Flavobacterium frigidarium sp. nov., an aerobic, psychrophilic, xylanolytic and laminarinolytic bacterium from Antarctica.
Humphry, D. R., George, A., Black, G. W. & Cummings, S. P. (2001). International Journal of Systematic and Evolutionary Microbiology, 51(4), 1235-1243.
A psychrophilic, aerobic bacterium designated A2iT was isolated from marine sediment recovered from shallow waters surrounding Adelaide Island, Antarctica (67° 34' S, 68° 07' W). The organism exhibited xylanolytic and laminarinolytic activity and was halotolerant. Basic characterization showed that it was gram-negative, non-motile, yellow-pigmented (β, β-carotene-3,3'-diol) and positive for oxidase and catalase synthesis. Analysis of the 16S rDNA sequence suggests that the organism belongs to the Flexibacter-Cytophaga-Bacteroides phylum. On the basis of its 16S rDNA sequence, the bacterium is 96.8% similar to Flavobacterium columnare ATCC 43622 - its closest relation. The genomic DNA G+C content was 35 mol%. Growth on xylan occurs optimally at 15°C, though growth also occurs at 0°C, and the doubling times are 9.6 and 34.8 h, respectively. The maximum growth temperature on xylan is at 24°C. The bacterium is a neutrophile, growing across the pH range 5.6-8.4 and having an optimum at pH 7.5. Analysis of the 16S rDNA sequence, together with phenotypic characterization, suggests that the organism is a member of the genus Flavobacterium. DNA-DNA hybridization experiments have shown that it is a novel species; it is proposed, therefore, that the organism be designated as the type strain of Flavobacterium frigidarium sp. nov. (= ATCC 700810T = NCIMB 13737T).
Cloning and characterization of an endo-β-1,3(4) glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938.
Bang, M. L., Villadsen, I. & Sandal, T. (1999). Applied Microbiology and Biotechnology, 51(2), 215-222.
We describe the identification and expression cloning of two novel enzymes, a β-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-β-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0–6.0, and a temperature activity optimum around 40°C. Both enzymes show only low sequence identity to other known enzymes.
Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen.
Groudieva, T., Kambourova, M., Yusef, H., Royter, M., Grote, R., Trinks, H. & Antranikian, G. (2004). Extremophiles, 8(6), 475-488.
The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4°C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses α and γ of Proteobacteria, the Bacillus–Clostridium group, the order Actinomycetales, and the Cytophaga–Flexibacter–Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria γ subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25°C. Only a few strains were psychrophilic, with an optimal growth at 10–15°C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4°C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, α-amylase and β-galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10°C, and almost no production was detected at higher temperatures (20–30°C). Catalytic activity was detected even below the freezing point of water (at −5°C), demonstrating the unique properties of these enzymes.
Identification, cloning and characterization of Dictyoglomus turgidum CelA, an endoglucanase with cellulase and mannanase activity.
Brumm, P. J., Hermanson, S., Luedtke, J. & Mead, D. A. (2011). Journal of Life Sciences, 5, 488-496.
The discovery of new, highly active, biomass-degrading enzymes is important to the development of a sustainable biofuels industry. Dictyoglomus turgidum, a thermophilic, anaerobic eubacterium that ferments cellulose and produces ethanol and hydrogen, was chosen as a candidate to screen for novel enzymes. A novel thermostable endoglucanase, CelA, was identified and purified during screening of a shotgun library of Dictyoglomus turgidum and subsequently subcloned and expressed in E. coli. The celA gene coding for a 312 amino acid protein showed low homology to proteins outside the genus Dictoglomi and lacked an apparent signal peptide. CelA had a broad substrate range, possessing both endo and exo activity on soluble and insoluble β-(1, 4)-linked glucose-containing substrates as well as endo activity on soluble and insoluble β-(1, 4)-linked mannose containing substrates. The specific activity of CelA was 226 U/mg using β-glucan, 66 U/mg using glucomannan, and 63 U/mg using CMC as substrates. The high temperature optimum of 70°C to 80°C and wide substrate range of the enzyme might make it an excellent tool for biomass degradation at high temperature.
