Characterization and functional insights of the novel RC-type plasmid pAnox1 from Anoxybacillus gonensis 05S15.

The plasmid pAnox1, isolated from Anoxybacillus gonensis 05S15, was sequenced and characterized as a circular, double-stranded DNA molecule of 1592 base pairs with a GC content of 40.01 %. Despite its cryptic nature and small genome, bioinformatic analyses identified conserved motifs associated with replication-related proteins, though BLAST searches revealed no significant homology with other plasmids. The plasmid genome contains five putative Open Reading Frames (ORFs), four palindromic sequences, and two direct repeats on both strands, suggesting regulatory roles. Electron microscopy and Southern hybridization studies confirmed that pAnox1 follows a Rolling Circle (RC) replication mode. The study further demonstrated that the plasmid encodes three distinct transcripts: ORF-1 and ORF-3 are oriented in the same direction, while ORF-5 is on the opposite strand. RACE and LACE analyses revealed transcript lengths of 903 bp for ORF1, 499 bp for ORF3, and 211 bp for ORF5. Quantitative real-time PCR estimated the relative copy number of pAnox1 at 127 ± 2 copies per chromosomal equivalent. This novel RC-type plasmid in the Anoxybacillus genome holds promise as a cloning and expression vector for biotechnological applications and in vivo protein engineering.

Genome-Based Reclassification of Anoxybacillus geothermalis Filippidou et al. 2016 as a Later Heterotypic Synonym of Anoxybacillus rupiensis Derekova et al. 2007.

In this study, our aim was to elucidate the relationship between Anoxybacillus rupiensis DSM 17127T and Anoxybacillus geothermalis GSsed3T through whole-genome phylogenetic analysis. The obtained 16S rRNA gene sequence from the genome of A. rupiensis DSM 17127T exhibited a 99.8% similarity with A. geothermalis GSsed3T. In the phylogenetic trees constructed using whole-genome sequences and 16S rRNA gene sequences, A. rupiensis DSM 17127T and A. geothermalis GSsed3T were observed to form a clade, indicating a close relationship between them. Moreover, the average amino acid identity, average nucleotide identity, and digital DNA-DNA hybridization values calculated between A. rupiensis DSM 17127T and A. geothermalis GSsed3T exceeded the threshold values typically used for species demarcation. Furthermore, the phylogenomic analysis based on the core genome of the strains in question provided additional support for the formation of a monophyletic clade by A. rupiensis DSM 17127T and A. geothermalis GSsed3T. Most phenotypic and chemotaxonomic features between both strains were almost identical except for a few exceptions. These findings suggest that both strains should be classified as belonging to the same species, and we propose that A. geothermalis GSsed3T is a later heterotypic synonym of A. rupiensis DSM 17127T.

Genome-based reclassification of Anoxybacillus salavatliensis Cihan et al. 2011 as a later heterotypic synonym of Anoxybacillus gonensis Belduz et al. 2003.

In the present study, we aim to clarify the taxonomic positions of Anoxybacillus salavatliensis DSM 22626T and Anoxybacillus gonensis G2T by using whole genome phylogenetic analysis, biochemical and chemotaxonomic characteristics. The genome sequences of A. salavatliensis DSM 22626T was not available in any database, so it was sequenced in this study. In phylogenetic trees drawn using whole genome sequences and 16S rRNA gene sequences, A. salavatliensis DSM 22626T and A. gonensis G2T clade together and showed high sequence similarity (99.3%) based on 16S rRNA gene. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between A. salavatliensis DSM 22626T and A. gonensis G2T were found to be greater than the threshold values for species demarcation. Further, the phylogenomic analysis based on the core genome of the strains under study confirmed that A. salavatliensis DSM 22626T and A. gonensis G2T formed a monophyletic clade. Most phenotypic and chemotaxonomic features between both strains were almost identical except for a few exceptions. The present results show that A. salavatliensis DSM 22626T is a later heterotypic synonym of A. gonensis G2T.

