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.
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.
Activity of arabinoxylan hydrolyzing enzymes during mashing with barley malt or barley malt and unmalted wheat.
Debyser, W., Delvaux, F. & Delcour, J. A. (1998). Journal of Agricultural and Food Chemistry, 46(12), 4836-4841.
Pilot scale brews were prepared either with 100% barley malt (BM100) or 60% barley malt and 40% unmalted wheat (BM60W40). Arabinoxylan and β-glucan hydrolyzing enzyme activities were determined during mashing using two temperature profiles. The measured enzymic activities increased for the BM100 and BM60W40 mashes in the early stages of mashing. The endoxylanase and α-L-arabinofuranosidase activities remained constant at 50°C but rapidly decreased above 50°C. At 72°C, the endoxylanase and α-L-arabinofuranosidase activities were almost completely lost. The β-D-xylosidase activity only decreased slowly at 63°C. The β-glucanase activity decreased rapidly at 50°C and was completely lost after 15 min at 50°C. From the xylose (Xyl) levels (a measure for arabinoxylan content) in the BM100 worts (1.28−1.33 g/L), a solubilization of 0.23−0.26% Xyl (% of cereal dry matter) during mashing was calculated. The Xyl levels in the BM60W40 worts (0.92−1.11 g/L) corresponded with a solubilization of 0.12 to 0.15% Xyl during mashing.
Purification, characterization and structural analysis of an abundant β-1, 3‐glucanase from banana fruit.
Peumans, W. J., Barre, A., Derycke, V., Rougé, P., Zhang, W., May, G. D., Delcour, J. A., Van Leuven, F. & Van Damme, E. J. (2000). European Journal of Biochemistry, 267(4), 1188-1195.
An abundant, catalytically active β-1,3-endoglucanase (EC 188.8.131.52) has been isolated from the pulp ripe of bananas. Biochemical analysis of the purified protein, molecular modelling, and molecular cloning of the corresponding gene indicate that this banana enzyme closely resembles previously characterized plant β-glucanases with respect to its amino-acid sequence, structure and biological activity. The results described in this paper demonstrate both the occurrence of an abundant active β-1,3-endoglucanases in fruits and also readdress the question of the possible involvement of these enzymes in the ripening and/or softening process.
Tenacibaculum skagerrakense sp. nov., a marine bacterium isolated from the pelagic zone in Skagerrak, Denmark.
Frette, L., Jørgensen, N. O. G., Irming, H. & Kroer, N. (2004). International Journal of Systematic and Evolutionary Microbiology, 54(2), 519-524.
A number of bacteria were isolated from sea water in Skagerrak, Denmark, at 30 m depth. Two of the isolates, strains D28 and D30T, belonged to the Flavobacteriaceae within the Cytophaga–Flavobacterium–Bacteroides group. Sequencing of 16S rRNA genes of the two strains indicated strongly that they belonged to the genus Tenacibaculum and that they showed greatest similarity to the species Tenacibaculum amylolyticum and Tenacibaculum mesophilum. DNA–DNA hybridization values, DNA base composition and phenotypic characteristics separated the Skagerrak strains from the other species within Tenacibaculum. Thus, it is concluded that the strains belong to a novel species within the genus Tenacibaculum, for which the name Tenacibaculum skagerrakense sp. nov. is proposed, with strain D30T (=ATCC BAA-458T=DSM 14836T) as the type strain.
Evaluation of cellulolytic and hemicellulolytic abilities of fungi isolated from coffee residue and sawdust composts.
Eida, M. F., Nagaoka, T., Wasaki, J. & Kouno, K. (2011). Microbes Environ, 26(3), 220-227.
