scholarly journals Cloning, purification, and characterization of GH3 β-glucosidase, MtBgl85, from Microbulbifer thermotolerans DAU221

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7106 ◽  
Author(s):  
Hyo-Min Pyeon ◽  
Yong-Suk Lee ◽  
Yong-Lark Choi
Keyword(s):  
Enzyme Stability ◽  
Cellulose Degradation ◽  
Glycosyl Hydrolase ◽  
Bacterial Species ◽  
Whole Genome Sequence ◽  
Potential Candidate ◽  
Polar Solvents ◽  
Deep Sea Sediment ◽  
Physiological Properties ◽  
Glycosyl Hydrolase Family 3

Background β-Glucosidases have attracted considerable attention due to their important roles in various biotechnological processes such as cellulose degradation to make energy and hydrolysis of isoflavone. Microbulbifer thermotolerans (M. thermotolerans) is isolated from deep-sea sediment and has not been researched much yet. As a potential candidate for a variety of biotechnological industries, β-glucosidases from the novel bacterial species should be researched extensively. Methods β-Glucosidase, MtBgl85, from M. thermotolerans DAU221 was purified by His-tag affinity chromatography and confirmed by SDS-PAGE and zymogram. Its biochemical and physiological properties, such as effects of temperature, pH, metal ions, and organic solvents, substrate specificity, and isoflavone hydrolysis, were investigated. Results M. thermotolerans DAU221 showed β-glucosidase activity in a marine broth plate containing 0.1% esculin and 0.25% ammonium iron (III) citrate. The β-glucosidase gene, mtbgl85, was isolated from the whole genome sequence of M. thermotolerans DAU221. The β-glucosidase gene was 2,319 bp and encoded 772 amino acids. The deduced amino acid sequence had a 43% identity with OaBGL84 from Olleya aquimaris and 35% and 32% identity with to CfBgl3A and CfBgl3C from Cellulomonas fimi among bacterial glycosyl hydrolase family 3, respectively. The optimal temperature of MtBgl85 was 50 °C and the optimum pH was 7.0. MtBgl85 activity was strongly reduced in the presence of Hg2+ and Cu2+ ions. As a result of measuring the activity at various concentrations of NaCl, it was confirmed that the activity was maintained up to the concentration of 1 M, but gradually decreased with increasing concentration. MtBgl85 showed higher enzyme stability at non-polar solvents (high Log Pow) than polar solvents (low Log Pow). The hydrolyzed products of isoflavone glycosides and arbutin were analyzed by HPLC.

scholarly journals Growth of Azospirillum irakense KBC1 on the Aryl β-Glucoside Salicin Requires either salA or salB

1999 ◽  
Vol 181 (10) ◽  
pp. 3003-3009 ◽  
Author(s):  
Denis Faure ◽  
Jos Desair ◽  
Veerle Keijers ◽  
My Ali Bekri ◽  
Paul Proost ◽  
...  
Keyword(s):  
Microbial Growth ◽  
Glycosyl Hydrolase ◽  
Cosmid Library ◽  
Glycosyl Hydrolases ◽  
Amino Acid Similarity ◽  
Functional Homology ◽  
Low Degree ◽  
Genetic Implication ◽  
Glycosyl Hydrolase Family 3 ◽  
Hydrolase Family

ABSTRACT The rhizosphere nitrogen-fixing bacteriumAzospirillum irakense KBC1 is able to grow on pectin and β-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring β-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl β-glucosides. A Δ(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB′ subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this β-glucosidase family in the utilization of β-glucosides for microbial growth.

scholarly journals Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

2020 ◽  
Vol 86 (16) ◽  
Author(s):  
Mario Viñambres ◽  
Marta Espada ◽  
Angel T. Martínez ◽  
Ana Serrano
Keyword(s):  
Hydrogen Peroxide ◽  
Heterologous Expression ◽  
Half Life ◽  
Enzyme Stability ◽  
Bacterial Species ◽  
Building Blocks ◽  
Enzymatic Production ◽  
Content Type ◽  
Furandicarboxylic Acid ◽  
Total Turnover