1,3,4-Thiadiazole and 1,2,4-triazole-5-thione derivatives bearing 2-pentyl-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-one ring: Synthesis, molecular docking, urease inhibition, and crystal structure.

Two series of 1,3,4-thiadiazole (40a-o) and 1,2,4-triazole-5-thione (41a-l) derivatives bearing a 2-pentyl-5-phenyl-1,2,4-triazole-3-one ring were synthesized and then studied for their urease inhibitory activities using thiourea as a standard drug. Among the two groups, the first group (40a-o) did not show good activity while the second group (41a-l) showed excellent activity. Compound 41j (1091.24 ± 14.02 µM) of the second series of compounds showed lower activity than thiourea, while the remaining 11 compounds (41a-i, k, and l) showed better activity than thiourea (183.92 ± 13.14 µM). Among the 11 compounds, 41b (15.96 ± 2.28 µM) having the 3-F group on the phenyl ring showed the highest inhibitory activity. Urease kinetic studies of 41b, which is the most active compound, determined it to have an un-competitive inhibition potential. Moreover, in silico analysis against urease from jack bean with 27 new heterocyclic compounds and the reference molecule was carried out to see the necessary interactions responsible for urease activity. The docking calculations of all compounds supported stronger binding to the receptor than the reference molecule, with high inhibition constants. In addition, compound 40m was characterized by single-crystal X-ray diffraction analysis. X-ray analysis reveals that the structures of the compound 40m crystallize in the monoclinic P21/c space group with the cell parameters: a = 10.2155(9) Å, b = 22.1709(18) Å, c = 21.4858(17) Å, ß = 99.677(8)°, V = 4797.0(7) Å3 . X-ray diffraction analyses were also performed to gain insights into the role of weak intermolecular interactions and C-H…X (halogen) interactions in compound 40m that influence the crystal packing.

Genome-based reclassification of Anoxybacillus kamchatkensis Kevbrin et al. 2005 as a later heterotypic synonym of Anoxybacillus ayderensis Dulger et al. 2004.

In this study, we aimed to clarify the taxonomic positions of Anoxybacillus kamchatkensis DSM 14988T and Anoxybacillus ayderensis AB04T using whole-genome phylogenetic analysis, biochemical and chemotaxonomic characteristics. In phylogenetic trees drawn using whole-genome sequences and 16S rRNA gene sequences, A. kamchatkensis DSM 14988T and A. ayderensis AB04T clade together and showed high sequence similarity (99.6%) based on 16S rRNA gene. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between A. kamchatkensis DSM 14988T and A. ayderensis AB04T were found to be greater than the threshold values for species demarcation. Most phenotypic and chemotaxonomic features between both species were almost identical except for a few exceptions. The present results show that A. kamchatkensis DSM 14988T is a later heterotypic synonym of A. ayderensis AB04T.

Genome-based reclassification of Anoxybacillus karvacharensis Panosyan et al. as a later heterotypic synonym of Anoxybacillus kestanbolensis Dulger et al. 2004; A. flavithermus subsp. yunnanensis CCTCC AB2010187T Dai et al. 2011 as a later heterotypic synonym of A. tengchongensis DSM 23211T Zhang et al. 2011.

In the present study, we attempted to clarify the taxonomic positions of Anoxybacillus karvacharensis K1T, Anoxybacillus kestanbolensis NCIMB 13971T, Anoxybacillus flavithermus subsp. yunnanensis CCTCC AB2010187T, and Anoxybacillus tengchongensis DSM 23211T using whole-genome phylogenetic analysis. The genome sequence of A. kestanbolensis NCIMB13971T was not available in any database, so it was sequenced in this study. The 16S rRNA gene sequence obtained from the genome of A. kestanbolensis NCIMB13971T had 99.93% similarity with A. karvacharensis K1T. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (DDH) values between A. karvacharensis K1T and A. kestanbolensis NCIMB13971T and between A. flavithermus subsp. yunnanensis CCTCCAB 2010187T and A. tengchongensis DSM 23211T were greater than the threshold values for species demarcation. The present results indicate that A. karvacharensis K1T is a later heterotypic synonym of A. kestanbolensis NCIMB13971T; A. flavithermus subsp. yunnanensis CCTCCAB 2010187T is a later heterotypic synonym of A. tengchongensis DSM 23211T.

