Endoglucanase gene of M42 aminopeptidase/endoglucanase family from thermophilic Bacillus sp. PW1 and PW2 isolated from Tattapani hot spring, Himachal Pradesh, India

Abstract Background Thermostable cellulases are in constant demand for several biotechnological applications. Two thermophilic bacterial strains PW1 and PW2 isolated from Tattapani hot spring were found to have cellulolytic activity. Subsequently, PW1 and PW2 were identified and mined for genes encoding cellulase activity. Results Sequencing of the 16S rDNA of PW1 and PW2 identified them as Bacillus sp. PW1 (Acc no. KU711837) and Bacillus sp. PW2 (Acc no. KU711838), respectively, which clustered in the clades containing thermophilic members of Bacillus sp. and Geobacillus species. Phylogenetic analysis revealed that despite the morphological and sequence identities, Bacillus sp. PW1 and Bacillus sp. PW2 are different at the genetic level. The cellulase genes (~ 1.1 kb) of the two bacterial strains were amplified using primers designed against related thermophilic cellulases. Sequencing of the cellulase gene amplicons of PW1 and PW2 revealed that they encode proteins of 280 and 206 amino acid residues, respectively. Sequence and domain analysis of the protein products of PW1 and PW2 revealed that they belong to M42 family of aminopeptidase/endoglucanase. The PW2 endoglucanase coding sequence was submitted to Genbank under accession no. MH049504. The structures of putative endoglucanases of PW1 and PW2 were generated using 1VHE.A as template, which showed the presence of vast proportion of random coils. Molecular docking of the modeled endoglucanase proteins with various substrates and products of cellulases showed that carboxymethyl cellulose and maltose exhibit the highest binding affinity, while xylan and glucose the least. Conclusions The two thermophilic bacteria PW1 and PW2 and their endoglucanase gene can be further utilized for recombinant production of thermostable cellulases for their application in industries.

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Isolation and Characterization of Acid-Tolerant, Thermophilic Bacteria for Effective Fermentation of Biomass-Derived Sugars to Lactic Acid

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3228-3235.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3228-3235 ◽

Cited By ~ 128

Author(s):

Milind A. Patel ◽

Mark S. Ou ◽

Roberta Harbrucker ◽

Henry C. Aldrich ◽

Marian L. Buszko ◽

...

Keyword(s):

Lactic Acid ◽

Pentose Phosphate Pathway ◽

Thermophilic Bacteria ◽

Cellulase Activity ◽

Pentose Phosphate ◽

Bacterial Strains ◽

16S Rrna Sequence ◽

Cost Component ◽

Isolation And Characterization ◽

Fungal Cellulase

ABSTRACT Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50ï¿½C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [13C]lactate accounted for more than 90% of the total 13C-labeled products from [13C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.

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Isolation, Biochemical Characterisation and Identification of Thermotolerant and Cellulolytic Paenibacillus lactis and Bacillus licheniformis

Food Technology and Biotechnology ◽

10.17113/ftb.59.03.21.7096 ◽

2021 ◽

Vol 59 (3) ◽

Author(s):

Krzysztof Makowski ◽

Martyna Leszczewicz ◽

Natalia Broncel ◽

Lidia Lipińska-Zubrycka ◽

Adrian Głębski ◽

...

Keyword(s):

Bacillus Licheniformis ◽

Hot Spring ◽

Growth Curves ◽

Maximum Growth ◽

Cellulolytic Activity ◽

Bacterial Strains ◽

Thermophilic Microorganisms ◽

Biochemical Characteristics ◽

Cmcase Activity ◽

Biochemical Characterisation

Research background. Cellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that, the polymer molecule will be degraded to smaller particles, used as a carbon source by microorganisms. Because of the frequently applied methods of pre-treatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable. Experimental approach. We were looking for cellulolytic microorganisms able to grow at 50 °C. We described their morphological features and biochemical characteristics based on CMCase activity and the api®ZYM. The growth curves, during incubation at 50 °C, were examined using the microbioreactor BioLector®. Results and conclusions. 40 bacterial strains were isolated from fermenting hay, geothermal karst spring, hot spring and geothermal pond at 50 °C. The vast majority of the bacteria were Gram-positive and rod-shaped with the maximum growth temperature of at least 50 °C. We also demonstrated a large diversity of biochemical characteristics among the microorganism. The CMCase activity was confirmed for 27 strains. However, the hydrolysis capacities (HC) were significant in bacterial strains: BBLN1, BSO6, BSO10, BSO13 and BSO14, and reached 2.74, 1.62, 1.30, 1.38 and 8.02 respectively. Rapid and stable growth was presented, among others, by BBLN1, BSO10, BSO13 and BSO14. The strains fulfilled the selection conditions and were identified based on the 16S rDNA sequences. BBLN1, BSO10, BSO13 were classified as Bacillus licheniformis, whereas BSO14 as Paenibacillus lactis. Novelty and scientific contribution. We described cellulolytic activity and biochemical characteristics of many bacteria isolated from hot environments. We are also the first to report the cellulolytic activity of thermotolerant P.s lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of the circular bioeconomy.

