scholarly journals Man/Cel5B, a Bifunctional Enzyme Having the Highest Mannanase Activity in the Hyperthermic Environment

2021 ◽  
Vol 9 ◽  
Author(s):  
Beenish Sadaqat ◽  
Chong Sha ◽  
Parveen Fatemeh Rupani ◽  
Hongcheng Wang ◽  
Wanbing Zuo ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Methyl Cellulose ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Mannanase Activity ◽  
Encoding Protein ◽  
Layer Chromatography ◽  
Carboxy Methyl

Thermotoga maritima (Tma) contains genes encoding various hyperthermophilic enzymes with great potential for industrial applications. The gene TM1752 in Tma genome has been annotated as cellulase gene encoding protein Cel5B. In this work, the gene TM1752 was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Interestingly, the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxy methyl cellulose, which is the highest among thermophilic mannanases. However, the putative enzyme did not show sequence homology with any of the previously reported mannanases; therefore, the enzyme Cel5B was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B. Man/Cel5B exhibited maximum activity at 85°C and pH 5.5. This enzyme retained more than 50% activity after 5 h of incubation at 85°C, and retained up to 80% activity after incubated for 1 h at pH 5–8. The Km and Vmax of Man/Cel5B were observed to be 4.5 mg/mL galactomannan and 769 U/mg, respectively. Thin layer chromatography depicted that locust bean gum could be efficiently degraded to mannobiose, mannotriose, and mannooligosaccharides by Man/Cel5B. These characteristics suggest that Man/Cel5B has attractive applications for future food, feed, and biofuel industries.

scholarly journals Prospects of Biocatalyst Purification Enroute Fermentation Processes

2021 ◽  
Author(s):  
Michael Bamitale Osho ◽  
Sarafadeen Olateju Kareem
Keyword(s):  
Fermentation Process ◽  
Colloidal Particles ◽  
Methyl Cellulose ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Inorganic Materials ◽  
Organic Substrates ◽  
Ammonium Sulphate Precipitation ◽  
Sodom Apple ◽  
Microbial Agents

Biotransformation of broth through fermentation process suffers a major setback when it comes to disintegration of organic substrates by microbial agents for industrial applications. These biocatalysts are in crude/dilute form hence needs to be purified to remove colloidal particles and enzymatic impurities thus enhancing maximum activity. Several contractual procedures of concentrating dilute enzymes and proteins had been reported. Such inorganic materials include ammonium sulphate precipitation; salting, synthetic polyacrylic acid; carboxy-methyl cellulose, tannic acid, edible gum and some organic solvents as precipitants etc. The emergence of organic absorbents such as sodom apple (Calostropis procera) extract, activated charcoal and imarsil had resulted in making significant impact in industrial circle. Various concentrations of these organic extracts have been used as purifying agents on different types of enzyme vis: lipase, amylase, protease, cellulase etc. Purification fold and stability of the enzyme crude form attained unprecedented results.

scholarly journals Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis

2010 ◽  
Vol 192 (6) ◽  
pp. 1624-1633 ◽  
Author(s):  
Chantal Fernandes ◽  
Vitor Mendes ◽  
Joana Costa ◽  
Nuno Empadinhas ◽  
Carla Jorge ◽  
...  
Keyword(s):  
Sequence Homology ◽  
Thermotoga Maritima ◽  
Functional Characterization ◽  
Compatible Solute ◽  
Homologous Gene ◽  
Gene Encoding ◽  
Alternative Pathways ◽  
Cell Extracts ◽  
Genes Encoding

ABSTRACT The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.

scholarly journals Colocation of Genes Encoding a tRNA-mRNA Hybrid and a Putative Signaling Peptide on Complementary Strands in the Genome of the Hyperthermophilic Bacterium Thermotoga maritima

2006 ◽  
Vol 188 (19) ◽  
pp. 6802-6807 ◽  
Author(s):  
Clemente I. Montero ◽  
Derrick L. Lewis ◽  
Matthew R. Johnson ◽  
Shannon B. Conners ◽  
Elizabeth A. Nance ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Normal Growth ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Transcriptional Responses ◽  
Gene Encoding ◽  
Hyperthermophilic Bacterium ◽  
Genes Encoding ◽  
Original Genome ◽  
Opposite Strand

ABSTRACT In the genome of the hyperthermophilic bacterium Thermotoga maritima, TM0504 encodes a putative signaling peptide implicated in population density-dependent exopolysaccharide formation. Although not noted in the original genome annotation, TM0504 was found to colocate, on the opposite strand, with the gene encoding ssrA, a hybrid of tRNA and mRNA (tmRNA), which is involved in a trans-translation process related to ribosome rescue and is ubiquitous in bacteria. Specific DNA probes were designed and used in real-time PCR assays to follow the separate transcriptional responses of the colocated open reading frames (ORFs) during transition from exponential to stationary phase, chloramphenicol challenge, and syntrophic coculture with Methanococcus jannaschii. TM0504 transcription did not vary under normal growth conditions. Transcription of the tmRNA gene, however, was significantly up-regulated during chloramphenicol challenge and in T. maritima bound in exopolysaccharide aggregates during methanogenic coculture. The significance of the colocation of ORFs encoding a putative signaling peptide and tmRNA in T. maritima is intriguing, since this overlapping arrangement (tmRNA associated with putative small ORFs) was found to be conserved in at least 181 bacterial genomes sequenced to date. Whether peptides related to TM0504 in other bacteria play a role in quorum sensing is not yet known, but their ubiquitous colocalization with respect to tmRNA merits further examination.