Investigation of potential inhibitor properties of violacein against HIV-1 RT and CoV-2 Spike RBD:ACE-2.

A violacein-producing bacterium was isolated from a mud sample collected near a hot spring on Kümbet Plateau in Giresun Province and named the GK strain. According to the phylogenetic tree constructed using 16S rRNA gene sequence analysis, the GK strain was identified and named Janthinobacterium sp. GK. The crude violacein pigments were separated into three different bands on a TLC sheet. Then violacein and deoxyviolacein were purified by vacuum liquid column chromatography and identified by NMR spectroscopy. According to the inhibition studies, the HIV-1 RT inhibition rate of 1 mM violacein from the GK strain was 94.28% and the CoV-2 spike RBD:ACE2 inhibition rate of 2 mM violacein was 53%. In silico studies were conducted to investigate the possible interactions between violacein and deoxyviolacein and three reference molecules with the target proteins: angiotensin-converting enzyme 2 (ACE2), HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain. Ligand violacein binds strongly to the receptor ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of -9.94 kcal/mol, -9.32 kcal/mol, and -8.27 kcal/mol, respectively. Deoxyviolacein strongly binds to the ACE2, HIV-1 reverse transcriptase, and SARS-CoV-2 spike receptor binding domain with a binding energy of -10.38 kcal/mol, -9.50 kcal/mol, and -8.06 kcal/mol, respectively. According to these data, violacein and deoxyviolacein bind to all the receptors quite effectively. SARS-CoV-2 spike protein and HIV-1-RT inhibition studies with violacein and deoxyviolacein were performed for the first time in the literature.

Investigation of potential inhibitor properties of ethanolic propolis extracts against ACE-II receptors for COVID-19 treatment by molecular docking study.

The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE-II, is a type I integral membrane protein of 805 amino acids that contains 1 HEXXH-E zinc binding consensus sequence. ACE-II has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). In this study, the potential of some flavonoids presents in propolis to bind to ACE-II receptors was calculated with in silico. Binding constants of ten flavonoids, caffeic acid, caffeic acid phenethyl ester, chrysin, galangin, myricetin, rutin, hesperetin, pinocembrin, luteolin and quercetin were measured using the AutoDock 4.2 molecular docking program. And also, these binding constants were compared to reference ligand of MLN-4760. The results are shown that rutin has the best inhibition potentials among the studied molecules with high binding energy - 8.04 kcal/mol, and it is followed by myricetin, quercetin, caffeic acid phenethyl ester and hesperetin. However, the reference molecule has binding energy of - 7.24 kcal/mol. In conclusion, the high potential of flavonoids in ethanolic propolis extracts to bind to ACE-II receptors indicates that this natural bee product has high potential for COVID-19 treatment, but this needs to be supported by experimental studies.

Biochemical characterization of a novel thermostable feruloyl esterase from Geobacillus thermoglucosidasius DSM 2542T.

The ferulic acid esterase (FAE) gene from Geobacillus thermoglucosidasius DSM 2542T was cloned into pET28a(+) expression vector and characterized and is being reported in this study for the first time in Geobacillus. The enzyme, designated as GthFAE, was purified by heat shock and ion-exchange column chromatography. In addition, a second clone containing a Histidine tag was expressed and purified by affinity column chromatography demonstrating future potential for scale-up. FAE gene contains an open reading frame (ORF) of 759-bp encoding a hypothetical 252 amino acid protein, a molecular mass of 28.11 kDa and an isoelectric point of 5.53. From this study it was found that GthFAE had optimal activity at 50 °C and pH of 8.5. Furthermore, the enzyme has been found to retain 64% of its activity after two days incubation at 50 °C and exhibited a high level of functionality with p-nitrophenyl caprylate (C8). Km, Vmax, kcat and kcat/Km values for p-nitrophenyl caprylate were determined as 0.035 mM, 11,735 µmol/min/mg protein, 5491 (1/s) and 156,885 s-1 mM-1 respectively. The combination of higher activity and stability compared to previously reported FAEs makes GthFAE a potential candidate for use in the paper manufacturing industry.