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Autochthonous Gut Bacteria in Two Indian Air-breathing Fish, Climbing Perch (Anabas testudineus) and Walking Catfish (Clarias batrachus): Mode of Association, Identification and Enzyme Producing Ability

Polish Journal of Microbiology ◽

10.5604/17331331.1185234 ◽

2015 ◽

Vol 64 (4) ◽

pp. 361-368 ◽

Cited By ~ 6

Author(s):

Goutam Banerjee ◽

Suhas K. Dan ◽

Ankita Nandi ◽

Pinki Ghosh ◽

Arun K. Ray

Keyword(s):

Cellulase Activity ◽

Clarias Batrachus ◽

Cellulolytic Activity ◽

Bacterial Cells ◽

Gi Tract ◽

Bacterial Strains ◽

Anabas Testudineus ◽

Activity Maximum ◽

Air Breathing ◽

Climbing Perch

Scanning electron microscopy (SEM) was used to define the location of epithelium-associated bacteria in the gastrointestinal (GI) tract of two Indian air-breathing fish, the climbing perch (Anabas testudineus) and walking catfish (Clarias batrachus). The SEM examination revealed substantial numbers of rod shaped bacterial cells associated with the microvillus brush borders of enterocytes in proximal (PI) and distal regions (DI) of the GI tract of both the fish species. Ten (two each from the PI and DI of climbing perch and three each from the PI and DI of walking catfish) isolated bacterial strains were evaluated for extracellular protease, amylase and cellulase production quantitatively. All the bacterial strains exhibited high cellulolytic activity compared to amylolytic and proteolytic activites. Only two strains, CBH6 and CBH7, isolated from the DI of walking catfish exhibited high proteolytic activity. Maximum cellulase activity was exhibited by the strain, CBF2, isolated from the PI of climbing perch. Six most promising enzyme-producing adherent bacterial strains were identified by 16S rDNA gene sequence analysis. The strain ATH1 (isolated from climbing perch) showed high similarity to Bacillus amyloliquefaciens whereas, the remaining five strains (isolated from walking catfish) were most closely related to Bacillus licheniformis.

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Decomposition of Rice Chaff Using a Cocultivation System of Thermobifida fusca and Ureibacillus thermosphaericus

Proceedings ◽

10.3390/proceedings2020066031 ◽

2021 ◽

Vol 66 (1) ◽

pp. 31

Author(s):

Sachiko Nakamura ◽

Norio Kurosawa

Keyword(s):

Lignin Peroxidase ◽

Culture Medium ◽

Reducing Sugars ◽

Thermophilic Bacteria ◽

Thermobifida Fusca ◽

Bacterial Strains ◽

Basal Salt Medium ◽

Moderately Thermophilic ◽

Fuels And Chemicals ◽

Ureibacillus Thermosphaericus

Lignocellulosic biomass comprises cellulose, hemicellulose, and lignin and is a potential source of fuels and chemicals. Although this complex biomass is persistent, it can be cooperatively decomposed by a microbial consortium in nature. In this study, a coculture of the moderately thermophilic bacteria Thermobifida fusca and Ureibacillus thermosphaericus was used for biodegradation of rice chaff. The bacterial strains were incubated in modified Brock’s basal salt medium (pH 8.0) supplemented with yeast extract and rice chaff at 50 °C for 7 days. The concentration of reducing sugars and the enzymatic activities of laccase, lignin peroxidase, cellulase, and xylanase in the supernatant of the culture medium were measured every day. The concentrations of reducing sugars in solo cultures of T. fusca and U. thermosphaericus and a mixed culture of the two strains after 7 days of incubation were 0.047, 0.040, and 0.195 mg/mL, respectively, indicating that the decomposition of rice chaff was enhanced in the coculture. Based on the results, it is thought that the lignin surrounding the cellulose was decomposed by laccase and lignin peroxidase secreted from U. thermosphaericus, resulting in cellulose and hemicellulose in the rice chaff being easily decomposed by enzymes from T. fusca.

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Identification and Genomic Characterization of Pathogenic Bacillus altitudinis from Common Pear Trees in Morocco

Agronomy ◽

10.3390/agronomy11071344 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1344

Author(s):

Naima Lemjiber ◽

Khalid Naamani ◽

Annabelle Merieau ◽

Abdelhi Dihazi ◽

Nawal Zhar ◽

...

