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.
Characterization of the Erwinia chrysanthemi gan locus, involved in Galactan catabolism.
Delangle, A., Prouvost, A. F., Cogez, V., Bohin, J. P., Lacroix, J. M. & Cotte-Pattat, N. H. (2007). Journal of Bacteriology, 189(19), 7053-7061.
β-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK2. Degradation of galactans would be catalyzed by the periplasmic 1,4-β-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-β-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny.
Effect of pH, temperature and diet on digestive enzyme profiles in the mud crab, Scylla serrata.
Pavasovic, M., Richardson, N. A., Anderson, A. J., Mann, D. & Mather, P. B. (2004). Aquaculture, 242(1), 641-654.
Commercial farming of the mud crab Scylla serrata is a significant industry throughout South East Asia. The limited scientific knowledge of mud crab nutritional requirements and digestive processes, however, is recognised as a major constraint to the future growth of this industry. To better understand the mechanisms of digestion in the mud crab we have analysed the diversity of digestive enzymes from the midgut (MG) gland. Significant protease, amylase, cellulase and xylanase activities were detected in soluble extracts from this organ. Temperature profiles for all enzymes were basically similar with optimal activities observed at 50°C. Examination of pH tolerances revealed optimal activities for protease and amylase at pH 7 while maximum cellulase and xylanase activities were observed at pH 5.5. Under optimum conditions, protease and amylase activities were approximately two orders of magnitude greater than those seen for either cellulase or xylanase. Interestingly, MG extracts were able to liberate glucose from either starch or carboxymethyl (CM)-cellulose suggesting that a range of carbohydrates may be utilised as energy sources. The effects of dietary carbohydrates on feed digestibility, digestive enzyme levels and growth were also studied by inclusion of additional starch or CM-cellulose at the expense of casein in formulated diets. It was shown that amylase, cellulase and xylanase activities in extracts from the midgut gland were highest in mud crabs fed diets containing 47% carbohydrate. Based on these findings, we suggest that the ability of the mud crab to modulate digestive enzyme activities may represent a mechanism to maximise access to essential nutrients when the dietary profile changes.
Towards a molecular understanding of symbiont function: identification of a fungal gene for the degradation of xylan in the fungus gardens of leaf-cutting ants.
Schiøtt, M., Licht, H. H. D. F., Lange, L. & Boomsma, J. J. (2008). BMC Microbiology, 8(1), 40.
Background: Leaf-cutting ants live in symbiosis with a fungus that they rear for food by providing it with live plant material. Until recently the fungus' main inferred function was to make otherwise inaccessible cell wall degradation products available to the ants, but new studies have shed doubt on this idea. To provide evidence for the cell wall degrading capacity of the attine ant symbiont, we designed PCR primers from conserved regions of known xylanase genes, to be used in PCR with genomic DNA from the symbiont as template. We also measured xylanase, cellulase and proteinase activities in the fungus gardens in order to investigate the dynamics of degradation activities. Results: We cloned a xylanase gene from the mutualistic fungus of Acromyrmex echinatior, determined its protein sequence, and inserted it in a yeast expression vector to confirm its substrate specificity. Our results show that the fungus has a functional xylanase gene. We also show by lab experiments in vivo that the activity of fungal xylanase and cellulase is not evenly distributed, but concentrated in the lower layer of fungus gardens, with only modest activity in the middle layer where gongylidia are produced and intermediate activity in the newly established top layer. This vertical distribution appears to be negatively correlated with the concentration of glucose, which indicates a directly regulating role of glucose, as has been found in other fungi and has been previously suggested for the ant fungal symbiont. Conclusion: The mutualistic fungus of Acromyrmex echinatior has a functional xylanase gene and is thus presumably able to at least partially degrade the cell walls of leaves. This finding supports a saprotrophic origin of the fungal symbiont. The observed distribution of enzyme activity leads us to propose that leaf-substrate degradation in fungus gardens is a multi-step process comparable to normal biodegradation of organic matter in soil ecosystems, but with the crucial difference that a single fungal symbiont realizes most of the steps that are normally provided by a series of microorganisms that colonize fallen leaves in a distinct succession.
Influence of dietary protein on digestive enzyme activity, growth and tail muscle composition in redclaw crayfish, Cherax quadricarinatus (von Martens).
Pavasovic, A., Anderson, A. J., Mather, P. B. & Richardson, N. A. (2007). Aquaculture Research, 38(6), 644-652.
