This study aims to determine the diversity of culturable thermophilic bacteria isolated from eigh... more This study aims to determine the diversity of culturable thermophilic bacteria isolated from eight terrestrial hot springs in Northeastern of Algeria using the conventional methods, SDS-PAGE fingerprinting of whole-cell proteins and 16S rRNA gene sequencing. In addition, their hydrolytic enzyme activities were also investigated. A total of 293 strains were isolated from the hot springs' water and sediment using different culture media. Overall, five distinct bacterial groups were characterized by whole-cell protein pattern analysis. Based on the 16S rRNA gene sequencing of 100 selected strains, the isolates were assigned to the following three major phyla: Firmicutes (93%), Deinococcus-Thermus (5%), and Actinobacteria (2%), which included 27 distinct species belonging to 12 different phylotypes, Aeribacillus, Aneurinibacillus, Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, Laceyella, Meiothermus, Saccharomonospora, Thermoactinomyces, Thermobifida, and Thermus. The screening for nine extracellular enzymes showed that 65.87% of the isolates presented at least five types of enzyme activities, and 6.48% of strains combined all tested enzymes (amylase, cellulase, pectinase, esculinase, protease, gelatinase, lipase, lecithinase, and nuclease). It was found that Bacillus, Anoxybacillus, Aeribacillus, and Aneurinibacillus were the genera showing the highest activities. Likewise, the study showed an abundant and diverse thermophilic community with novel taxa presenting a promising source of thermozymes with important biotechnological applications. This study showed that a combined identification method using SDS-PAGE profiles of whole-cell proteins and subsequent 16S rRNA gene sequence analysis could successfully differentiate thermophilic bacteria from Algerian hot springs.
Journal of Enzyme Inhibition and Medicinal Chemistry, Oct 26, 2015
A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter s... more A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co 2+ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the b-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 C and pH of 8.3: k cat of 4.8 Â 10 5 s À1 and k cat /K m of 5.6 Â 10 7 M À1 Â s À1. This activity was potently inhibited by acetazolamide which showed a K I of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO 3 biomineralization processes.
16SrRNA analyses of the clones obtained from a composite water sample, the bacterial community wa... more 16SrRNA analyses of the clones obtained from a composite water sample, the bacterial community was determined to consist of Acidithiobacillus ferrivorans, Ferrovum myxofaciens, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans as iron-oxidising bacteria, Acidocella facilis, Acidocella aluminiidurans, Acidiphilium cryptum and Acidiphilium multivorum as iron-reducing bacteria, and Acidithiobacillus ferrivorans, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidiphilium cryptum as sulphur-oxidising bacteria. This association of bacteria with varying roles was interpreted as evidence of a concomitant occurrence of sulphur and iron cycles during the generation of AMD along the Acısu effluent draining the Karaerik mine. Keywords Acid mine drainage • 16S rRNA • Acidithiobacillus ferrivorans • Leptospirillum ferrooxidans • Acidiphilium cryptum • Acidocella facilis • Ferrovum myxofaciens Communicated by H. Atomi.
Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization d... more Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization due to its heterogeneous structure. Degradation of lignin can be achieved by the use of potential lignin-degrading bacteria. The current study was designed to evaluate the degradation efficiency of newly isolated Bacillus altitudinis SL7 from pulp and paper mill effluent. The degradation efficiency of B. altitudinis SL7 was determined by color reduction, lignin content, and ligninolytic activity from degradation medium supplemented with alkali lignin (3 g/L). B. altitudinis SL7 reduced color and lignin content by 26 and 44%, respectively, on the 5th day of incubation, as evident from the maximum laccase activity. Optimum degradation was observed at 40°C and pH 8.0. FT-IR spectroscopy and GC-MS analysis confirmed lignin degradation by emergence of the new peaks and identification of low-molecular-weight compounds in treated samples. The identified compounds such as vanillin, 2-methyoxyhenol, 3-methyl phenol, oxalic acid and ferulic acid suggested the degradation of coniferyl and sinapyl groups of lignin. Degradation efficiency of B. altitudinis SL7 towards high lignin concentration under alkaline pH indicated the potential application of this isolate in biological treatment of the lignin-containing effluents.