Keyword(s):

16S Rrna Gene Sequencing ◽

Genomic Islands ◽

Rrna Gene ◽

In Planta ◽

Bacterial Strains ◽

Bacillus Altitudinis ◽

Genes Encoding ◽

Bacillus Spp ◽

Pear Trees ◽

Rrna Gene Sequencing

Bacterial burn is one of the major diseases affecting pear trees worldwide, with serious impacts on producers and economy. In Morocco, several pear trees (Pyrus communis) have shown leaf burns since 2015. To characterize the causal agent of this disease, we isolated fourteen bacterial strains from different parts of symptomatic pear trees (leaves, shoots, fruits and flowers) that were tested in planta for their pathogenicity on Louise bonne and Williams cultivars. The results showed necrotic lesions with a significant severity range from 47.63 to 57.77% on leaves of the Louise bonne cultivar inoculated with isolate B10, while the other bacterial isolates did not induce any disease symptom. 16S rRNA gene sequencing did not allow robust taxonomic discrimination of the incriminated isolate. Thus, we conducted whole-genome sequencing (WGS) and phylogenetic analyzes based on gyrA, gyrB and cdaA gene sequences, indicating that this isolate belongs to the Bacillus altitudinis species. This taxonomic classification was further confirmed by the Average Nucleotide Identity (ANI) and the in silico DNA-DNA hybridization (isDDH) analyzes compared to sixty-five Bacillus spp. type strains. The genome was mined for genes encoding carbohydrate-active enzymes (CAZymes) known to play a role in the vegetal tissue degradation. 177 candidates with functions that may support the in planta phytopathogenicity results were identified. To the best of our knowledge, this is the first data reporting B. altitudinis as agent of leaf burn in P. communis in Morocco. Our dataset will improve our knowledge on spread and pathogenicity of B. altitudinis genotypes that appears as emergent phytopathogenic agent, unveiling virulence factors and their genomic location (i.e., within genomic islands or the accessory genome) to induce trees disease.

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PRODUCTION AND CHARACTERIZATION OF CHITINASE ENZYMES FROM SULILI HOT SPRING IN SOUTH SULAWESI, Bacillus sp. HSA,3-1a

Indonesian Journal of Chemistry ◽

10.22146/ijc.21470 ◽

2010 ◽

Vol 10 (2) ◽

pp. 256-260 ◽

Cited By ~ 6

Author(s):

Hasnah Natsir ◽

Abd. Rauf Patong ◽

Maggy Thenawidjaja Suhartono ◽

Ahyar Ahmad

Keyword(s):

Hot Spring ◽

Thermophilic Bacteria ◽

Extracellular Enzyme ◽

Colloidal Chitin ◽

Bacillus Sp ◽

Optimum Temperature ◽

South Sulawesi ◽

Sds Page ◽

Physiological Analysis

Chitinase is an extracellular enzyme which is capable in hydrolyzing insoluble chitin to its oligomeric and monomeric components. The enzyme produced by thermophilic bacteria was screened and isolated from Sulili hot spring in Pinrang, South Sulawesi, Indonesia. The gram positive spore forming rod shape bacteria was identified as Bacillus sp. HSA,3-1a through morphological and physiological analysis. The production of chitinase enzyme was conducted at various concentration of colloidal chitin at a pH of 7.0 and a temperature of 55 °C. The bacteria optimally was produced the enzyme at a colloidal chitin concentration of 0.5% after 72 h of incubation. The optimum temperature, pH and substrate concentration of chitinase were 60 °C, 7.0 and 0.3%, respectively. The enzyme was stable at a pH of 7.0 and a temperature of 60 °C after 2 h of incubation. The chitinase activities was increased by addition of 1 mM Mg2+, Ca2+ and Mn2+ ions, whereas the activities were decreased by 1 mM Co2+, Fe2+ and Zn2 ions. The molecular weight of the crude enzyme was determined using SDS-PAGE analysis. Five protein fractions were obtained from SDS-PAGE, with MWs of 79, 71, 48, 43 and 22 kDa. Keywords: colloidal chitin, thermophilic bacteria, chitinase

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First Report of Black Soft Rot of Indian Gooseberry Caused by Syncephalastrum racemosum

Plant Disease ◽

10.1094/pdis.2004.88.5.575c ◽

2004 ◽

Vol 88 (5) ◽

pp. 575-575 ◽

Cited By ~ 2

Author(s):

Neelima Garg ◽

Om Prakash ◽

B. K. Pandey ◽

B. P. Singh ◽

G. Pandey

Keyword(s):