Improved pulp bleaching potential of Bacillus subtilis WB800 through overexpression of three lignolytic enzymes from various bacteria.

A chemical bleaching process of paper pulps gives off excessive amount of chlorinated organic wastes mostly released to environment without exposing complete bioremediaton. Recent alternative and eco-friendly approaches toward pulp bleaching appear more responsive to environmental awareness. Here we report, direct use of a recombinant Bacillus subtilis bacterium for pulp bleaching, endowed with three ligninolytic enzymes from various bacteria. In addition, efficient bleaching performance from glutathione-S-transferase (GST) biocatalyst tested for the first time in pulp bleaching applications was also achieved. Simultaneous and extracellular overproduction of highly active GST, laccase, and lignin peroxidase catalysts were also performed by Bacillus cells. Both enhanced bleaching success and improved delignification rates were identified when enzyme combinations tested on both pine kraft and waste paper pulps, ranging from 69.75% to 79.18% and 60.89% to 74.65%, respectively. Furthermore, when triple enzyme combination applied onto the papers from pine kraft and waste pulps, the best ISO brightness values were identified as 66.45% and 64.67%, respectively. The delignification rates of pulp fibers exposed to various enzymatic bleaching sequences were comparatively examined under SEM. In conclusion, the current study points out that in near future, a more fined-tuned engineering of pulp-colonizing bacteria may become a cost-effective and environmentally friendly alternative to chemical bleaching.

Thermus anatoliensis sp. nov., a thermophilic bacterium from geothermal waters of Buharkent, Turkey.

A Gram-stain-negative, lack of motility, catalase- and oxidase- positive bacterium (strain MT1(T)) was isolated from Buharkent hot spring in Aydin, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain was able to grow at 45-80 °C, pH 5.5-10.5 and with a NaCI tolerance up to 2.0% (w/v). Strain MT1(T) was able to utilize d-mannitol and l-arabinose, not able to utilize d-cellobiose as sole carbon source. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Thermus; strain MT1(T) detected low-level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. The predominant fatty acids of strain MT1(T) were iso-C(15:0) (43.0%) and iso-C(17:0) (27.4%). Polar lipid analysis revealed a major phospholipid, one major glycolipid, one major aminophospholipid, two minor aminolipids, one minor phospholipid, and several minor glycolipids. The major isoprenoid quinone was MK-8. The DNA G+C content of MT1(T) was 69.6 mol%. On the basis of a taxonomic study using a polyphasic approach, strain MT1(T) is considered to represent a novel species of the genus Thermus, for which the name Thermus anatoliensis sp. nov. is proposed. The type strain is MT1(T) (=NCCB 100425(T) =LMG 26880(T)).

Bis-1,2,4-triazol derivatives: Synthesis, characterization, DFT, antileishmanial activity and molecular docking study.

