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 184.108.40.206) 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 220.127.116.11). 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.
Comparison of endolytic hydrolases that depolymerise 1,4-β-D-mannan, 1,5-α-L-arabinan and 1,4-β-D-galactan.
McCleary, B. V. (1991). “Enzymes in Biomass Conversion”, (M. E. Himmel and G. F. Leatham, Eds.), ACS Symposium Series 460, Chapter 34, pp. 437-449. American Chemical Society, Washington.
Hydrolysis of mannan-type polysaccharides by β-mannanase is dependent on substitution on and within the main-chain as well as the source of the β-mannanase employed. Characterisation of reaction products can be used to define the sub-site binding requirements of the enzymes as well as the fine-structures of the polysaccharides. Action of endo-arabinanase and endo-galactanase on arabinans and arabinogalactans is described. Specific assays for endo-arabinanase and arabinan (in fruit-juice concentrates) are reported.
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.
Measurement of polysaccharide degrading enzymes using chromogenic and colorimetric substrates.
McCleary, B. V. (1991). Chemistry in Australia, 58, 398-401.
Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.
Optimising the response.
Acamovic, T. & McCleary, B. V. (1996). Feed Mix, 4, 14-19.
A fine balance exists between enzyme activity and the adverse effects associated with feed processing. Accurate estimation of enzyme activity in the feed is a pre-requisite to optimising the response.
Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post‐translocational protein folding and degradation.
Vitikainen, M., Hyyryläinen, H. L., Kivimäki, A., Kontinen, V. P. & Sarvas, M. (2005). Journal of Applied Microbiology, 99(2), 363-375.
Aims: To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation. Methods and Results: Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of α-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1•5-fold). Increasing the net negative charge of the cell wall because of lack of the D-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1•5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases. Conclusions: Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner. Significance and Impact of the Study: The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.
Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence.
Sakamoto, Y., Watanabe, H., Nagai, M., Nakade, K., Takahashi, M. & Sato, T. (2006). Plant Physiology, 141(2), 793-801.
Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising β-1,3-glucan with β-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited β-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.
Thermostabilities of grain β-amylase and β-glucanase in finnish landrace barleys and their putative past adaptedness.
Ahokas, H. & Manninen, M. L. (2000). Hereditas, 132(2), 111-118.
Thermostability of β-amylase activity was a general feature in a sample of 32 Finnish barley landraces. One of two Finnish landraces probably contributed the thermostability to cv. ‘Pirkka’ in crosses performed about 70 years ago. The stability is less evolved in β-glucanase activity although the most tolerant types appeared in landraces and in Pirkka with a Finnish landrace background. Selection pressure for thermostability in grains may have been a feature of traditional crop management practices among Finns in the past: drying grain crops, including premature barley, above an oven in a special drying house at temperatures exceeding 55°C, and germination in black, sunlit slash-and-burn soils, with a measured surface temperature of 63°C. A positive, though small correlation between the thermotolerance ratios of the two enzymes may be a remnant of their common long selection pressure ending tens of generations prior to collection in the 1960s and 1970s.