scholarly journals Expression and Characterization ofGeobacillus stearothermophilusSR74 Recombinantα-Amylase inPichia pastoris

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Sivasangkary Gandhi ◽  
Abu Bakar Salleh ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Siti Nurbaya Oslan
Keyword(s):  
Specific Activity ◽  
Thermophilic Bacteria ◽  
Recombinant Expression ◽  
Expression System ◽  
Alcohol Oxidase ◽  
Product Yield ◽  
Inducible Promoter ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Gene Encoding

Geobacillus stearothermophilusSR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactiveα-amylase. Increased production and commercialization of thermostableα-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostableα-amylase inG. stearothermophilusSR74 was amplified, sequenced, and subcloned intoP. pastorisGS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinantα-amylase SR74 achieved in shake flask was 28.6 U mL−1at 120 h after induction. The recombinant 59 kDaα-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH ofα-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostableα-amylase SR74 fromG. stearothermophilusSR74 would be beneficial for industrial applications, especially in liquefying saccrification.

DPV UL41 gene encoding protein induces host shutoff activity and affects viral replication

2021 ◽  
Vol 255 ◽  
pp. 108979
Author(s):  
Tianqiong He ◽  
Mingshu Wang ◽  
Anchun Cheng ◽  
Qiao Yang ◽  
Renyong Jia ◽  
...  
Keyword(s):  
Viral Replication ◽  
Gene Encoding ◽  
Host Shutoff ◽  
Encoding Protein

scholarly journals Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 427
Author(s):  
Yoshiko Okamura ◽  
Hirokazu Takahashi ◽  
Atsuyuki Shiida ◽  
Yuto Hirata ◽  
Haruko Takeyama ◽  
...  
Keyword(s):  
Immune System ◽  
Genomic Sequence ◽  
Maximum Activity ◽  
Associated Bacteria ◽  
Metagenome Library ◽  
Peptide Synthetase ◽  
Sponge Associated Bacteria ◽  
Genes Encoding ◽  
Chemotactic Factors ◽  
By Products

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 104 clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 999-1007
Author(s):  
R G Gregerson ◽  
L Cameron ◽  
M McLean ◽  
P Dennis ◽  
J Strommer
Keyword(s):  
Chromosomal Location ◽  
Higher Plants ◽  
Gene Encoding ◽  
Genomic Libraries ◽  
Regulatory Strategy ◽  
Adh1 Gene ◽  
Adh Genes ◽  
Genes Encoding ◽  
Adh Gene ◽  
Polymerase Chain

Abstract In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

scholarly journals Non-Syndromic Dentinogenesis Imperfecta Caused by Mild Mutations in COL1A2

2021 ◽  
Vol 11 (6) ◽  
pp. 526
Author(s):  
Yejin Lee ◽  
Youn Jung Kim ◽  
Hong-Keun Hyun ◽  
Jae-Cheoun Lee ◽  
Zang Hee Lee ◽  
...  
Keyword(s):  
Collagen Type ◽  
Molecular Genetic ◽  
Collagen Type I ◽  
Type I ◽  
Dentinogenesis Imperfecta ◽  
Gene Encoding ◽  
Korean Families ◽  
Whole Exome ◽  
Genes Encoding ◽  
Candidate Gene Sequencing

Hereditary dentin defects can be categorized as a syndromic form predominantly related to osteogenesis imperfecta (OI) or isolated forms without other non-oral phenotypes. Mutations in the gene encoding dentin sialophosphoprotein (DSPP) have been identified to cause dentinogenesis imperfecta (DGI) Types II and III and dentin dysplasia (DD) Type II. While DGI Type I is an OI-related syndromic phenotype caused mostly by monoallelic mutations in the genes encoding collagen type I alpha 1 chain (COL1A1) and collagen type I alpha 2 chain (COL1A2). In this study, we recruited families with non-syndromic dentin defects and performed candidate gene sequencing for DSPP exons and exon/intron boundaries. Three unrelated Korean families were further analyzed by whole-exome sequencing due to the lack of the DSPP mutation, and heterozygous COL1A2 mutations were identified: c.3233G>A, p.(Gly1078Asp) in Family 1 and c.1171G>A, p.(Gly391Ser) in Family 2 and 3. Haplotype analysis revealed different disease alleles in Families 2 and 3, suggesting a mutational hotspot. We suggest expanding the molecular genetic etiology to include COL1A2 for isolated dentin defects in addition to DSPP.

scholarly journals CAU-1, a Subclass B3 Metallo-β-Lactamase of Low Substrate Affinity Encoded by an Ortholog Present in the Caulobacter crescentus Chromosome

2002 ◽  
Vol 46 (6) ◽  
pp. 1823-1830 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Fabrizio Pantanella ◽  
Francesco Giuliani ◽  
Maria Cristina Thaller ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Hypothetical Protein ◽  
Exchange Chromatography ◽  
Substrate Affinity ◽  
Gene Encoding ◽  
Native Form ◽  
Significant Similarity ◽  
Expression Studies ◽  
Genes Encoding ◽  
Isoelectric Ph

ABSTRACT The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Km values were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, the bla CAU determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the class Proteobacteria.

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