In this study, triazol derivatives, 4,4'-(((1E, 1E')-1,2-phenylenebis (methanylyidene)) bis (azanylidene)) bis (5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (2), 4,4'-(((1E, 1E')-1,3-phenylenebis (methanylyidene)) bis (azanylidene)) bis (5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (3) and 4,4'-(((1E, 1E')-1,4-phenylene bis (methanyl yidene)) bis (azanylidene)) bis (5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (4) were synthesized from the reaction of 4-amino-5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one and phthalaldehyde/isophthalaldehyde/terephthalaldehyde, respectively. Compounds 2-4 were characterized by Fourier transform infrared (FTIR), proton and carbon-13 nuclear magnetic resonance (1H- and 13C- NMR) spectroscopic methods. Theoretical study for compounds 2-4 were carried out by DFT/B3LYP/6-311++G(d,p). Structural and spectroscopic parameters were determined theoreticaly and compared with experimental ones. Also, the molecular electrostatic potential (MEP) maps of compounds were obtained. Leishmanicidal activity of compounds 2-4 against to Leishmania infantum was determined by microdilution broth method containing alamar blue. As a result of the study, compounds 2-4 were found to be effective against the specie of Leishmania. Molecular docking analysis against Trypanothione Reductase (TRe) with compound 2 was carried out to see the necessary interactions responsible for antileishmanial activity. The docking calculations of compound 2 supported the antileishmanial activity exhibiting high inhibition constant.Communicated by Ramaswamy H. Sarma.

HIV-1-RT inhibition activity of Satureja spicigera (C.KOCH) BOISS. Aqueous extract and docking studies of phenolic compounds identified by RP-HPLC-DAD.

AIDS is a global disease caused by HIV, affecting millions of people and causing death. The current limitations of antiretroviral therapy used in the therapy of HIV/AIDS have led to the need to search for new and effective drugs from natural products, especially plants. Herewith, using the present study, the detection of HIV-1-RT inhibition of aqueous extract of Satureja spicigera (C.KOCH) BOISS. was performed for the first time. Besides, total phenolic content (TPC), analysis of phenolic constituents by RP-HPLC-DAD and antioxidant capacity by DPPH and Ferric reducing antioxidant power (FRAP) methods were determined for the first time. In addition, molecular docking studies were carried out between HIV-1-RT and phenolic substances, the presence of which was determined in the aqueous extract, for the determination of the phenolics that may be responsible for HIV-1-RT activity. HIV-1-RT inhibition was defined as IC50 : 22.83 µg/ml. Benzoic acid, vanillin, rutin, and chlorogenic acid were present as main phenolics in quantities of 621.96, 505.87, 349.33, and 323.23 µg phenolic/g extract, respectively. Further, TPC, DPPH, and FRAP were calculated as in the order of 151.69 mg GAE/g extract, 23.77 µg/ml, and 445.7 µmol TE/g extract. Chlorogenic acid (-8.48 kcal/mol) was found to be the most effective ligand in docking studies, with a value close to positive standard nevirapine (-9.35 kcal/mol). Hereby, although the aqueous extract of S. spicigera can be used as a natural antioxidant, the crude extract or its phenolics have the potential to be used in the treatment of AIDS due to its high HIV-1-RT activity. PRACTICAL APPLICATIONS: In this study, anti-HIV-1-RT and antioxidant activity and total phenolic content of Satureja spicigera aqueous extract were determined. In addition, HPLC analysis of some phytochemicals and the activities of these phytochemicals against HIV-1-RT enzyme was determined by molecular docking studies. The results showed that the aqueous extract of S. spicigera and some of the phytochemicals it contains have the potential to be used as a natural product against HIV infection or in the treatment of AIDS.

New chalcone derivative, ethyl 2-(4-(3-(benzo[b]thiophen-2-yl)acryloyl)phenoxy)acetate: synthesis, characterization, DFT study, enzyme inhibition activities and docking study.

