymethyl groups to increase solubility. Preparation of this substrate required the development of an improved procedure for the extraction and purification of barley β-glucan. Assays based on the use of the described chromogenic substrate at pH 6•5 are sensitive and specific for enzymes active on barley β-glucan.
Problems caused by barley beta-glucans in the brewing industry.
McCleary, B. V. (1986). Chemistry in Australia, 53, 306-308.
Brewing, the oldest application of bio-technology is now a mix of trade art and modern science. This article describes new applications of enzyme chemistry to trouble-shooting in beer production.
Assay of malt β-glucanase using azo-barley glucan: an improved precipitant.
McCleary, B. V. & Shameer, I. (1987). Journal of the Institute of Brewing, 93(2), 87-90.
A procedure recently described for the assay of malt β-glucanase, which employs a dye-labelled and chemically-modified barley β-glucan substrate, has been improved by changing the precipitant solution used to terminate the reaction. The new precipitant solution contains 0•4% (w/v) zinc acetate and 4% (w/v) sodium acetate dissolved in 80% (v/v) aqueous methyl cellosolve. With this precipitant the procedure can be directly applied to the assay of cellulase activity, and with minor modification, to the assay of lichenase activity.
Effect of high hydrostatic pressure-temperature combinations on the activity of β-Glucanase from barley malt.
Buckow, R., Heinz, V. & Knorr, D. (2005). Journal Institute Brewing, 111(3), 282–289.
β-Glucanase from barley malt is known to be thermolabile but important in the mashing process. Therefore, the potential of increasing the thermostability of β-glucanase in ACES buffer (0.1 M, pH 5.6) by high hydrostatic pressure has been investigated. Inactivation of the enzyme as well as changes of the conversion rate in response to combined pressure-temperature treatments in the range of 0.1–900 MPa and 30–75°C were assessed by analyzing the kinetic rate constants. A significant stabilization of β-glucanase against temperature-induced inactivation was detected at 400 MPa. With increasing pressure up to 600 MPa the catalytic activity of β-glucanase was progressively decelerated. However, for the overall depolymerization reaction of β-glucans in ACES buffer (0.1 M, pH 5.6) a maximum was identified at 215 MPa and 55°C yielding approximately 2/3 higher degradation of β-glucan after 20 min as compared to the maximum at ambient pressure (45°C).
Effect of the use of dilute alkaline prior to Bacillus subtilis based biocontrol steeping and germination conditions on red sorghum malt β-glucanase activities and residual β-glucans.
Bwanganga Tawaba, J. C., Destain, J., Malumba Kamba, P., Béra, F. & Thonart, P. (2013). Journal of Cereal Science, 58(1), 148-155.
Malting is the ideal stage to deal with β-glucans. Their hydrolysis is very important as the diffusion of both hormones and hydrolytic enzymes in the endosperm of germinated grain depend on it. A high malt β-glucanase activity is not a guarantee of an extensive hydrolysis of β-glucans. When Bacillus subtilis is used to control mould growth, red sorghum malt β-glucanase activity (measured using carboxymethylcellulose as the substrate) was improved without significantly affecting the hydrolysis of malt β-glucans. Thus, in order to reduce the residual β-glucans content, soaking in 0.2% NaOH was combined with a biocontrol. Soaking in 0.2% NaOH is recognized as capable of improving grain hydration by opening-up the endosperm cell walls. The combined use of 0.2% NaOH with B. subtilis-based biocontrol treatments during red sorghum malting, leads to malt with increased β-glucanase activity and a significant reduction of residual β-glucans when compared with the 16 h biocontrol steeping without prior steeping in 0.2% NaOH. β-glucanase activity increases with increased germination temperature and time while, conversely, the residual β-glucans content of the malts decreases. Indeed, while the level of β-glucanase was not vastly different between the malts obtained after steeping in distilled water and those obtained after 8 h steeping in 0.2% NaOH followed by 8 h resteeping in distilled water (NaOH + H2O treatment), their residual β-glucans levels differ significantly. B. subtilis-based treatment leads to malt with improved β-(1-3)- and β-(1-4)-glucanase activities without significantly improved malt β-(1-3),(1-4)-glucanase activity. While malts obtained after 84 h germination weren't significantly different in terms of malt β-(1-3),(1-4)-glucanase activities for all steeping treatments, the use of 0.2% NaOH steeping prior to resteeping led to malts with improved β-glucans content. Combining the steeping in dilute alkaline and biocontrol enables taking advantage of the dilute alkaline effect on residual β-glucans content, due probably to the opening-up of the cell walls and the improvement of water uptake, and that of the biocontrol (improvement of β-glucanase synthesis).