This study focused on the evaluation of cellulolytic and hemicellulolytic fungi isolated from sawdust compost (SDC) and coffee residue compost (CRC). To identify fungal isolates, the ITS region of fungal rRNA was amplified and sequenced. To evaluate enzyme production, isolates were inoculated onto wheat bran agar plates, and enzymes were extracted and tested for cellulase, xylanase, β-glucanase, mannanase, and protease activities using different azurine cross-linked (AZCL) substrates. In total, 18 isolates from SDC and 29 isolates from CRC were identified and evaluated. Four genera (Aspergillus, Galactomyces, Mucor, and Penicillium) and five genera (Aspergillus, Coniochaeta, Fusarium, Penicillium, and Trichoderma/Hypocrea) were dominant in SDC and CRC, respectively. Penicillium sp., Trichoderma sp., and Aspergillus sp. displayed high cellulolytic and hemicellulolytic activities, while Mucor isolates exhibited the highest β-glucanase and mannanase activities. The enzyme analyses revealed that Penicillium, Aspergillus, and Mucor isolates significantly contributed to the degradation of SDC, whereas Penicillium, Aspergillus, and Trichoderma isolates had a dominant role in the degradation of CRC. Notably, isolates SDCF5 (P. crustosum), CRCF6 (P. verruculosum), and CRCF2 and CRCF16 (T. harzianum/H. lixii) displayed high activity regarding cellulose and hemicellulose degradation, which indicates that these species could be beneficial for the improvement of biodegradation processes involving lignocellulosic materials.
Patterns of functional enzyme activity in fungus farming ambrosia beetles.
Licht, H. H. D. F. & Biedermann, P. H. W. (2012). Frontiers in Zoology, 9(1), 13.
Introduction: In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results: We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion: Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only in fungi) indicates that only larvae (pre-) digest plant cell wall structures. This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.
Evolutionary transitions in enzyme activity of ant fungus gardens.
De Fine Licht, H. H., Schiøtt, M., Mueller, U. G. & Boomsma, J. J. (2010). Evolution, 64(7), 2055-2069.
Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.
Aspergillus hancockii sp. nov., a biosynthetically talented fungus endemic to southeastern Australian soils.
Pitt, J. I., Lange, L., Lacey, A. E., Vuong, D., Midgley, D. J., Greenfield, P., Bradbury, M. I., Lacey, E., Busk, P. K., Pilgaard, B., Chooi, Y. H. & Piggott, A. M. (2017). PloS One, 12(4), e0170254.
Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.
Diversity of microbial carbohydrate-active enzymes in Danish anaerobic digesters fed with wastewater treatment sludge.
Wilkens, C., Busk, P. K., Pilgaard, B., Zhang, W. J., Nielsen, K. L., Nielsen, P. H. & Lange, L. (2017). Biotechnology for Biofuels, 10(1), 158.
Background: Improved carbohydrate-active enzymes (CAZymes) are needed to fulfill the goal of producing food, feed, fuel, chemicals, and materials from biomass. Little is known about how the diverse microbial communities in anaerobic digesters (ADs) metabolize carbohydrates or which CAZymes that are present, making the ADs a unique niche to look for CAZymes that can potentiate the enzyme blends currently used in industry. Results: Enzymatic assays showed that functional CAZymes were secreted into the AD environments in four full-scale mesophilic Danish ADs fed with primary and surplus sludge from municipal wastewater treatment plants. Metagenomes from the ADs were mined for CAZymes with Homology to Peptide Patterns (HotPep). 19,335 CAZymes were identified of which 30% showed 50% or lower identity to known proteins demonstrating that ADs make up a promising pool for discovery of novel CAZymes. A function was assigned to 54% of all CAZymes identified by HotPep. Many different α-glucan-acting CAZymes were identified in the four metagenomes, and the most abundant family was glycoside hydrolase family 13, which contains α-glucan-acting CAZymes. Cellulytic and xylanolytic CAZymes were also abundant in the four metagenomes. The cellulytic enzymes were limited almost to endoglucanases and β-glucosidases, which reflect the large amount of partly degraded cellulose in the sludge. No dockerin domains were identified suggesting that the cellulytic enzymes in the ADs studied operate independently. Of xylanolytic CAZymes, especially xylanases and β-xylosidase, but also a battery of accessory enzymes, were present in the four ADs. Conclusions: Our findings suggest that the ADs are a good place to look for novel plant biomass degrading and modifying enzymes that can potentiate biological processes and provide basis for production of a range of added-value products from biorefineries.
Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter.
Huang, Y., Yi, Z., Jin, Y., Huang, M., He, K., Liu, D., Luo, H., Zhao, D., He, H., Fang, Y. & Zhao, H. (2017). Frontiers in Microbiology, 8, 1747.
Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.