Description and genomic characterization of Cohnella caldifontis sp. nov., isolated from hot springs in Yunnan province, south-west China.

A bacterial strain, Gram staining positive, strictly aerobic, rod-shaped, motile bacterium with flagellum and endospore-forming, designated strain YIM B05605T, was isolated from soil sampled in Hamazui hot springs, Tengchong City, Yunnan province, China. Optimum growth for the strain occurred at pH 7.0 and 45 °C. MK-7 was the main menaquinone in the strain YIM B05605T. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PME), unidentified glycolipid (GL), three unknown aminophospholipids (APLs) and unidentified polarlipid (PL) were part of the polar lipid profile. The major fatty acids were anteiso-C15:0 and iso-C16:0. The DNA G + C content of the type strain was 58.76%. Genome-based phylogenetic analysis confirmed that strain YIM B05605T formed a distinct phylogenetic cluster within the genus Cohnella. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strain YIM B05605T with the most related species C. fontinalis YT-1101T were 73.42% and 15.7%. Functional analysis by NR, Swiss-prot, Pfam, eggNOG, GO, KEGG databases revealed that strain YIM B05605T has 13 genes related to the sulfur cycle, 2 genes related to the nitrogen cycle. Based on phylogenomic and phylogenetic analyses coupled with phenotypic and chemotaxonomic characterizations, strain YIM B05605T could be classified as a novel species of the genus Cohnella, for which the name Cohnella caldifontis sp. nov., is proposed. The type strain is YIM B05605T (= CGMCC 1.60052T = KCTC 43462T = NBRC 115921T).

Cohnella mopanensis sp. nov., a new Gram-negative bacterium isolated from soil in Xinping Mountain National Forest Park.

A Gram-stain-negative, aerobic, rod-shaped bacteria strain, named YIM B01951T, was isolated from a forest soil sample collected from Mopan Mountain National Forest Park, Xinping City, Yunnan Province, southwest PR China (101°58'06" N, 23°03'02" E). Growth occurred at 15-40 °C (optimum, 30 °C), pH 5.0-8.0 (optimum, pH 6.5) and with up to ≤ 3.0% (w/v) NaCl on Nutrient Agar plates. The results of 16S rRNA gene sequence similarity analysis showed that strain YIM B01951T was closely related to the type strain of Cohnella arctica M9-62T (96.5%) and Cohnella lupini RLAHU4BT (96.3%). YIM B01951T contains anteiso-C15:0 and iso-C16:0 as the major cellular fatty acids; the main polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), lysylphosphatidylglycerol (PGL) and five aminophospholipids (APL). The MK-7 is the major respiratory quinone and the DNA G + C content is 49.2 mol%. Based on these phenotypic, chemotaxonomic and phylogenetic analyses, strain YIM B01951T is considered to be a novel species of the genus Cohnella, and named Cohnella mopanensis sp. nov. The type strain is YIM B01951T (= NBRC 115331T = KCTC 43370T).

Cohnella herbarum sp. nov., isolated from wild grass fermentation broth.

A novel strictly aerobic, Gram-stain-positive, rod-shaped, motile, endospore-forming, white-coloured bacterium, designated strain MFER-1T, was isolated from a fermented liquor of wild grasses sampled in the Republic of Korea. The respiratory quinone of strain MFER-1T was menaquinone-7 and its major cellular fatty acids were anteiso-C15 : 0 (55.3 %), iso-C16 : 0 (17.5 %) and C16 : 0 (12.1 %). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids and an unidentified phospholipid. The 16S rRNA gene sequence of strain MFER-1T showed similarity of 98.1 % to 'Cohnella cholangitidis' 1 605-214T and below 98.0 % sequence similarity to the other Cohnella species. The phylogenomic tree indicated that strain MFER-1T formed a reliable cluster with several Cohnella species. The estimated genome size of strain MFER-1T was 8.52 Mb. Genomic DNA G+C content was 50.7mol%. The orthologous average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values of strain MFER-1T with the most closely related species 'Cohnella cholangitidis' 1 605-214T were 78.7, 23.0 and 79.6 %, respectively. Based on the phenotypic, chemotaxonomic and phylogenetic results, strain MFER-1T should represent a novel species of the genus Cohnella, for which the name Cohnella herbarum sp. nov. is proposed, with strain MFER-1T (=KACC 21 257T=NBRC 114 628T) as the type strain.