Cellulase Activity ◽

Basal Medium ◽

Petri Dish ◽

Soft Rot ◽

Cellulolytic Activity ◽

Academic Press ◽

First Report ◽

Syncephalastrum Racemosum ◽

Indian Gooseberry ◽

Pathogenicity Tests

Indian gooseberry (Emblica officinalis Gaertn.) is a medicinal plant with high nutraceutical value. During November and December 2003, soft rot was noticed on harvested and stored (20 ± 5°C and 65 ± 5% relative humidity) fruits at the experimental farm in Rehmanhera, Lucknow, India (26°50′N, 80°54′E). These fruits had numerous, minute brown necrotic lesions showing white mycelial growth. A pronounced halo of water-soaked, faded tissue surrounded the lesion between the fringe of mycelium and healthy tissues. The rotted surface was covered with a black, powdery layer of spores. On Czapek yeast extract agar, fungal colonies were blackish grey, moderately dense, and covered the entire petri dish. The fungus produced aseptate mycelium. The sporangial heads were 30 to 50 μm in diameter with sporangiospores found linearly within cylindrical sacs (merosporangia) borne on spicules around the columella. Sporangiospores, spherical to cylindrical in shape and borne in chains, measured 3.0 to 5.0 μm long. The fungus was morphologically and physiologically identified as Syncephalastrum racemosum Schr. (2). For pathogenicity tests, healthy fruits (10 replicates) were surface sterilized and punctured inoculated aseptically with 1.0 × 106 conidia and incubated at 20 ± 5°C Typical symptoms of the disease appeared after 4 days. The fungus exhibited a strong level of cellulolytic activity as indicated by prolific growth on Indian gooseberry fiber waste under solid-state fermentation conditions. The level of cellulase activity (1) was 21 filter paper activity unit per ml at 72 hr in culture supernatant of basal medium having carboxymethyl cellulose as the carbon source. The fungus showed resistance to tannins (as much as 2%), since it could grow well in liquid growth medium (Czapek Dox broth) with 2% tannins and aonla juice with 1.8% tannins. Since Indian gooseberry is rich in fiber (2.5 to 3.4%) and tannins (1.5 to 2.0%), this may be an important pathogen. To our knowledge, this is the first report of the occurrence of Syncephalastrum racemosum on Indian gooseberry fruits. References: (1) T. K. Ghose. Pure Appl. Chem. 59(2):257, 1987. (2) J. I. Pitt and A. D. Hocking. Fungi and Food Spoilage. Academic Press. North Ryde, Australia, 1985.

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Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.688-695.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 688-695 ◽

Cited By ~ 46

Author(s):

Jan Bohuslavek ◽

Jason W. Payne ◽

Yong Liu ◽

Harvey Bolton ◽

Luying Xun

Keyword(s):

Gene Cluster ◽

Genomic Library ◽

Regulatory Protein ◽

Chelating Agent ◽

Environmental Pollutant ◽

Flavin Mononucleotide ◽

Bacterial Strains ◽

System A ◽

Monooxygenase Gene ◽

Genes Encoding

ABSTRACT EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed inEscherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. TheemoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.

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Cultivation and Genomic, Nutritional, and Lipid Biomarker Characterization of Roseiflexus Strains Closely Related to Predominant In Situ Populations Inhabiting Yellowstone Hot Spring Microbial Mats

Journal of Bacteriology ◽

10.1128/jb.01610-09 ◽

2010 ◽

Vol 192 (12) ◽

pp. 3033-3042 ◽

Cited By ~ 46

Author(s):

Marcel T. J. van der Meer ◽

Christian G. Klatt ◽

Jason Wood ◽

Donald A. Bryant ◽

Mary M. Bateson ◽

...

Keyword(s):

National Park ◽

Microbial Mats ◽

Hot Spring ◽

Small Subunit ◽

Small Subunit Rrna ◽

Anoxygenic Phototrophs ◽

Lipid Biomarker ◽

Genes Encoding

ABSTRACT Roseiflexus sp. strains were cultivated from a microbial mat of an alkaline siliceous hot spring in Yellowstone National Park. These strains are closely related to predominant filamentous anoxygenic phototrophs found in the mat, as judged by the similarity of small-subunit rRNA, lipid distributions, and genomic and metagenomic sequences. Like a Japanese isolate, R. castenholzii, the Yellowstone isolates contain bacteriochlorophyll a, but not bacteriochlorophyll c or chlorosomes, and grow photoheterotrophically or chemoheterotrophically under dark aerobic conditions. The genome of one isolate, Roseiflexus sp. strain RS1, contains genes necessary to support these metabolisms. This genome also contains genes encoding the 3-hydroxypropionate pathway for CO2 fixation and a hydrogenase, which might enable photoautotrophic metabolism, even though neither isolate could be grown photoautotrophically with H2 or H2S as a possible electron donor. The isolates exhibit temperature, pH, and sulfide preferences typical of their habitat. Lipids produced by these isolates matched much better with mat lipids than do lipids produced by R. castenholzii or Chloroflexus isolates.

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