Purification and characterization of a neutral serine protease with nematicidal activity from Hirsutella rhossiliensis.
Wang, B., Wu, W. & Liu, X. (2007). Mycopathologia, 163(3), 169-176.
Serine protease plays an important role in fungal infection to invertebrate hosts. An extracellular protease (Hnsp) was detected in liquid culture of Hirsutella rhossiliensis OWVT-1 with nematodes (Panagrellus redivivus) as the unique nitrogen source and purified to homogeneity by ammonium sulphate precipitation, anion exchange chromatography and gel filtration. Its molecular mass was about 32 kDa, and the optimal reaction pH value and temperature were pH 7 and 40°C, respectively. The Hnsp activity was stable at pH 6–8 and decreased radically at 50°C for 10 min. Hnsp was highly sensitive to inhibitor of PMSF and well decomposed the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, suggesting that it belonged to the chymotrypsin/subtilisin of serine proteases. The N-terminal amino acid sequence of Hnsp was SVTDQQGADCGLARISHRE, which showed high homology with other serine proteases from nematophagous fungi. Ability to kill nematode and degrade its cuticle in vitro indicated that Hnsp could be involved in the infection of nematode.
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).
Purification, characterization, and gene cloning of an alkaline serine protease from a highly virulent strain of the nematode-endoparasitic fungus Hirsutella rhossiliensis.
Wang, B., Liu, X., Wu, W., Liu, X. & Li, S. (2009). Microbiological Research, 164(6), 665-673.
Hirsutella rhossiliensis OWVT-1 has substantial potential as a biocontrol agent against plant-parasitic nematodes. Serine proteases have emerged as a potentially useful factor in the nematode–fungus interactions. When grown in liquid culture with the nematode Panagrellus redivivus as the sole nitrogen source, an extracellular alkaline protease (Hasp) was produced by the OWVT-1. The purified Hasp killed the juveniles of the soybean-cyst nematode (Heterodera glycines) and degraded proteins of the nematode cuticle. The molecular mass of Hasp was estimated to be 33 kDa. The optimum pH and temperature for enzyme activity were pH 9 and 75°C. The amino acid sequence obtained by the N-terminal sequence analysis was applied for the primer design to isolate the Hasp cDNA gene, which consists of 1170 bp open reading frame. Analysis of the cDNA and corresponding genomic sequence revealed that Hasp included four exons (279, 186, 513, and 192 bp) divided by three introns (65, 99, and 93 bp). Southern blotting showed that Hasp was a single-copy gene in the genome. The deduced amino acid sequence was very similar to other serine proteases of endoparasitic and egg-parasitic fungi of nematodes and of entomopathogenic fungi but was less similar to the serine proteases of nematode-trapping fungi. In a phylogenetic analysis of the amino acid sequences of serine proteases, the serine protease of H. rhossiliensis OWVT-1 clustered with the serine proteases of parasites of nematode eggs rather than with those of the trapping fungi.
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.
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.
Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species.
Dammannagoda, L. K., Pavasovic, A., Hurwood, D. A. & Mather, P. B. (2015). Aquaculture Research, 46(3), 626-636.
The current study evaluated the effect of soluble dietary cellulose on growth, survival and digestive enzyme activity in three endemic, Australian freshwater crayfish species (redclaw: Cherax quadricarinatus, marron: C. tenuimanus, yabby: C. destructor). Separate individual feeding trials were conducted for late-stage juveniles from each species in an automated recirculating freshwater, culture system. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch, over a 12-week period. Redclaw fed with RD showed significantly higher (P < 0.05) specific growth rates (SGR) compared with animals fed the TD, while SGR of marron and yabby fed the two diets were not significantly different. Expressed cellulase activity levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD (P < 0.05). Amylase and protease activity in all three species were significantly higher in the animals fed with RD (P < 0.05). These results indicate that test animals of all three species appear to utilize starch more efficiently than soluble dietary cellulose in their diet. The inclusion of 20% soluble cellulose in diets did not appear, however, to have a significant negative effect on growth rates.
Taxonomic and functional diversity of pseudomonads isolated from the roots of field‐grown canola.
Misko, A. L. & Germida, J. J. (2002). FEMS Microbiology Ecology, 42(3), 399-407.
Among the most important rhizosphere bacteria are the pseudomonads, which are aggressive colonizers and utilize a wide range of substrates as carbon sources. The objective of this study was to determine if the taxonomic or metabolic diversity of pseudomonads differed among field-grown canola cultivars. Bacteria (n=2257) were isolated from the rhizosphere and root interior of six cultivars of field-grown canola, including three transgenic varieties. The bacteria were identified by fatty acid methyl ester (FAME) analysis, and about 35% were identified as Pseudomonas species. The most abundant species were Pseudomonas putida and Pseudomonas chlororaphis. Dendrograms based on FAME analysis revealed that many pseudomonad strains were found in all of the canola cultivars. Pseudomonads of the same strain were found in both the rhizosphere and the root interior of canola plants, suggesting that endophytic bacteria were a subset of the rhizosphere community. Because metabolic profiling provides more useful information than taxonomy, P. putida and P. chlororaphis isolates were characterized for their ability to utilize carbon substrates and produce several enzymes. Bacteria isolated from different plant cultivars had different carbon utilization profiles, but when only carbon substrates found in root exudates were analyzed, the cultivar effect was less pronounced. These characterizations also demonstrated that bacteria that were determined by FAME to be the same strain were metabolically different, suggesting functional redundancy among Pseudomonas isolates. The results of this study suggest that pseudomonads were functionally diverse. They differed in their metabolic potential among the canola cultivars from which they were isolated. Because bacteria capable of using many substrates can effectively adapt to new environments, these results have implications for the use of pseudomonads as biofertilizers, biological control agents and plant growth-promoting bacteria in canola.
Cold-adaptation and alkaline hydrolytic proprieties of the polar streptomycetes prediction on plate assay, based on insoluble chromogenic substrates with azurine cross-linked.
Cotarlet, M., Negoită, T., Bahrim, G. & Stougaard, P. (2008). Annals of the University Dunarea de Jos of Galati. Fascicle VI--Food Technology, 1(31).
A semi-qualitative screening based on protease and amylase activity evaluation in a basal agar medium supplemented with insoluble chromogenic substrates based on AZCL (Azurine-Crosslinked with amylose or casein) using a plate assay was used for selecting the polar streptomycetes able to produce cold actives and alkaline amylases and proteases. This technique provides a specific and rapid simultaneous detection of high active hydrolase producing strains based on the visible solubilization of small particles of AZCL and the formation of haloes on plates. It has a great potential in increasing the efficacy of screening streptomycetes able to produce hydrolytic enzymes. This study revealed the potential of the selected streptomycetes isolated from polar soils to biosynthesize amylases and proteases cold-adapted at low temperatures (from 5 to 20°C) and alkaline pH values (8 to 9).
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.