Retinol-induced secretion of human retinol-binding protein in yeast.
Reppe, S., Smeland, S., Moskaugb, J. & Blomhof, R. (1998). FEBS Letters, 427(2), 213–219.
Retinol-binding protein (RBP) functions as a transporter for retinol (vitamin A) in plasma in higher eukaryotes. We have successfully expressed human RBP in Saccharomyces cerevisiae, and its secretion was found to be induced by retinol also in this lower eukaryote. Reduced induction of secretion by retinol in a temperature-sensitive sec18-1 mutant that is blocked in secretion at the restricted temperature suggests that as in mammalian cells, RBP can be released from the endoplasmic reticulum upon addition of retinol. Thus, the molecular mechanism involved in retinol-dependent secretion of RBP appears to be conserved in yeast, and this points to yeast as a putative model system for studying retinol-regulated secretion of RBP. RBP purified from yeast was found to be indistinguishable from RBP purified from human plasma in several functional assays.
Supplements of transgenic malt or grain containing (1, 3-1, 4)-β-glucanase increase the nutritive value of barley-based broiler diets to that of maize.
Von Wettstein, D., Warner, J. & Kannangara, C. G. (2003). British Poultry Science, 44(3), 438-449.
1. A diet with addition to normal barley of malt from transgenic barley expressing a protein engineered, thermotolerant Bacillus (1,3-1,4)-β-glucanase during germination has previously been demonstrated to provide a broiler chicken weight gain comparable to maize diets. It also reduced dramatically the number of birds with adhering sticky droppings, but did not entirely eliminate sticky droppings. One of the objectives of the broiler chicken trials reported here was to determine if higher concentrations of transgenic malt could alleviate the sticky droppings. 2. Another aim was to investigate the feasibility of using mature transgenic grain containing the thermotolerant (1,3-1,4)-β-glucanase as feed addition and to compare diets containingtransgenic grain to a diet with the recommended amount of a commercial β-glucanase-based product. 3. Inclusion of 75 or 151 g/kg transgenic malt containing 4·7 or 98 mg/kg thermotolerant (1,3-1,4)-β-glucanase with 545 or 469 g/kg non-transgenic barley instead of maize yielded a weight gain in Cornish Cross broiler chickens indistinguishable from presently used maize diets. The gene encoding the enzyme is expressed in the aleurone with a barley α-amylase gene promoter and the enzyme is synthesised with a signal peptide for secretion into the endosperm of the malting grain. 4. Equal weight gain was achieved, when the feed included 39 g/kg transgenic barley grain [containing 66 mg/kg thermotolerant (1,3-1,4)-β-glucanase] and 581 g/kg non-transgenic barley instead of maize. In this case, the gene encoding the enzyme has been expressed with the D-hordein gene (Hor3-1) promoter during grain maturation. The enzyme is synthesised as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeted into the storage vacuoles. Deposition of the enzyme in the prolamin storage protein bodies of the endosperm protects it from degradation during the programmed cell death of the endosperm in the final stages of grain maturation and provides extraordinaryheat stability. The large amount of highly active (1,3-1,4)-β-glucanase in the mature grain allowed the reduction of the transgenic grain ingredient to 0·2 g/kg diet, thus making the ingredient comparable to that of the trace minerals added to standard diets. 5. A direct comparison using transgenic grain supplement at the level of 1 g/kg of feed with the standard recommended addition of the commercial enzyme preparation Avizyme 1100® at 1 g/kg yielded equal weight gain, feed consumption and feed efficiency in birds fed a barley-based diet. 6. The production of sticky droppings characteristic of broilers fed on barley diets was avoided with all 9 experimental diets and reduced to the level observed with a standard maize diet by supplementation With transgenic barley. 7. The excellent growth and normal survival of the 400 broilers tested on barley diets supplemented with transgenic grain or malt showed the grain and malt not to be toxic. 8. The barley feed with added transgenic grain or malt containingthermotolerant (1,3-1,4)-β-glucanase provides an environmentally friendly alternative to enzyme additives, as it uses photosynthetic energy for production of the enzyme in the grain and thus avoids use of non-renewable energy for fermentation. The deposition of the enzyme in the protein bodies of the grain in the field makes coating procedures for stabilisation of enzyme activity superfluous. 9. Barley feed with the small amount of transgenic grain as additive to normal barley provides an alternative for broiler feed in areas where grain maize cannot be grown for climatic reasons or because of unsuitable soil and thus has to be imported.