ABSTRACT The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF) has gained interest in recent years, as FDCA is a renewable precursor of poly(ethylene-2,5-furandicarboxylate) (PEF). 5-Hydroxymethylfurfural oxidases (HMFOs) form a flavoenzyme family with genes annotated in a dozen bacterial species but only one enzyme purified and characterized to date (after heterologous expression of a Methylovorus sp. HMFO gene). This oxidase acts on both furfuryl alcohols and aldehydes and, therefore, is able to catalyze the conversion of HMF into FDCA through 2,5-diformylfuran (DFF) and 2,5-formylfurancarboxylic acid (FFCA), with only the need of oxygen as a cosubstrate. To enlarge the repertoire of HMFO enzymes available, genetic databases were screened for putative HMFO genes, followed by heterologous expression in Escherichia coli. After unsuccessful trials with other bacterial HMFO genes, HMFOs from two Pseudomonas species were produced as active soluble enzymes, purified, and characterized. The Methylovorus sp. enzyme was also produced and purified in parallel for comparison. Enzyme stability against temperature, pH, and hydrogen peroxide, three key aspects for application, were evaluated (together with optimal conditions for activity), revealing differences between the three HMFOs. Also, the kinetic parameters for HMF, DFF, and FFCA oxidation were determined, the new HMFOs having higher efficiencies for the oxidation of FFCA, which constitutes the bottleneck in the enzymatic route for FDCA production. These results were used to set up the best conditions for FDCA production by each enzyme, attaining a compromise between optimal activity and half-life under different conditions of operation. IMPORTANCE HMFO is the only enzyme described to date that can catalyze by itself the three consecutive oxidation steps to produce FDCA from HMF. Unfortunately, only one HMFO enzyme is currently available for biotechnological application. This availability is enlarged here by the identification, heterologous production, purification, and characterization of two new HMFOs, one from Pseudomonas nitroreducens and one from an unidentified Pseudomonas species. Compared to the previously known Methylovorus HMFO, the new enzyme from P. nitroreducens exhibits better performance for FDCA production in wider pH and temperature ranges, with higher tolerance for the hydrogen peroxide formed, longer half-life during oxidation, and higher yield and total turnover numbers in long-term conversions under optimized conditions. All these features are relevant properties for the industrial production of FDCA. In summary, gene screening and heterologous expression can facilitate the selection and improvement of HMFO enzymes as biocatalysts for the enzymatic synthesis of renewable building blocks in the production of bioplastics.

scholarly journals Antimicrobial resistance genetic factor identification from whole-genome sequence data using deep feature selection

2019 ◽  
Vol 20 (S15) ◽  
Author(s):  
Jinhong Shi ◽  
Yan Yan ◽  
Matthew G. Links ◽  
Longhai Li ◽  
Jo-Anne R. Dillon ◽  
...  
Keyword(s):  
Feature Selection ◽  
Antimicrobial Resistance ◽  
Genome Sequence ◽  
Sequence Data ◽  
Bacterial Species ◽  
Clinical Diagnostics ◽  
New Drugs ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genome Sequence Data

Abstract Background Antimicrobial resistance (AMR) is a major threat to global public health because it makes standard treatments ineffective and contributes to the spread of infections. It is important to understand AMR’s biological mechanisms for the development of new drugs and more rapid and accurate clinical diagnostics. The increasing availability of whole-genome SNP (single nucleotide polymorphism) information, obtained from whole-genome sequence data, along with AMR profiles provides an opportunity to use feature selection in machine learning to find AMR-associated mutations. This work describes the use of a supervised feature selection approach using deep neural networks to detect AMR-associated genetic factors from whole-genome SNP data. Results The proposed method, DNP-AAP (deep neural pursuit – average activation potential), was tested on a Neisseria gonorrhoeae dataset with paired whole-genome sequence data and resistance profiles to five commonly used antibiotics including penicillin, tetracycline, azithromycin, ciprofloxacin, and cefixime. The results show that DNP-AAP can effectively identify known AMR-associated genes in N. gonorrhoeae, and also provide a list of candidate genomic features (SNPs) that might lead to the discovery of novel AMR determinants. Logistic regression classifiers were built with the identified SNPs and the prediction AUCs (area under the curve) for penicillin, tetracycline, azithromycin, ciprofloxacin, and cefixime were 0.974, 0.969, 0.949, 0.994, and 0.976, respectively. Conclusions DNP-AAP can effectively identify known AMR-associated genes in N. gonorrhoeae. It also provides a list of candidate genes and intergenic regions that might lead to novel AMR factor discovery. More generally, DNP-AAP can be applied to AMR analysis of any bacterial species with genomic variants and phenotype data. It can serve as a useful screening tool for microbiologists to generate genetic candidates for further lab experiments.

scholarly journals Diversity of Bacteria and Glycosyl Hydrolase Family 48 Genes in Cellulolytic Consortia Enriched from Thermophilic Biocompost

2010 ◽  
Vol 76 (11) ◽  
pp. 3545-3553 ◽  
Author(s):  
Javier A. Izquierdo ◽  
Maria V. Sizova ◽  
Lee R. Lynd
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Cellulose Degradation ◽  
Glycosyl Hydrolase ◽  
Rrna Gene ◽  
Cellulolytic Activity ◽  
The Novel ◽  
Glycosyl Hydrolase Family ◽  
Enrichment Cultures ◽  
Hydrolase Family

ABSTRACT The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.

scholarly journals Cellulose- and Xylan-Degrading Thermophilic Anaerobic Bacteria from Biocompost

2011 ◽  
Vol 77 (7) ◽  
pp. 2282-2291 ◽  
Author(s):  
M. V. Sizova ◽  
J. A. Izquierdo ◽  
N. S. Panikov ◽  
L. R. Lynd
Keyword(s):  
Enrichment Culture ◽  
Cellulose Degradation ◽  
Phylogenetic Analyses ◽  
Glycosyl Hydrolase ◽  
Anaerobic Bacteria ◽  
Xylanase Activity ◽  
Cellulase Activity ◽  
Lag Phase ◽  
Fermentation Products ◽  
Clostridium Clariflavum