Chalcone derivative, ethyl 2-(4-(3-(benzo[b]thiophen-2yl)acryloyl)phenoxy)acetate (I), was synthesized. Compound I was characterized by proton and carbon-13 nuclear magnetic resonance (1H- and 13C- NMR), fourier transform infrared (FTIR) and mass (LC-ESI-MS/MS) spectroscopic methods. Density Functional Theory (DFT) calculations for compound I were performed at B3LYP/6-311++G(d,p) level. Optimized geometry, frontier molecular orbitals (HOMO; highest occupied molecular orbital; LUMO: lowest unoccupied molecular orbital), IR and NMR parameters of compound I were obtained. The evaluations reveal that the calculation results support the experimental results. The inhibition effects of compound I on cholinesterases and GST enzyme were investigated. Ki and inhibition concentration (IC50) values were calculated separately. Ki values of compound I were found for GST 14.19 ± 2.15, for AChE 11.13 ± 1.22 and for BChE 8.74 ± 0.76 recpectively. The docking analysis of compound I supported the enzym inhibition activity exhibiting high inhibition constant and binding energy for three receptors. Compound I is strongly bound to AChE, huBChE and Glutathione S-transferase with binding energies -11.24, -8.56 and -10.39 kcal/mol, respectively.Communicated by Ramaswamy H. Sarma.

3-(5-(1H-imidazol-1-yl) pent-1-en-1-yl)-9-ethyl-9H-carbazole: synthesis, characterization (IR, NMR), DFT, antimicrobial-antioxidant activities and docking study.

3-(5-(1H-imidazol-1-yl) pent-1-en-1-yl)-9-ethyl-9H-carbazole called as compound 1 was synthesized and characterized by proton and carbon-13 nuclear magnetic resonance (1H- and 13C- NMR) and Fourier transform infrared (FTIR) spectroscopic methods. Density Functional Theory/Becke, 3-parameter (DFT/B3LYP), for compound 1 were performed with 6-311++G(d,p) method. Optimized geometry, frontier molecular orbitals (HOMO; highest occupied molecular orbital; LUMO: lowest unoccupied molecular orbital), IR and NMR parameters of compound 1 were obtained. The evaluations reveal that the calculation results support the experimental results. In addition, the antimicrobial (a microwell dilution method) and antioxidant activities (2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) ferric ion reducing antioxidant power (FRAP) of compound 1 were evaluated. According to the results obtained, it showed higher antimicrobial activity (Minimal inhibition concentration (MIC): 78.12 µg/mL) against B. subtilis subsp. Spizizenii. Morever, molecular docking studies were carried out to investigate the interactions of an antimicrobial agent on some important enzymes played important roles in nucleic acid (Deoxyribo nucleic acid (DNA) synthesis, cell wall synthesis, protein synthesis, and metabolism etc. The compound 1 was strongly bound to tyrosyl-tRNA synthetase enzyme (binding energy: -11.18 and Ki: 6.37 nM) and Beta-Ketoacyl-Acp Synthase III enzyme (binding energy: -10.29 and Ki: 28.47 nM).Communicated by Ramaswamy H. Sarma.

Targeting CoV-2 spike RBD and ACE-2 interaction with flavonoids of Anatolian propolis by in silico and in vitro studies in terms of possible COVID-19 therapeutics.

Propolis is a multi-functional bee product rich in polyphenols. In this study, the inhibitory effect of Anatolian propolis against SARS-coronavirus-2 (SARS-CoV-2) was investigated in vitro and in silico. Raw and commercial propolis samples were used, and both samples were found to be rich in caffeic acid, p-coumaric acid, ferulic acid, t-cinnamic acid, hesperetin, chrysin, pinocembrin, and caffeic acid phenethyl ester (CAPE) at HPLC-UV analysis. Ethanolic propolis extracts (EPE) were used in the ELISA screening test against the spike S1 protein (SARS-CoV-2): ACE-2 interaction for in vitro study. The binding energy values of these polyphenols to the SARS-CoV-2 spike and ACE-2 protein were calculated separately with a molecular docking study using the AutoDock 4.2.6 program. In addition, the pharmacokinetics and drug-likeness properties of these eight polyphenols were calculated according to the SwissADME tool. The binding energy value of pinocembrin was highest in both receptors, followed by chrysin, CAPE, and hesperetin. Based on the in silico modeling and ADME (absorption, distribution, metabolism, and excretion) behaviors of the eight polyphenols, the compounds exhibited the potential ability to act effectively as novel drugs. The findings of both studies showed that propolis has a high inhibitory potential against the Covid-19 virus. However, further studies are now needed.