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.
Nucleotide sequence of a β-1, 3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis.
Ferrer, P., Halkier, T., Hedegaard, L., Savva, D., Diers, I. & Asenjo, J. A. (1996). Journal of Bacteriology, 178(15), 4751-4757.
The nucleotide sequence of the βgIIIA gene, encoding the extracellular β-1,3-glucanase IIA (βgIIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined.Sequence comparison shows that the βgIIIA enzyme has over 80% identity to the βgIII isoenzyme, an endo-β-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The βgIIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in β-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that βgIIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).
Pseudoalteromonas arctica sp. nov., an aerobic, psychrotolerant, marine bacterium isolated from Spitzbergen.
Al Khudary, R., Stößer, N. I., Qoura, F. & Antranikian, G. (2008). International Journal of Systematic and Evolutionary Microbiology, 58(9), 2018-2024.
A novel aerobic, psychrotolerant marine bacterium was isolated at 4°C from seawater samples collected from Spitzbergen in the Arctic. The strain was a polar-flagellated, Gram-negative bacterium that grew optimally at 10–15°C and pH 7–8 in media containing 2–3 % NaCl (w/v), using various carbohydrates and organic acids as substrates. The main fatty acid components included 16 : 0 (12.7 % of total fatty acids), straight-chain saturated fatty acid methyl ester (FAME) and 16 : 1ω7c (40.2 %) monounsaturated FAME. Phylogenetic analysis revealed a close relationship (99 % 16S rRNA gene sequence similarity) between the novel isolate and Pseudoalteromonas elyakovii KMM 162T and some other species of the genus Pseudoalteromonas. The DNA G+C content of the novel strain was 39 mol%. DNA–DNA hybridization showed only 47.6 % DNA–DNA relatedness with P. elyakovii KMM 162T, 44.2 % with Pseudoalteromonas distincta KMM 638T and 22.6 % with Pseudoalteromonas nigrifaciens NCIMB 8614T Based on phylogenetic and phenotypic characteristics, this isolate represents a novel species of the genus Pseudoalteromonas for which the name Pseudoalteromonas arctica is proposed; the type strain is A 37-1-2T (=LMG 23753T=DSM 18437T).
Mining Dictyoglomus turgidum for enzymatically active carbohydrases.
Brumm, P., Hermanson, S., Hochstein, B., Boyum, J., Hermersmann, N., Gowda, K. & Mead, D. (2011). Applied Biochemistry and Biotechnology, 163(2), 205-214.
The genome of Dictyoglomus turgidum was sequenced and analyzed for carbohydrases. The broad range of carbohydrate substrate utilization is reflected in the high number of glycosyl hydrolases, 54, and the high percentage of CAZymes present in the genome, 3.09% of its total genes. Screening a random clone library generated from D. turgidum resulted in the discovery of five novel biomass-degrading enzymes with low homology to known molecules. Whole genome sequencing of the organism followed by bioinformatics-directed amplification of selected genes resulted in the recovery of seven additional novel enzyme molecules. Based on the analysis of the genome, D. turgidumdoes not appear to degrade cellulose using either conventional soluble enzymes or a cellulosomal degradation system. The types and quantities of glycosyl hydrolases and carbohydrate-binding modules present in the genome suggest that D. turgidum degrades cellulose via a mechanism similar to that used by Cytophaga hutchinsonii and Fibrobacter succinogenes.
Patterns of functional enzyme activity in fungus farming ambrosia beetles.
Licht, H. H. D. F. & Biedermann, P. H. (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.
The use of plant cell wall degrading enzymes from a newly isolated Penicillium ochrochloron Biourge for viscosity reduction in ethanol production with fresh sweet potato tubers as feedstock.
Huang, Y., Jin, Y., Shen, W., Fang, Y., Zhang, G. & Zhao, H. (2013). Biotechnology and Applied Biochemistry, 61(4), 480-491.