This study was conducted to evaluate the effects of dietary protein on digestive enzyme profiles, growth and tail muscle composition in the freshwater redclaw crayfish, Cherax quadricarinatus. Crayfish were fed five diets that consisted of a commercial crayfish pellet and experimental diets containing 13%, 18%, 25% or 32% crude protein (CP), for a period of 12 weeks. Analysis of digestive enzyme profiles from the midgut gland (MG) revealed a positive correlation between protease, amylase and cellulase activities and dietary protein level. For all treatments, carbohydrase activity levels (cellulase and amylase) were significantly higher than those detected for protease. As dietary protein was elevated, there was a general increase in specific growth rate (SGR), with the highest SGR (0.58 ± 0.06) values observed in crayfish fed the diet containing 25% CP. Feed conversion ratio (FCR) ranged between 5.84 and 6.97 and did not differ significantly among the treatment groups including the reference diet, with the exception of the low-protein diet (13% CP) which showed an FCR of 9.31. Finally, regression analysis revealed a strong positive correlation between the level of dietary protein and CP content in the tail muscle (P=0.004; r2) =0.99).
A novel antifungal Pseudomonas fluorescens isolated from potato soils in Greenland.
Michelsen, C. F. & Stougaard, P. (2011). Current Microbiology, 62(4), 1185-1192.
A rhizobacterium with high antifungal activity was isolated from a potato field at Inneruulalik, South Greenland. Phylogenetic analysis based on multi locus sequence typing showed that the bacterium was affiliated with strains of Pseudomonas fluorescens. The bacterium, denoted as Pseudomonas fluorescens In5, inhibited in vitro a broad range of phytopathogenic fungi, and the antifungal activity increased with decreasing temperature. Microcosm experiments demonstrated that P. fluorescens In5 protected tomato seedlings from Rhizoctonia solani. Transposon mutagenesis showed that the major cause for the antifungal activity of P. fluorescens In5 was a novel non-ribosomal peptide synthase (NRPS) gene. In addition, transposon mutagenesis showed that P. fluorescens In5 also contained a putative quinoprotein glucose dehydrogenase gene, which was involved in growth inhibition of phytopathogenic fungi. Although P. fluorescens In5 contained the capacity to synthesize hydrogen cyanide, β-1,3-glucanase, protease, and chitinase, these did not seem to play a role in the in vitro and microcosm antifungal assays.
Characterization of a new oxidant-stable serine protease isolated by functional metagenomics.
Biver, S., Portetelle, D. & Vandenbol, M. (2013). SpringerPlus, 2(1), 410.
A novel serine protease gene, SBcas3.3, was identified by functional screening of a forest-soil metagenomic library on agar plates supplemented with AZCL-casein. Overproduction in Escherichia coli revealed that the enzyme is produced as a 770-amino-acid precursor which is processed to a mature protease of ~55 kDa. The latter was purified by affinity chromatography for characterization with the azocasein substrate. The enzyme proved to be an alkaline protease showing maximal activity between pH 9 and 10 and at 50°C. Treatment with the chelating agent ethylenediaminetetraacetic acid irreversibly denatured the protease, whose stability was found to depend strictly on calcium ions. The enzyme appeared relatively resistant to denaturing and reducing agents, and its activity was enhanced in the presence of 10 ml/l nonionic detergent (Tween 20, Tween 80, or Triton X-100). Moreover, SBcas3.3 displayed oxidant stability, a feature particularly sought in the detergent and bleaching industries. SBcas3.3 was activated by hydrogen peroxide at concentrations up to 10 g/l and it still retained 30% of activity in 50 g/l H2O2.
Cloning and relational analysis of 15 novel fungal endoglucanases from family 12 glycosyl hydrolase.
Goedegebuur, F., Fowler, T., Phillips, J., van der Kley, P., van Solingen, P., Dankmeyer, L. & Power, S. D. (2002). Current Genetics, 41(2), 89-98.
Cellulases belong to the large family of glycosyl hydrolases (GHs) and are produced by a variety of bacteria and fungi. These extracellular enzymes act as endoglucanases (EGs), cellobiohydrolases or β-glucosidases. In this paper, we describe molecular screening for EGs from the GH family 12. Using three homologous sequence boxes deduced from five previously known members of the family, we analysed 22 cellulase-producing fungal strains obtained from a diverse area of the fungal kingdom. Polymerase chain reactions using degenerate primers designed to the homologous protein boxes were used to identify the family 12 homologues. Several fungi showed the presence of multiple versions of the gene, while amino acid sequence analysis showed diversity in 15 novel members of the family, ranging from 26% to 96% similarity. Our sequence analysis shows that the phylogenetic tree of family 12 EGs can be divided into four subfamilies: 12-1 (fungal group I), 12-2 (fungal group II), 12-3 (Streptomyces group in which Rhodothermus marinus fits) and 12-4 (Thermophiles group). Erwinia carotovora may form a new subgroup.
Digestive enzyme spectra in crustacean decapods (Paleomonidae, Portunidae and Penaeidae) feeding in the natural habitat.