Cohnella cholangitidis sp. nov., a novel species of the genus Cohnella isolated from a clinical specimen in Korea.

A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214T, was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214T belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270T (GQ214052). DNA-DNA hybridization, digital DNA-DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214T were mainly comprised of anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214T contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214T was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214T was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed.

Cohnella pontilimi sp. nov., isolated from tidal-flat mud.

A Gram-positive, aerobic, endospore-forming, rod-shaped bacterial strain, CAU 1483 T, was isolated from tidal-flat mud in the Republic of Korea. It grew optimally at 30 °C, in a pH 7.0 medium with 2% (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain CAU 1483 T formed a separate clade within Paenibacillaceae together with members of the genus Cohnella. Strain CAU 1483 T exhibited the highest 16S rRNA gene sequence similarity (97.1%) to C. candidum 18JY8-7 T. Whole genome of strain CAU 1483 T was 4.29 Mb in size with a 53.7 mol% G + C content, and included 4046 coding sequences and included 4046 coding sequences, some of which associated with stress response. The average nucleotide identity and digital DNA-DNA hybridization similarity between strain CAU 1483 T and related members of the genus Cohnella were 71.8-74.9% and 22.6-33.9%, respectively. The major respiratory quinone present in this strain was menaquinone-7. Strain CAU 1483 T contained anteiso-C15:0 and iso-C16:0 as the major fatty acids, while its polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, lysyl-phosphatidylglycerol, phosphatidylcholine, three unidentified aminophospholipids, two unidentified lipids and an unidentified phospholipid. Peptidoglycan type was A1γ meso-Dpm. On the basis of taxonomic characterization, strain CAU 1483 T constitutes a novel species, for which the name Cohnella pontilimi sp. nov. is proposed. The type strain of this novel species is CAU 1483 T (= KCTC 43047 T = NBRC 113953 T).

Cohnella terricola sp. nov., isolated from soil.

A Gram-positive, aerobic, flagellated, endospore-forming, rod-shaped strain, designated as G13T, was isolated from soil. The results of 16S rRNA gene sequence analysis led to the conclusion that strain G13T was phylogenetically related to Cohnella boryungensis BR29T (97.5 %) and Cohnella phaseoli CECT 7287T (96.9 %) with digital DNA-DNA hybridization values of 21.0 and 21.4 %, and distantly related to Cohnella thermotolerans CCUG 47242T (94.8 %), type species of the genus Cohnella, at 19.0 %. The genome size of strain G13T was 5 387 258 bp, with 51.3 mol% G+C content. The predominant fatty acids were summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), anteiso-C17 : 0, iso-C17 : 0 and iso-C15 : 0. The predominant quinone was menaquinone-7 and the major polar lipids were diphosphatidyglycerol, phosphatidylethanolamine, phosphatidylglycerol, lysylphosphatidylglycerol, three aminophospholipids, two phosphoglycolipids, three aminolipids and two unidentified lipids. Based on the data from phenotypic tests and the genotypic differences between strain G13T and its close phylogenetic relatives, strain G13T represents a new species belonging to the genus Cohnella, for which the name Cohnella terricola sp. nov. (=KACC 19905T=NBRC 113748T) is proposed.

Endo-xylanases from Cohnella sp. AR92 aimed at xylan and arabinoxylan conversion into value-added products.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-ß-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

Description of Cohnella zeiphila sp. nov., a bacterium isolated from maize callus cultures.