Cloning, expression, biochemical characterization, and molecular docking studies of a novel glucose tolerant ß-glucosidase from Saccharomonospora sp. NB11.

Most of the presently known ß-glucosidases are sensitive to end-product inhibition by glucose, restricting their potential use in many industrial applications. Identification of novel glucose tolerant ß-glucosidase can prove a pivotal solution to eliminate end-product inhibition and enhance the overall lignocellulosic saccharification process. In this study, a novel gene encoding ß-glucosidase BglNB11 of 1405bp was identified in the genome of Saccharomonospora sp. NB11 and was successfully cloned and heterologously expressed in E. coli BL21 (DE3).The presence of conserved amino acids; NEPW and TENG indicated that BglNB11 belonged to GH1 ß-glucosidases. The recombinant enzyme was purified using a Ni-NTA column, with the molecular mass of 51 kDa, using SDS-PAGE analysis. BglNB11 showed optimum activity at 40 °C and pH 7 and did not require any tested co-factors for activation. The kinetic values, Km, Vmax, kcat, and kcat/Km of purified enzyme were 0.4037 mM, 5735.8 µmol/min/mg, 5042.16 s-1 and 12487.71 s-1 mM-1, respectively. The enzyme was not inhibited by glucose to a concentration of 4 M but was slightly stimulated in the presence of glucose. Molecular docking of BglNB11 with glucose suggested that the relative binding position of glucose in the active site channel might be responsible for modulating end product tolerance and stimulation. ß-glucosidase from BglNB11 is an excellent enzyme with high catalytic efficiency and enhanced glucose tolerance compared to many known glucose tolerant ß-glucosidases. These unique properties of BglNB11 make it a prime candidate to be utilized in many biotechnological applications.

Cloning, biochemical characterization and molecular docking of novel thermostable ß-glucosidase BglA9 from Anoxybacillus ayderensis A9 and its application in de-glycosylation of Polydatin.

This study reports a novel BglA9 gene of 1345 bp encoding ß-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using pNPG as substrate were Km (0.28 mM), Vmax (43.8 µmol/min/mg), kcat (38.43 s-1) and kcat/Km (135.5 s-1 mM-1). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 µmol/min/mg, 18.28 s-1and 3.27 s-1 mM-1 for Km, Vmax, kcat, and kcat/Km values, respectively. The Ki value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin.

A novel cryptic and theta type plasmid (pHIG22) from Thermus scodotuctus sp. K6.

A cryptic plasmid pHIG22 from Thermus scotoductus sp. K6, an isolate from the Alangullu Hot Spring (Aydin, Turkey), was sequenced and characterized. The pHIG22 plasmid is a multicopy, double stranded and 2222 bp circular molecule with 62.78% GC content, which shows a characteristical nucleotide sequence without any homology to other known plasmids. Five open reading frames were predicted based on the nucleotide sequence analysis. The deduced amino acid sequence of all predicted ORFs didn't show any similarity with any known proteins. Three palindroms were detected and two promoter sequences were predicted in both strands. With electron microscopy (TEM) analysis, the replication intermediates were seen as typical Q-shaped molecules that committing pHIG22 replicates via the Theta replication mechanism. A 2012 bp region among 387 and 614 bp of pHIG22 was determined as minimal replicon which carries the elements necessary for plasmid replication and ori region. Furthermore, quantitative real-time PCR showed that the relative copy number of pHIG22 was estimated to be 148.2 ±â€¯4.7 copies per chromosome equivalent. The new Theta type plasmid would be useful and beneficial to build vectors for cloning of thermophilic genes and in vivo protein engineering.

C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5.

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70°C versus 60°C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60-80°C and 6.0-9.0 versus 40-60°C and 5.0-8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3h at 80°C as compared to wild -type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.