New chromogenic substrates for the assay of alpha-amylase and (1→4)-β-D-glucanase.
McCleary, B. V. (1980). Carbohydrate Research, 86(1), 97-104.
New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1→4)-β-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1→4)-β-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.
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 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.
Xyr1 receives the lactose induction signal and regulates lactose metabolism in Hypocrea jecorina.
Stricker, A., R., Steiger, M., G. & Mach, R., L. (2007). FEBS Letters, 581(21), 3915-3920.
This study reports the vital regulatory influence of Xyr1 (xylanase regulator 1) on the transcription of hydrolytic enzyme-encoding genes and hydrolase formation on lactose in Hypocrea jecorina. While the transcription of the xyr1 gene itself is achieved by release of carbon catabolite repression, the transcript formation of xyr1 (xylanase 1) is regulated by an additional induction mechanism mediated by lactose. Xyr1 has an important impact on lactose metabolism by directly activating xyr1 (xylose reductase 1) transcription and indirectly influencing transcription of bga1 (β-galactosidase 1). The latter is achieved by regulating the conversion of D-galactose to the inducing carbon source galactitol.
Scouring of flax rove with the aid of enzymes.
Ossola, M. & Galante, Y. M. (2004). Enzyme and Microbial Technology, 34(2), 177-186.
Linen is the yarn or the fabrics made from fibres of the flax plant (Linum usitatissumum), which, like other bast fibre crops, can be grown in moderate climates and needs low input of agrochemicals to give high yields. Before cotton took over as the main plant-derived textile material, linen was one of the most important source of textile fibres. Following a few decades of almost abandonment, flax fibres are being re-evaluated thanks to the unique features of freshness, comfort and elegance of linen apparels, sheets, towels and other household textile items. However, flax processing into yarn essentially still follows traditional methodologies. We have reinvestigated the effects of several well characterized, mostly recombinant, industrial enzymes on raw flax rove (on a pilot scale from four times 1 kg spools up to 130 kg of material) as an alternative to chemical scouring, followed by a single bleaching step and by yarn wet mechanical spinning. After spinning, all relevant yarn parameters were measured and evaluated (e.g., resistance, stretching, percentage of neps, etc.). In the present work, we demonstrate the advantages of scouring with the enzymes tested, used under mild reaction conditions, in comparison with traditional chemical scouring. The decreasing order of effectiveness was: pectinase>xylanase=galactomannanase=protease>lipase>or=laccase. A simple and straightforward scheme of rove biopreparation and bleaching is proposed, followed by wet spinning to yield a high quality linen yarn.
Efficient yeast cell-surface display of exo- and endo-cellulase using the SED1 anchoring region and its original promoter.
Inokuma, K., Hasunuma, T. & Kondo, A.(2014). Biotechnology for Biofuels, 7(1):8.
Background: The recombinant yeast strains displaying the heterologous cellulolytic enzymes on the cell surface using the glycosylphosphatidylinositol (GPI) anchoring system are considered promising biocatalysts for direct conversion of lignocellulosic materials to ethanol. However, the cellulolytic activities of the conventional cellulase-displaying yeast strains are insufficient for the hydrolysis of cellulose. In this study, we constructed novel gene cassettes for the efficient cellulose utilization by cellulase-displaying yeast strains. Results: The novel gene cassettes for the cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reeseii endoglucanase II (EGII) were constructed using the promoter and the GPI anchoring region derived from Saccharomyces cerevisiae SED1. The gene cassettes were integrated into the S. cerevisiae genome, then the β-glucosidase activity of these recombinant strains was evaluated. We revealed that simultaneous utilization of the SED1 promoter and Sed1 anchoring domain in a gene cassette enabled highly-efficient enzyme integration into the cell wall. The β-glucosidase activity of recombinant yeast cells transduced with the novel gene cassette was 8.4-fold higher than that of a conventional strain. The novel EGII-displaying strain also achieved 106-fold higher hydrolysis activity against the water-insoluble cellulose than a conventional strain. Furthermore, direct ethanol production from hydrothermally processed rice straw was improved by the display of T. reeseii EGII using the novel gene cassette. Conclusions: We have developed novel gene cassettes for the efficient cell-surface display of exo- and endo-type cellulolytic enzymes. The results suggest that this gene cassette has the wide applicability for cell-surface display and that cellulase-displaying yeasts have significant potential for cost-effective bioethanol production from lignocellulosic biomass.
Enhanced features of Dictyoglomus turgidum Cellulase A engineered with carbohydrate binding module 11 from Clostridium thermocellum.
Cattaneo, C., Cesaro, P., Spertino, S., Icardi, S. & Cavaletto, M. (2018). Scientific Reports, 8(1), 4402.
Lignocellulosic biomass (LCB) is a low-cost and abundant source of fermentable sugars. Enzymatic hydrolysis is one of the main ways to obtain sugars from biomass, but most of the polysaccharide-degrading enzymes are poorly efficient on LCB and cellulases with higher performances are required. In this study, we designed a chimeric protein by adding the carbohydrate binding module (CBM) of the cellulosomal enzyme CtLic26A-Cel5E (endoglucanase H or CelH) from Clostridium (Ruminiclostridium) thermocellum to the C-terminus of Dtur CelA, an interesting hyperthermostable endoglucanase from Dictyoglomus turgidum. The activity and binding rate of both native and chimeric enzyme were evaluated on soluble and insoluble polysaccharides. The addition of a CBM resulted in a cellulase with enhanced stability at extreme pHs, higher affinity and activity on insoluble cellulose.