A Gram-stain positive, aerobic, motile, rod-shaped bacterium designated as strain CBP-2801T was isolated as a contaminant from a culture containing maize callus in Peoria, Illinois, United States. The strain is unique relative to other Cohnella species due to its slow growth and reduced number of sole carbon sources. Phylogenetic analysis using 16S rRNA indicated that strain CBP-2801T is a Cohnella bacterium and showed the highest similarity to Cohnella xylanilytica (96.8%). Genome-based phylogeny and genomic comparisons based on average nucleotide identity confirmed the strain to be a novel species of Cohnella. Growth occurs at 15-45 °C (optimum 40 °C), pH 5-7 (optimum pH 6) and with 0-1% NaCl. The predominant fatty acids are anteiso-15:0 and 18:1 ω6c. Genome mining for secondary metabolites identified a putative biosynthetic cluster that encodes for a novel lasso peptide. In addition, this study contributes five new genome assemblies of type strains of Cohnella species, a genus with less than 30% of the type strains sequenced. The DNA G + C content is 58.7 mol %. Based on the phenotypic, phylogenetic and biochemical data strain CBP-2801T represents a novel species, for which the name Cohnella zeiphila sp. nov. is proposed. The type strain is CBP-2801T (= DSM 111598 = ATCC TSD-230).

Cohnella fermenti sp. nov., isolated from a fermentation process.

A novel Gram-stain-positive, aerobic, motile with peritrichous flagella, rod-shaped bacterium, designated CC-MHH1044T, was isolated from a preserved vegetable sample. A polyphasic taxonomic approach was applied to the isolates in order to clarify its taxonomic position. Growth of the strain CC-MHH1044T occurred at 15-50 °C (optimum, 30 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-2.0 % (w/v) NaCl (optimum, 1 %, w/v). The genome of strain CC-MHH1044T consisted of 8.5 Mb and the genomic DNA G+C content was 58.5 mol%. Comparison of the 16S rRNA gene sequences showed that CC-MHH1044T belonged to the genus Cohnella and showed a close relationship with the type strains of Cohnella damuensis (96.2 %) and Cohnella panacarvi (95.9 %), and lower sequence similarity to other species. Average nucleotide identity values calculated from whole-genome sequencing data proved that CC-MHH1044T represents a distinct Cohnella species. The dominant cellular fatty acids (>5 %) included iso-C14 : 0(7.4 %), iso-C15 : 0 (6.4 %), anteiso-C15 : 0(40.3 %), C16 : 0 (6.6 %) and iso-C16 : 0 (27.0 %). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids, one unidentified phospholipid and glycolipid. The major polyamine was spermidine. The predominant isoprenoid quinone was menaqinone 7 (MK-7). Based on its distinct phylogenetic, phenotypic and chemotaxonomic traits, together with results of comparative 16S rRNA gene sequence, average nucleotide identity and digital DNA-DNA hybridization analyses, we conclude that strain CC-MHH1044T represents a novel member of the genus Cohnella, for which the name Cohnella fermenti sp. nov. is proposed. The type strain is CC-MHH1044T (=BCRC 81147T=JCM 32834T).

Draft genome and description of Cohnella massiliensis sp. nov., a new bacterial species isolated from the blood culture of a hemodialysis patient.

Strain 6021052837T was isolated from the blood culture of a hemodialysis patient on Chocolat PolyViteX medium at 37 °C after 2 days of incubation. Colonies could not be identified by our systematic MALDI-TOF Mass Spectrometry screening. The16S rRNA gene sequencing showed that the strain had 96% sequence identity with Cohnella formosensis (Genbank accession number JN806384), the phylogenetic closely related type strain of a species with standing in nomenclature, which putatively classifies it as a new species. The colonies cultivated on Columbia agar with 5% sheep blood medium at 37 °C after 24 h of incubation, are white pigmented, their size varied from 1.5 to 2 mm in diameter. Strain 6021052837T is an aerobic, Gram-negative, motile, spore forming rod, which cannot grow microaerophilically or under anaerobic conditions. The major fatty acids are branched saturated fatty acids: 14-methyl-pentadecanoic acid (34%) and 12-methyl-tetradecanoic acid (31%). The 6.328 Mb long genome, composed of 25 contigs, has a G+C content of 57.24%. Out of the 5710 predicted genes, 5646 were protein-coding genes and 64 were RNAs. A total of 3239 genes (57.37%) were assigned as putative function (by COGs) and 288 genes were identified as ORFans (5.1%). Average genomic identity of orthologous gene sequences (AGIOS) of strain 6021052837T against genomes of the type strains of related species ranged between 58.26 and 79.63%, respectively. According to our taxonogenomics results, we propose the creation of Cohnella massiliensis sp. nov. that contains the type strain 6021052837T (= CSUR P2659, =DSM103435).