Penicillium ochrochloron Biourge, which was isolated from rotten sweet potato, can produce plant cell wall degrading enzymes (PCWDEs) with high viscosity reducing capability for ethanol production using fresh sweet potato tubers as feedstock. The enzyme preparation was characterized by a broad enzyme spectrum including 13 kinds of enzymes with the activity to hydrolyze cellulose, hemicellulose, pectin, starch and protein. The maximum viscosity reducing capability was observed when the enzyme preparation was obtained after five days fermentation using 20 g/L corncob as sole carbon source, 4.5 g/L NH4NO3 as sole nitrogen source, and an initial medium pH of 6.5. The sweet potato mash treated with the enzyme preparation exhibited much higher fermentation efficiency (92.58%) compared with commercial cellulase (88.06%) and control (83.5%). The enzyme production was then scaled up to the 0.5, 5, and 100 L, and the viscosity reducing rates were found to be 85%, 90%, and 91%, respectively. Thus, P. ochrochloron Biourge displays potential viscosity reducing capability for ethanol production.
The optimization of some extracellular enzymes biosynthesis by Aspergillus niger 377-4. Wikiera, A., Mika, M., Janiszewska,
A. S. & Zyla, K. (2015). Journal of Scientific & Industrial Research, 74, 145-149.
The effect of initial solid and moisture contents, temperature and time of incubation on the production of polygalacturonase, phytase, acid phosphatase, xylanase and β-glucanase by Aspergillus niger 377-4 during solid state fermentation was studied. Parameters of enzyme synthesis were optimized using statistical experimental designs. It was shown that the capacity of strain to synthesize the aforementioned enzymes could be modified within a wide range by culture parameters selection. The optimal polygalacturonase production efficiency was achieved with the initial medium mass of 19.9 g and humidity of 59.9%, after 77.7 h of incubation at 28.9°C. The best combination of culture parameters for phytase synthesis was: initial medium mass 19.9 g, moistures 50%, temperature 33°C and incubation time 83.9 h. The highest activity of acid phosphatase was obtained after 81.3 h of incubation at 27°C, with initial substrate mass of 17.8 g and moistness content of 60%. The initial solid and moisture contents to synthesize xylanase were 19.9 g and 50%, respectively, with incubation time of 73 h at 29.6°C. The highest efficiency of β-glucanase biosynthesis was obtained when A. niger 377-4 was cultivated for 80.4 h at 27°C on a initial medium mass of 20 g and initial level of moistness 59.9%.
Structural and functional characterization of a novel family GH115 4-O-methyl-α-glucuronidase with specificity for decorated arabinogalactans.
Aalbers, F., Turkenburg, J. P., Davies, G. J., Dijkhuizen, L. & van Bueren, A. L. (2015). Journal of Molecular Biology, 427(24), 3935-3946.
Glycoside hydrolases are clustered into families based on amino acid sequence similarities, and belonging to a particular family can infer biological activity of an enzyme. Family GH115 contains α-glucuronidases where several members have been shown to hydrolyze terminal α-1,2-linked glucuronic acid and 4-O-methylated glucuronic acid from the plant cell wall polysaccharide glucuronoxylan. Other GH115 enzymes show no activity on glucuronoxylan, and therefore, it has been proposed that family GH115 may be a poly-specific family. In this study, we reveal that a putative periplasmic GH115 from the human gut symbiont Bacteroides thetaiotaomicron, BtGH115A, hydrolyzes terminal 4-O-methyl-glucuronic acid residues from decorated arabinogalactan isolated from acacia tree. The three-dimensional structure of BtGH115A reveals that BtGH115A has the same domain architecture as the other structurally characterized member of this family, BoAgu115A; however the position of the C-terminal module is altered with respect to each individual enzyme. Phylogenetic analysis of GH115 amino sequences divides the family into distinct clades that may distinguish different substrate specificities. Finally, we show that BtGH115A α-glucuronidase activity is necessary for the sequential digestion of branched galactans from acacia gum by a galactan-β-1,3-galactosidase from family GH43; however, while B. thetaiotaomicron grows on larch wood arabinogalactan, the bacterium is not able to metabolize acacia gum arabinogalactan, suggesting that BtGH115A is involved in degradation of arabinogalactan fragments liberated by other microbial species in the gastrointestinal tract.
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.