ABSTRACTNine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to aClostridium clariflavumstrain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.

scholarly journals c-di-AMP, a likely master regulator of bacterial K+ homeostasis machinery, activates a K+ exporter

2021 ◽  
Vol 118 (14) ◽  
pp. e2020653118
Author(s):  
Tatiana B. Cereija ◽  
João P. L. Guerra ◽  
João M. P. Jorge ◽  
João H. Morais-Cabral
Keyword(s):  
Bacterial Species ◽  
Structural Data ◽  
Regulatory Function ◽  
Dna Integrity ◽  
Master Regulator ◽  
Quantitative Evidence ◽  
Physiological Properties ◽  
The Impact

bis-(3′,5′)-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.

scholarly journals Whole Genome Sequence Analysis of Porcine Astroviruses Reveals Novel Genetically Diverse Strains Circulating in East African Smallholder Pig Farms

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1262
Author(s):  
Joshua O. Amimo ◽  
Eunice M. Machuka ◽  
Edward O. Abworo ◽  
Anastasia N. Vlasova ◽  
Roger Pelle
Keyword(s):  
Genetic Diversity ◽  
Nucleotide Sequence ◽  
Epitope Prediction ◽  
Whole Genome Sequence ◽  
Diagnostic Tools ◽  
Whole Genome ◽  
Potential Candidate ◽  
Whole Genome Analysis ◽  
Porcine Astrovirus ◽  
Sequence Identities

Astroviruses (AstVs) are widely distributed and are associated with gastroenteritis in human and animals. The knowledge of the genetic diversity and epidemiology of AstVs in Africa is limited. This study aimed to characterize astroviruses in asymptomatic smallholder piglets in Kenya and Uganda. Twenty-four samples were randomly selected from a total of 446 piglets aged below 6 months that were initially collected for rotavirus study and sequenced for whole genome analysis. Thirteen (13/24) samples had contigs with high identity to genus Mamastrovirus. Analysis of seven strains with complete (or near complete) AstV genome revealed variable nucleotide and amino acid sequence identities with known porcine astrovirus (PoAstV) strains. The U083 and K321 strains had nucleotide sequence identities ranging from 66.4 to 75.4% with the known PoAstV2 strains; U460 strain had nucleotide sequence identities of 57.0 to 65.1% regarding the known PoAstV3; and K062, K366, K451, and K456 strains had nucleotide sequence identities of 63.5 to 80% with the known PoAstV4 strains. The low sequence identities (<90%) indicate that novel genotypes of PoAstVs are circulating in the study area. Recombination analysis using whole genomes revealed evidence of multiple recombination events in PoAstV4, suggesting that recombination might have contributed to the observed genetic diversity. Linear antigen epitope prediction and a comparative analysis of capsid protein of our field strains identified potential candidate epitopes that could help in the design of immuno-diagnostic tools and a subunit vaccine. These findings provide new insights into the molecular epidemiology of porcine astroviruses in East Africa.

scholarly journals Whole-Genome Sequence of Chlamydia gallinacea Type Strain 08-1274/3

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Martin Hölzer ◽  
Karine Laroucau ◽  
Heather Huot Creasy ◽  
Sandra Ott ◽  
Fabien Vorimore ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Type Strain ◽  
Bacterial Species ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Protein Coding ◽  
Coding Sequences ◽  
Domestic Poultry ◽  
Avian Pathogen

The recently introduced bacterial species Chlamydia gallinacea is known to occur in domestic poultry and other birds. Its potential as an avian pathogen and zoonotic agent is under investigation. The whole-genome sequence of its type strain, 08-1274/3, consists of a 1,059,583-bp chromosome with 914 protein-coding sequences (CDSs) and a plasmid (p1274) comprising 7,619 bp with 9 CDSs.

scholarly journals Genes Involved in the Synthesis and Degradation of Matrix Polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae Biofilms

2004 ◽  
Vol 186 (24) ◽  
pp. 8213-8220 ◽  
Author(s):  
Jeffrey B. Kaplan ◽  
Kabilan Velliyagounder ◽  
Chandran Ragunath ◽  
Holger Rohde ◽  
Dietrich Mack ◽  
...  
Keyword(s):  
Glycosyl Hydrolase ◽  
Bacterial Species ◽  
Extracellular Polysaccharide ◽  
Actinobacillus Pleuropneumoniae ◽  
Intercellular Adhesion ◽  
Actinobacillus Actinomycetemcomitans ◽  
Respiratory Pathogen ◽  
Bacterial Cells ◽  
Abiotic Surfaces ◽  
Biofilm Matrix

ABSTRACT Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-d-glucosamine residues in β(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.

Sign in / Sign up

Export Citation Format

Share Document