Cohnella faecalis sp. nov., isolated from animal faeces in a karst cave.

A Gram-stain-positive, rod-shaped, endospore-forming, motile and aerobic bacterial isolate, designated strain K2E09-144T, was obtained from animal faeces that were collected from a karst cave in Guizhou province, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain K2E09-144T represents a novel member of the genus Cohnella within the family Paenibacillaceae of the phylum Firmicutes. Strain K2E09-144T was phylogenetically closely related to Cohnella nanjingensis D45T (16S rRNA gene sequence similarity 97.0 %). The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and C16 : 0. The major isoprenoid quinone was menaqinone 7 (MK-7). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified phospholipid, four unidentified aminophospholipids, one glycolipid and one unidentified lipid. The isomer type of diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The genome of strain K2E09-144T comprised 6.43 Mb, and encoded 6029 genes in total. The DNA G+C content of the genomic DNA was 53.3 mol%. Based on its phylogenetic, phenotypic and chemotaxonomic characteristics, strain K2E09-144T is considered to represent a novel species of the genus Cohnella, for which the name Cohnella faecalis sp. nov. is proposed. The type strain is K2E09-144T (=CGMCC 1.13587T=NBRC 113454T).

Cohnella endophytica sp. nov., a novel endophytic bacterium isolated from bark of Sonneratia apetala.

A Gram-stain-positive, aerobic, rod-shaped, endospore-forming bacterium, designated strain M2MS4P-1T, was isolated from surface-sterilized bark of Sonneratiaapetala sampled in Guangxi, China. The bacterium was characterized by a polyphasic approach to determine its taxonomic position. 16S rRNA gene sequence comparisons revealed that strain M2MS4P-1T belonged to the genus Cohnella and was most closely to Cohnella luojiensis HY-22RT (98.4 % similarity). The average nucleotide identity value and estimated DDH value between strain M2MS4P-1T and the type strain of C. luojiensis HY-22RT were 79.2 and 20.1 %, respectively. Neither substrate nor aerial mycelia were formed, and no diffusible pigments were observed on the media tested. Strain M2MS4P-1T grew in the pH range 6.0-9.0 (optimum, pH 7.0-8.0), at temperatures between 10-37 °C (30 °C) and in 0-1 % (w/v) NaCl (0 %). The predominant isoprenoid quinone in strain M2MS4P-1T was menaquinone-7. The major fatty acids were anteiso-C15 : 0 and iso-C16 : 0. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysyl-phosphatidylglycerol, four unidentified aminophospholipids and two unidentified phospholipids. The DNA G+C content was 51.5 mol%. According to the phylogenetic, phenotypic and chemotaxonomic evidence, strain M2MS4P-1T was clearly distinguishable from other species with validly published names in the genus Cohnella and should therefore be classified as a novel species, for which we suggest the name Cohnellaendophytica sp. nov. The type strain is M2MS4P-1T (=KCTC 43011T=CGMCC 1.13745T).

Cohnella candidum sp. nov., radiation-resistant bacterium from soil.

A Gram-stain positive, endospore-forming, circular, convex, cream colored, designated strain 18JY8-7T, was isolated from soil collected in Jeju Island, South Korea. Phylogenetic analysis using 16S rRNA gene sequences showed that strain 18JY8-7T formed a distinct lineage within the family Paenibacillaceae (order Bacillales, class Bacilli), and is closely related to Cohnella rhizosphaerae (96.1%, sequence similarity) and Cohnella xylanilytica (96.0%). Optimal growth occurred at 30 °C, pH 6.5 and in the absence of NaCl. The predominant cellular fatty acids were anteiso-C15:0 and iso-C16:0. The major respiratory quinone was MK-7. The polar lipids profile comprised of diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The DNA G + C content was 57.0 mol %. The genotypic and phenotypic analyses revealed the differentiation of strain 18JY8-7T from all recognized Cohenella species. The strain 18JY8-7T, therefore represents a novel bacterial species within the family Paenibacillaceae, for which the name Cohnella candidum sp. nov. is proposed. The type strain is 18JY8-7T (= KCTC 33969T = JCM 33199T).

