Expression, characterization, and activity optimization of a novel cellulase from the thermophilic bacteria Cohnella sp. A01

Mohammadi, Shabnam; Tarrahimofrad, Hossein; Arjmand, Sareh; Zamani, Javad; Haghbeen, Kamahldin; Aminzadeh, Saeed

doi:10.1038/s41598-022-14651-7

Cited by 12 publications

(3 citation statements)

References 83 publications

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“…The GH5 enzymes are involved in the hydrolysis of cellulose into cellobiose ( Wang et al, 2019 ), while GH3 enzymes break down cellobiose into glucose ( Agirre et al, 2016 ). Members of the GH5 superfamily are extensively dispersed across archaea, bacteria, and eukaryotes, and various enzyme functions related to biomass conversion have been discovered in this superfamily ( Mohammadi et al, 2022 ). In cellulose biomass degradation, β-glucosidase (GH3) activity is regarded as the rate-limiting factor, reducing cellobiose inhibition on endoglucanases and permitting more effective cellulolytic enzyme action ( Tiwari et al, 2017 ; Zhang et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Discovery of novel carbohydrate degrading enzymes from soda lakes through functional metagenomics

et al. 2022

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Extremophiles provide a one-of-a-kind source of enzymes with properties that allow them to endure the rigorous industrial conversion of lignocellulose biomass into fermentable sugars. However, the fact that most of these organisms fail to grow under typical culture conditions limits the accessibility to these enzymes. In this study, we employed a functional metagenomics approach to identify carbohydrate-degrading enzymes from Ethiopian soda lakes, which are extreme environments harboring a high microbial diversity. Out of 21,000 clones screened for the five carbohydrate hydrolyzing enzymes, 408 clones were found positive. Cellulase and amylase, gave high hit ratio of 1:75 and 1:280, respectively. A total of 378 genes involved in the degradation of complex carbohydrates were identified by combining high-throughput sequencing of 22 selected clones and bioinformatics analysis using a customized workflow. Around 41% of the annotated genes belonged to the Glycoside Hydrolases (GH). Multiple GHs were identified, indicating the potential to discover novel CAZymes useful for the enzymatic degradation of lignocellulose biomass from the Ethiopian soda Lakes. More than 73% of the annotated GH genes were linked to bacterial origins, with Halomonas as the most likely source. Biochemical characterization of the three enzymes from the selected clones (amylase, cellulase, and pectinase) showed that they are active in elevated temperatures, high pH, and high salt concentrations. These properties strongly indicate that the evaluated enzymes have the potential to be used for applications in various industrial processes, particularly in biorefinery for lignocellulose biomass conversion.

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Section: Discussionmentioning

confidence: 99%

Discovery of novel carbohydrate degrading enzymes from soda lakes through functional metagenomics

et al. 2022

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“…GraphPad Prism V.8 was used to perform non-linear regression to characterize the kinetic properetises (K m , V max , k cat , and k cat /K m ) of the pure KRLr1 in terms of Michaelis/Menten kinetic parameters ( Mohammadi et al, 2022 ). A variety of keratin substrate concentrations (10–70 mM) and the activity optimum temperature were used to measure the keratinase activity.…”

Section: Methodsmentioning

confidence: 99%

Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis (KRLr1)