Figueiredo, M. S. R. B. & Anderson, A. J. (2009). Aquaculture Research, 40(3), 282-291.
This work describes the profile of five proteases and four carbohydrases from the crustacean decapods Macrobrachium australiense (Holthuis), Scylla serrata (Forskal), Portunus pelagicus (Linnaeus), Penaeus esculentus, Penaeus plebejus (Hess) and Metapenaeus bennettae (Racck & Dall), feeding in the natural habitat, in order to provide an indication of their digestive capabilities. The results raised the following points. First, species from each family showed a particular suite of digestive enzymes. Second, the activity of cellulase from M. australiensis and S. serrata, using AZCL-HE cellulose as the substrate, was around 90% higher than that observed with AZO-CM cellulose. However, for P. pelagicus and P. esculentus, the enzyme activity was better with AZO-CM cellulose. Third, M. australiense displayed the highest ratio of amylase to protease activity. In contrast, Portunidae species, P. pelagicus and S. serrata showed the lowest ratios. Fourth, comparison of the laminarinase activity of M. bennettae and P. esculentus in October (Spring) and December (early Summer) showed a significant decrease in December. Finally, the wide distribution of digestive enzymes in these crustaceans may reflect different feeding habits and habitats.
Rationally selected single‐site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates.
Srikrishnan, S., Randall, A., Baldi, P. & Da Silva, N. A. (2012). Biotechnology and Bioengineering, 109(6), 1595-1599.
Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. Using a rational mutagenesis approach based on structural bioinformatics and evolutionary analysis, two positions (F16S and Y95F) were identified as priority sites for mutagenesis. The mutant and parent enzymes were expressed and secreted from Pichia pastoris and the single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in Kcat and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent enzyme, the two variants were active between pH 4.0 and 8.0 and showed optimal activity at temperature 70°C at pH 5.0. The purified enzymes were active at 50°C for over 12 h and retained at least 80% of initial activity for 2 h at 70°C. In contrast to the improved hydrolysis seen with the single mutation enzymes, no improvement was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.
A comparative study of cellulase and xylanase activity in freshwater crayfish and marine prawns.
Crawford, A. C., Richardson, N. R. & Mather, P. B. (2005). Aquaculture Research, 36(6), 586-592.
Cellulase and xylanase digestive enzyme activities were compared in four freshwater crayfish (Genus Cherax) and three marine prawn (Genus Penaeus) species. Temperature and pH profiles for cellulase (endoglucanase) were found to be very similar in all species, with maximum activity occurring at 60°C and pH 5.0. Temperature and pH profiles for xylanase (endoxylanase) were also very similar in all crayfish species, with maximum activity occurring at 50°C and pH 5.0. Xylanase activity was not detected in the three prawn species examined. In addition, in vitro studies showed that most species were able to liberate glucose from carboxymethyl cellulose, indicating that cellulose substrates can be a source of energy for both crayfish and prawn species.
Exo‐exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo‐lytic character of the enzymes.
Badino, S. F., Christensen, S. J., Kari, J., Windahl, M. S., Hvidt, S., Borch, K. & Westh, P. (2017). Biotechnology and Bioengineering, 9999: 1–9.
Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 103–104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2–3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs.
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.
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.
Cloning, molecular modeling and characterization of acidic cellulase from buffalo rumen and its applicability in saccharification of lignocellulosic biomass.
Dadheech, T., Shah, R., Pandit, R., Hinsu, A., Chauhan, P. S., Jakhesara, S., Kunjadiya, A., Rank, D. & Joshi, C. (2018). International Journal of Biological Macromolecules, 113, 73-81.
Cellulase hydrolyses the cellulose by cleaving the β-1,4-linkages to produce mono-, oligo- and shorter polysaccharide units. These enzymes have applications in various industries such as pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. In the present study we have cloned acid-cellulase gene (Cel-1) from the fosmid library of buffalo rumen metagenomic DNA and functionally expressed it in Escherichia coli. The ORF encoding cellulase consisted of 1176-bp, corresponding to protein of 391 amino acid and has catalytic domain belonging to glycosyl hydrolase family 5. The purified protein has a molecular weight of 43-kDa on SDS-PAGE and its expression was confirmed by western blotting. The tertiary structure of the cellulase (Cel-1) showed a classical (α/β) TIM-like barrel motif. Model surface charge of Cel-1 predicted that surface near active site was mostly negative which might be responsible for the stability of enzyme at lower pH. The pH and temperature for maximum enzyme activity were 4.5 and 45°C respectively. Various metal ions enhanced the enzyme activity and in presence of Mn+2 activity was significantly increased. Cel-1 hydrolyzed pre-treated wheat straw and released reducing sugars (62.60%). These desirable properties of Cel-1 make it attractive for the bioconversion of 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.