Molecular evidence that cellulolytic bacterial genus Cohnella is widespread among Neotropical Nasutitermitinae from NE Argentina.

Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2-99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.

Cohnella kolymensis sp. nov., a novel bacillus isolated from Siberian permafrost.

A facultative anaerobic, rod-shaped, endospore-forming and non-motile bacterium was isolated from permafrost sediment cores in the Kolyma lowland, Siberia, Russia. The permafrost isolate clustered with members of the genus Cohnella on the basis of 16S rRNA gene sequence analysis and showed the highest sequence similarity to Cohnella saccharovorans CJ22T (96.3 %), followed by Cohnella cellulosilytica FCN3-3T (96.0 %) and Cohnella panacarvi KCTC 13060T (96.0 %). The chemotaxonomic characteristics (quinone system, cellular fatty acids and polar lipid profile) of strain 20.16T were consistent with members of the genus Cohnella. The peptidoglycan diaminoacids included meso-diaminopimelic acid and a small amount of ll-diaminopimelic acid. The molar ratio and composition of major amino acids (meso-diaminopimelic acid, alanine, and glutamic acid) correspond to the peptydoglycan type A1γ. The estimated genome size of strain 20.16T is 4.34 Mb (lower than those in other Cohnella species). The genome has a G+C content of 50.5 mol% and encodes 4843 predicted genes, of these 4740 are protein-coding ones. The results of chemotaxonomic, physiological and biochemical characterization allowed clear differentiation of strain 20.16T from the closest Cohnella species. Based on data provided, a new species Cohnella kolymensis sp. nov. is proposed, with 20.16T (=VKM B-2846T=DSM 104983T) as the type strain.

Cohnella algarum sp. nov., isolated from a freshwater green alga Paulinella chromatophora.

A Gram-stain-positive, facultatively aerobic and endospore-forming bacterium, designated strain Pch-40T, was isolated from a freshwater green alga, Paulinella chromatophora. Cells were motile rods with a monotrichous polar flagellum showing catalase- and oxidase-positive reactions. Strain Pch-40T grew at 20-50 °C (optimum, 37-40 °C), at pH 5.0-11.0 (optimum, pH 7.0) and in the presence of 0-4.0 % (w/v) NaCl (optimum, 0 %). Menaquinone-7 was detected as the sole isoprenoid quinone. The genomic DNA G+C content of strain Pch-40T was 55.6 mol%. The major cellular fatty acids of strain Pch-40T were C16 : 0, iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Pch-40T clearly belonged to the genus Cohnella of the family Paenibacillaceae. Strain Pch-40T was most closely related to Cohnella rhizosphaerae CSE-5610T with a 96.1 % 16S rRNA gene sequence similarity. The phenotypic and chemotaxonomic features and the phylogenetic inference clearly suggested that strain Pch-40T represents a novel species of the genus Cohnella, for which the name Cohnellaalgarum sp. nov. is proposed. The type strain is strain Pch-40T (=KACC 19279T=JCM 32033T).

Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01.

Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5kDa on SDS-PAGE. The values of Km and Vmax, calculated by the Michaelis-Menten equation, were 1077µM and 61.94U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70°C and pH 8.5. Activities at 50, 55 and 60°C for 120min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5-10.0 for 180min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% α-helix, 12.8% ß-sheet, 22.7% ß-turn and 27% random coil.

Identification and biochemical characterization of a novel endo-type ß-agarase AgaW from Cohnella sp. strain LGH.