et al. 2023

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The keratin-degrading bacterium Bacillus licheniformis secretes a keratinase with potential industrial interest. Here, the Keratinase gene was intracellularly expressed in Escherichia coli BL21(DE3) using pET-21b (+) vector. Phylogenetic tree analysis showed that KRLr1 is closely related to Bacillus licheniformis keratinase that belongs to the serine peptidase/subtilisin-like S8 family. Recombinant keratinase appeared on the SDS-PAGE gel with a band of about 38 kDa and was confirmed by western blotting. Expressed KRLr1 was purified by Ni-NTA affinity chromatography with a yield of 85.96% and then refolded. It was found that this enzyme has optimum activity at pH 6 and 37°C. PMSF inhibited the KRLr1 activity and Ca2+ and Mg2+ increased the KRLr1 activity. Using keratin 1% as the substrate, the thermodynamic values were determined as Km 14.54 mM, kcat 912.7 × 10−3 (S−1), and kcat/Km 62.77 (M−1 S−1). Feather digestion by recombinant enzyme using HPLC method, showed that the amino acids cysteine, phenylalanine, tyrosine and lysine had the highest amount compared to other amino acids obtained from digestion. Molecular dynamics (MD) simulation of HADDOCK docking results exhibited that KRLr1 enzyme was able to interact strongly with chicken feather keratine 4 (FK4) compared to chicken feather keratine 12 (FK12). These properties make keratinase KRLr1 a potential candidate for various biotechnological applications.

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“…Cohnella species have been first isolated from a wide variety of environmental samples, most usually in soil (Kampfer et al, 2014 ), but also in plant tissue (Garcia-Fraile et al, 2008 ; Flores-Felix et al, 2014 ) and compost heaps (He et al, 2013 ). Species from this genus are used in industrial applications including fermentation, biofuel production, and wastewater treatment bioreactors (Lin et al, 2020 ; Hero et al, 2021 ; Chen et al, 2022 ; Mohammadi et al, 2022 ). As with the genus Paenibacillus , many members of the genus Cohnella are strongly associated with the plant rhizosphere environment, particularly that of legume species (Niang et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Comparative genomic analysis of Cohnella hashimotonis sp. nov. isolated from the International Space Station

et al. 2023

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A single strain from the family Paenibacillaceae was isolated from the wall behind the Waste Hygiene Compartment aboard the International Space Station (ISS) in April 2018, as part of the Microbial Tracking mission series. This strain was identified as a gram-positive, rod-shaped, oxidase-positive, catalase-negative motile bacterium in the genus Cohnella, designated as F6_2S_P_1T. The 16S sequence of the F6_2S_P_1T strain places it in a clade with C. rhizosphaerae and C. ginsengisoli, which were originally isolated from plant tissue or rhizosphere environments. The closest 16S and gyrB matches to strain F6_2S_P_1T are to C. rhizosphaerae with 98.84 and 93.99% sequence similarity, while a core single-copy gene phylogeny from all publicly available Cohnella genomes places it as more closely related to C. ginsengisoli. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values to any described Cohnella species are <89 and <22%, respectively. The major fatty acids for strain F6_2S_P_1T are anteiso-C15:0 (51.7%), iso-C16:0 (23.1%), and iso-C15:0 (10.5%), and it is able to metabolize a wide range of carbon compounds. Given the results of the ANI and dDDH analyses, this ISS strain is a novel species within the genus Cohnella for which we propose the name Cohnella hashimotonis, with the type strain F6_2S_P_1T (=NRRL B-65657T and DSMZ 115098T). Because no closely related Cohnella genomes were available, this study generated the whole-genome sequences (WGSs) of the type strains for C. rhizosphaerae and C. ginsengisoli. Phylogenetic and pangenomic analysis reveals that F6_2S_P_1T, C. rhizosphaerae, and C. ginsengisoli, along with two uncharacterized Cohnella strains, possess a shared set of 332 gene clusters which are not shared with any other WGS of Cohnella species, and form a distinct clade branching off from C. nanjingensis. Functional traits were predicted for the genomes of strain F6_2S_P_1T and other members of this clade.

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Expression, characterization, and activity optimization of a novel cellulase from the thermophilic bacteria Cohnella sp. A01

Cited by 12 publications

References 83 publications

Discovery of novel carbohydrate degrading enzymes from soda lakes through functional metagenomics

Discovery of novel carbohydrate degrading enzymes from soda lakes through functional metagenomics

Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis (KRLr1)

Comparative genomic analysis of Cohnella hashimotonis sp. nov. isolated from the International Space Station

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