Assessment of enzymatic endosperm modification of malting barley using individual grain analyses.
De Sá, R. M. & Palmer, G. H. (2004). Journal of the Institute of Brewing, 110(1), 43-50.
Enzymatic modification of the endosperm of malting barley is a main feature of the malting process. Uneven enzymatic modification of the endosperm (heterogeneity) can cause brewhouse problems. Although there is a general correlation between endosperm modification, beta-glucan breakdown and endo-beta-glucanase development, it is based on average results from sample analyses and may conceal heterogeneity. The primary aim of this work was to use individual grain analyses to investigate factors that control endosperm modification and beta-glucan breakdown. In terms of beta-glucan breakdown and physical modification, the barley variety Chariot malted faster than Decanter. However, both varieties showed similar distribution of endo-beta-glucanase in individual grains during malting. Further work on individual grains showed that the correlation between beta-glucan breakdown and endo-beta-glucanase activity was not significant. Surprisingly beta-glucan breakdown did not always correlate with the physical modification of the endosperm. Both these findings suggest that sample analyses of beta-glucan levels and malt beta-glucanase activities are not reliable indicators of the degrees of which malt samples are modified during malting. Since the distribution of beta-glucan in individual grains of the unmalted barley varieties was similar, the total beta-glucan levels of the original barley did not determine the rate at which beta-glucan was broken-down during malting. Although protein studies are at a preliminary stage, the rate of protein breakdown was not correlated with the rate at which beta-glucan was broken down in the malting grain. It is possible that the physico-chemical properties of endosperm storage proteins may limit the rate of beta-glucan breakdown during malting.
Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi.
Shen, H., Gilkes, N. R., Kilburn, D. G., Miller Jr, R. C. & Warren, R. A. (1995). Biochemical Journal, 311, 67-74.
The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a polypeptide of 1090 amino acids. Cellobiohydrolase B (CbhB) is 1037 amino acids long, with a calculated molecular mass of 109765 Da. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose-binding domain of 104 amino acids. The catalytic domain belongs to family 48 of glycosyl hydrolases. CbhB has a very low activity on CM-cellulose. Viscometric analysis of CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase. Cellobiose is the major product of hydrolysis of cellulose. In common with two other exoglycanases from C. fimi, CbhB has low but detectable endoglucanase activity. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases.
Production of a thermostable 1,3-1,4-β-glucanase mutant in Bacillus subtilis WB600 at a high fermentation capacity and its potential application in the brewing industry.
Niu, C., Liu, C., Li, Y., Zheng, F., Wang, J. & Li, Q. (2017). International Journal of Biological Macromolecules, In Press.
1,3-1,4-β-glucanase was an important biotechnological aid in the brewing industry. In a previous research, a Bacillus BglTO mutant (BglTO) with high tolerance towards high temperature and low-pH conditions was constructed and expressed in Escherichia coli. However, E. coli was not a suitable host for enzyme production in food industry. Therefore, the present work aimed to achieve the high-level expression of BglTO in Bacillus subtilis WB600 and to test its effect in Congress mashing. The β-glucanase mutant was successfully expressed in B. subtilis WB600 and favorable plasmid segregation and structural stability were observed. The maximal extracellular activity of β-glucanase in recombinant B. subtilis WB600 reached 4840.4 U mL−1 after cultivation condition optimization, which was 1.94-fold higher than that before optimization. The fermentation capacity of recombinant B. subtilis reached 242.02 U mL−1 h−1, which was the highest among all reported β-glucanases. The addition of BglTO in Congress mashing significantly reduced the filtration time and viscosity of mash by 29.7% and 12.3%, respectively, which was superior to two commercial enzymes. These favorable properties indicated that B. subtilis WB600 was a suitable host for production of BglTO, which was promising for application in the brewing industry.