Azo-Barley Glucan

We report herein the development of a novel assay procedure for the measurement of β-glucanase and lichenase (EC 3.2.1.73) 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 3.2.1.6). 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.

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.

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 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.

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.

Facing the critical issue of high production costs for cellulase, numerous studies have focused on improving the efficiency of cellulase production by potential cellulolytic microorganisms using agricultural wastes as substrates, extremophilic cellulases, in particular, are crucial in the biorefinery process because they can maintain activity under harsh environmental conditions. This study aims to investigate the ability of a potential carboxymethylcellulose-hydrolyzing bacterial strain H1, isolated from an Algerian saline soil and identified as Bacillus velezensis, to use untreated olive mill wastes as a substrate for the production of an endo-1,4-β-glucanase. The enzyme was purified 44.9 fold using only two steps: ultrafiltration concentration and ion exchange chromatography, with final recovery of 80%. Its molecular mass was estimated to be 26 kDa by SDS-PAGE. Enzyme identification by LC–MS analysis showed 40% identity with an endo-1,3-1,4-β-glucanase of GH-16 family. The highest enzymatic activity was significantly measured on barley β-glucan (604.5 U/mL) followed by lichenan and carboxymethylcellulose as substrates, confirming that the studied enzyme is an endo-1,4-β-glucanase. Optimal enzymatic activity was at pH 6.0-6.5 and at 60-65°C. It was fairly thermotolerant, retaining 76.9% of the activity at 70°C, and halotolerant, retaining 70% of its activity in the presence of 4 M NaCl. The enzyme had a V_max of 625 U/min/mL and a high affinity with barley β-glucan resulting a K_m of 0.69 mg/mL. It also showed a significant ability to release cello-oligosaccharides. Based on such data, the H1 endo-1,4-β-glucanase may have significant commercial values for industry, argo-waste treatment, and other biotechnological applications.

High stability at acidic environment is required for 1,3-1,4-β-glucanase to function in biofuel, brewing and animal feed industries. In this study, a mesophilic β-glucanase from Bacillus terquilensis CGX 5-1 was rationally engineered through sequence alignment and surface charge engineering to improve its acidic resistance ability. Nineteen singly-site variants were constructed and Q1E, I133L and V134A variants showed better acidic stability without the compromise of catalytic property and thermostability. Furthermore, four multi-site variants were constructed and one double-site variant Q1E/I133L with better stability at acidic environment and higher catalytic property was obtained. The fluorescence spectroscopy and structural analysis showed that more surface negative charge, decreased exposure degree of residue No.1, shifted side chain direction of residue No.133 and the lower total and folding free energy might be the reason for the improvement of acidic stability of Q1E/I133L variant. The obtained Q1E/I133L variant has potential applications in industries.

This study aimed at developing a complete miniaturized high‐throughput screening workflow for the evaluation of the Cell Wall‐Degrading Enzyme (CWDE) activities produced by any fungal strain directly cultivated on raw feedstock in a submerged manner. In this study, wheat straw was selected as model substrate as it represents an important carbon source but yet poorly valorised to yield high added value products. Fungi were grown in a microbioreactor in a high‐throughput (HT) way to replace the fastidious shaking flask cultivations. Both approaches were compared in order to validate our new methodology. The range of CWDE activities produced from the cultures was assayed using AZO‐died and pNP‐linked substrates in an SBS plate format using a Biomek FXp pipetting platform. As highlighted in this study, it was shown that the CWDE activities gathered from the microbioreactor cultivations were similar or higher to those obtained from shake flasks cultures, with a lower standard deviation on the measured values, making this new method much faster than the traditional one and suitable for HT CWDE production thanks to its pipetting platform compatibility. Also, the results showed that the enzymatic activities measured were the same when doing the assay manually or using the automated method.

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.

Wheat bran was fermented at 28 °C for 7 days under 70% humidity by Aspergillus niger, Aspergillus oryzae and Aspergillus awamori. Total phenolic content (TPC) of the unfermented sample was 1531.5 µg g^-1 wheat bran. After the fermentation of Aspergillus awamori, Aspergillus oryzae and Aspergillus niger, TPC reached 5362.1, 7462.6 and 10 707.5 µg g^-1, respectively. The antioxidant activity in the extractions of fermented wheat bran also increased significantly compared with the unfermented sample (P < 0.05). Aspergillus niger showed the greatest capacity to release bound ferulic acid (416.6 µg g^-1). Aspergillus oryzae and Aspergillus awamori had the advantages of releasing more chlorogenic acid (84.0 µg g^-1) and syringic acid (142.3 µg g^-1), respectively. The destructive effect of Aspergillus niger on wheat bran structure was the strongest, followed by that of Aspergillus oryzae. This effect of Aspergillus niger may be due to its higher cellulase, xylanase, arabinofuranosidase and β-xylosidase activities. Besides, Aspergillus oryzae possessed higher β-glucosidase activity, and Aspergillus awamori had higher α-amylase and feruloyl esterase activities. Aspergillus niger may be the best to release bound phenolic acids in the three Aspergillus species. These will provide the helpful information for understanding mechanism of the fermentation by Aspergillus species releasing bound phenolic in wheat bran.

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).

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).

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.

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.

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.

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.