An agar-degrading bacterium, strain LGH, was isolated and identified as Cohnella sp. This strain had a capability of utilizing agar as a sole carbon source for growth and showed a strong agarolytic activity. A novel endo-type ß-agarase gene agaW, encoding a primary translation product of 891 amino acids, including a 26 amino acid signal peptide, was cloned and identified from a genomic library of strain LGH. The AgaW belonged to the glycoside hydrolase (GH) GH50 family, with less than 39% amino acid sequence similarity with any known protein, and hydrolyzed agarose into neoagarotetraose as the major end product and neoagarobiose as the minor end product through other neoagarooligosaccharide intermediates, such as neoagarohexaose.

Expression optimization and characterization of a novel amylopullulanase from the thermophilic Cohnella sp. A01.

Amylopullulanase (EC. 3.2.1.41/1) is an enzyme that hydrolyzes starch and pullulan, capable of breaking (4 â†’ 1)-α and (6 â†’ 1)-α bonds in starch. Here, the Amy1136 gene (2166 base pairs) from the thermophilic bacterium Cohnella sp. A01 was cloned into the expression vector pET-26b(+) and expressed in Escherichia coli BL21. The enzyme was purified using heat shock at 90 °C for 15 min. The expression optimization of Amy1136 was performed using Plackett-Burman and Box-Behnken design as follows: temperature of 26.7 °C, rotational speed of 180 rpm, and bacterial population of 1.25. The Amy1136 displayed the highest activity at a temperature of 50 °C (on pullulan) and a pH of 8.0 (on starch) and, also exhibited stability at high temperatures (90 °C) and over a range of pH values. Ag+ significantly increased enzyme activity, while Co2+ completely inhibited amylase activity. The enzyme was found to be calcium-independent. The kinetic parameters Km, Vmax, kcat, and kcat/Km for amylase activity were 2.4 mg/mL, 38.650 µmol min-1 mg-1, 38.1129 S-1, and 0.09269 S-1mg mL-1, respectively, and for pullulanase activity were 173.1 mg/mL, 59.337 µmol min-1 mg-1, 1.586 S-1, and 1.78338 S-1mg mL-1, respectively. The thermodynamic parameters Kin, t1/2, Ea#, ΔH#, ΔG# and ΔS# were calculated equal to 0.20 × 10-2 (m-1), 462.09 (min), 16.87 (kJ/mol), 14.18 (kJ/mol), 47.34 (kJ/mol) and 102.60 (Jmol K-1), respectively. The stability of Amy1136 under high temperature, acidic and alkaline pH, surfactants, organic solvents, and calcium independence, suggests its suitability for industrial applications.

Identification of a novel xanthan-binding module of a multi-modular Cohnella sp. xanthanase.

A new strain of xanthan-degrading bacteria identified as Cohnella sp. has been isolated from a xanthan thickener for food production. The strain was able to utilize xanthan as the only carbon source and to reduce the viscosity of xanthan-containing medium during cultivation. Comparative analysis of the secretomes of Cohnella sp. after growth on different media led to the identification of a xanthanase designated as CspXan9, which was isolated after recombinant production in Escherichia coli. CspXan9 could efficiently degrade the ß-1,4-glucan backbone of xanthan after previous removal of pyruvylated mannose residues from the ends of the native xanthan side chains by xanthan lyase treatment (XLT-xanthan). Compared with xanthanase from Paenibacillus nanensis, xanthanase CspXan9 had a different module composition at the N- and C-terminal ends. The main putative oligosaccharides released from XLT-xanthan by CspXan9 cleavage were tetrasaccharides and octasaccharides. To explore the functions of the N- and C-terminal regions of the enzyme, truncated variants lacking some of the non-catalytic modules (CspXan9-C, CspXan9-N, CspXan9-C-N) were produced. Enzyme assays with the purified deletion derivatives, which all contained the catalytic glycoside hydrolase family 9 (GH9) module, demonstrated substantially reduced specific activity on XLT-xanthan of CspXan9-C-N compared with full-length CspXan9. The C-terminal module of CspXan9 was found to represent a novel carbohydrate-binding module of family CBM66 with binding affinity for XLT-xanthan, as was shown by native affinity polyacrylamide gel electrophoresis in the presence of various polysaccharides. The only previously known binding function of a CBM66 member is exo-type binding to the non-reducing fructose ends of the ß-fructan polysaccharides inulin and levan.