Sakarya University Journal of Science, Oct 20, 2022
In this study, esterase of Aneurinibacillus sp. PDF24 strain, a thermophilic bacteria, was purifi... more In this study, esterase of Aneurinibacillus sp. PDF24 strain, a thermophilic bacteria, was purified to homogenity (5.25 fold purification) by column chromotography, and characterized. The molecular weight of Aneurinibacillus sp. PDF24 esterase was determined about 40 kDa. The maximum activity of the purified esterase was analyzed at 55°C, pH 8.5. The esterase was found to be stable at 40ºC, 50ºC and 60ºC for 1 hour. Km and Vmax values for p-nitrophenyl butyrate were determined as 0.120 mM and 3164.8 U/mg, respectively. Considering Km values in the literature, Aneurinibacillus sp. PDF24 esterase was found to have a good Km value compared to other esterases. In the presence of 1 mM and 5 mM metal salts of Mg 2+ , Li + , Ca 2+ , K + , no significant change occured in enzyme activity. The activity of Aneurinibacillus sp PDF24 esterase was found to be stable also in the presence of ethanol, DMSO, EDTA, DTT and ß-mercaptoethanol. The data obtained suggest that the enzyme is a serine esterase, not a metalloprotein, and that disulfide bonds are not required to maintain enzyme conformation, and therefore, depending on its features, this esterase may be a suitable candidate for industrial applications.
Bilim adamları, dünyada hızla artan hastalıklara karşı kullanılacak ilaçların bulunması ve gelişt... more Bilim adamları, dünyada hızla artan hastalıklara karşı kullanılacak ilaçların bulunması ve geliştirilmesinde, besin maddesi olarak ihtiyaç duyulan bazı proteinlerin üretilmesinde ve çe
A cryptic plasmid pHIG22 from Thermus scotoductus sp. K6, an isolate from the Alangullu Hot Sprin... more 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 commiting 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 ± 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.
In order to characterize two α-Ð-arabinofuranosidases (α-Ð-AFases), Abf1Geo12 and Abf2Geo12, prod... more In order to characterize two α-Ð-arabinofuranosidases (α-Ð-AFases), Abf1Geo12 and Abf2Geo12, produced by Geobacillus stearothermophilus strain 12, the genes (abf 1 and abf 2) coding for these enzymes were cloned and sequenced. Based on the protein sequence similarities, approximately 57 kDa two α-Ð-AFases were assigned to the glycoside hydrolase family 51. To obtain pure enzymes, the abf 1 and abf 2 genes were cloned into pET28a+ expression vector and recombinant α-Ð-AFases were produced in E.coli BL21(DE3): pLysS. Characterization of recombinant α-Ð-AFases revealed that Abf1Geo12 and Abf2Geo12 were active in a broad temperature range from 50 to 85 • C and from 40 to 80 • C, respectively. Also, the Abf1Geo12 was active in a broad pH range from 5.0 to 9.0. The optimum pH and temperature for Abf1Geo12 were determined as pH 6.0 and 65 • C, respectively, whereas the optimum pH and temperature for Abf2Geo12 were determined as pH 5.5 and 60 • C, respectively. Based on characterization studies, it was determined that the Abf1Geo12 was more stable than Abf2Geo12 and previously identified α-Ð-AFases from G. stearothermophilus. Using p-nitrophenyl α-Ð-arabinofuranoside as a substrate, the Km and Vmax values for Abf1Geo12 and Abf2Geo12 were determined as 0.31 mM and 290 U/mg for the former enzyme and 0.19 mM and 213.2 U/mg for the latter enzyme, respectively. The activities of Abf1Geo12 and Abf2Geo12 were strongly inhibited by 1 mM Hg 2+. Interestingly, Cu 2+ and Co 2+ stimulated the activity of Abf1Geo12, but they reduced the activity of Abf2Geo12. The recombinant enzymes released Ð-arabinose from sugar beet arabinan, arabinobiose, arabinotriose, arabinotetraose and arabinopentaose. Consequently, these characterized two enzymes may be used in industrial fields since they are stable at high temperatures.
Highlights Purification of endo-chitinase (ChiA-Hh59) from a H. hirschii KB-DZ44 was carried ou... more Highlights Purification of endo-chitinase (ChiA-Hh59) from a H. hirschii KB-DZ44 was carried out. The molecular weight and the NH2-terminal sequence of the chitinase was determined. Optimum pH and temperature values for activity were pH 5.0 and 85 °C, respectively. The kinetic parameters and the TLC of hydrolysis products of the enzyme were studied. ChiA-Hh59 may be used as potential candidate for the enzymatic degradation of chitin.
Applied Biochemistry and Biotechnology, Sep 3, 2019
In order to bleach the eucalyptus kraft pulp, two enzyme treatments involving feruloyl esterase a... more In order to bleach the eucalyptus kraft pulp, two enzyme treatments involving feruloyl esterase and laccase were used in the TCF sequence. Hydroxycinnamic acids, which were released from lignin subunits by the activity of feruloyl esterase, were used as a natural mediator of laccase. The use of sequentially feruloyl esterase and laccase has much higher pulp bleaching effects than the individual enzymes. GthFAE, BmegLac and GthFAE+BmegLac treatments (X) reduced the kappa number of eucalyptus kraft pulps by indicating 9%, 18%, and 30% delignification rates, respectively. Just like in delignification rates, the highest brightness improvement was achieved from the GthFAE+BmegLac combination. The results of the present study indicated that the natural mediators, which are presented in the structure of lignin, could be used as laccase-mediators for pulp bleaching more efficiently and costeffectively.
Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potenti... more Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potential in paper pulp and bleaching processes. Here, we report rapid, cost-effective, and result-oriented combinatorial potential of in silico DNA swapping strategy to engineer the pH optimum of industrially crucial enzymes, particularly engineering of Geobacillus sp. TF16 endoxylanase for alkaline environments. The 3D structures of Geobacillus sp. TF16 and donor Bacillus halodurans C-125 endoxylanases were firstly predicted, analyzed, and compared for their similarities before any in silico design of mutants. Reasonably, to improve its alkaline pH tolerance, the corresponding regions in Geobacillus sp.TF16 endoxylanase were further engineered by swapping with negatively-charged amino acid-rich regions from B. halodurans C-125 endoxylanase. Through only two of four in silico-designed mutants, the optimum pH of GeoTF16 endoxylanase was improved from 8.5 to 10.0. Moreover, as compared to GeoTF16 parental enzyme, both GeoInt3 and GeoInt4 mutants revealed (i) enhanced biobleaching performance, (ii) improved adaptability to alkaline conditions, and (iii) better activity for broader pH range. Unlike GeoTF16 losing activity at pH 11.0 completely, GeoInt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, GeoInt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications. The current study represents an efficient protein engineering strategy to adapt industrial catalysts to diverse processing conditions. Further comprehensive and fine-tuned research efforts may result in biotechnologically more promising outcomes.
A newly identified ligninolytic Rhodococcus strain (Rhodococcus sp. T1) was isolated from forestr... more A newly identified ligninolytic Rhodococcus strain (Rhodococcus sp. T1) was isolated from forestry wastes (Trabzon/Turkey). The DyP type peroxidase of Rhodococcus sp. T1 (DyPT1) was cloned, characterized and paper treated for industrial applications. Molecular weight of the protein was about 38 kDa. The kinetic parameters were 0.94 mM and 1417.53 µmol/min/mg for Km and Vmax, respectively. The enzyme was active at the temperature range of 25-65 °C and optimum temperature was 35 °C, enzyme was stable up to 6 days at room temperature. Optimum pH of the DyPT1 was 4.0 and it was stable between pH 4.0-6.0 up to 8 days at room temperature. Effects of some metal ions, Hemin, and some chemical agents on DyPT1 were determined. Hemin has implemented protective effects on the stability and the activity of the enzyme in long time periods when added into growing medium. DyPT1 was applied to eucalyptus kraft pulp for analyzing the bleaching efficiency, physical and optical tests of the manufuctared paper were carried out. Application of lignin peroxidase to kraft pulp caused a decrease of 5.2 units for kappa number and an increase from 52.05 to 64.18% in the delignification rate.
Introduction Anoxybacillus is a relatively new genus compared to the well-studied genera Geobacil... more Introduction Anoxybacillus is a relatively new genus compared to the well-studied genera Geobacillus or Bacillus. The genus Anoxybacillus represents aerobic or facultatively anaerobic, neutrophilic, obligately thermophilic, endospore-forming bacteria (İnan et al., 2011). Most of the reported data have revealed that the members of this genus produce interesting enzymes that are thermostable and tolerant to alkaline pH. Some of the well-studied enzymes were discovered through partnerships with industry; for example, the raw starch-degrading amylase was discovered by a Novozyme team (Viksø-Nielsen et al., 2006), and the BfιI RE was discovered by New England Biolabs (D'Souza et al., 2004; Goh et al., 2013). L-ribulokinase (RK; EC 2.7.1.16) is 1 of 3 major enzymes of the arabinose catabolic pathway. L-arabinose is 1 of the major polysaccharide components in plant cell walls and among the most abundant monosaccharides in nature. Furthermore, its utilization pathway in bacteria has been investigated extensively (Zhang et al., 2012). The arabinose regulon is 1 of many gene systems in Escherichia coli and the regulon consists of 4 operons, araBAD, araC, araE, and araFGH, which are responsible for L-arabinose catabolism, gene regulation, low-affinity transport, and high-affinity transport, respectively (Englesberg and Wilcox, 1974; Lichenstein et al., 1987). In the lowaffinity transport system, the transporter, the araE gene product, is bound to the inner membrane and utilizes the electrochemical potential to transport arabinose. The araFGH genes encode arabinose-specific components of a high-affinity transport system, ABC transporters. These are 3 proteins of the ATP-binding cassette transporter family. AraF is the periplasmic arabinose-binding protein, AraG is the ATP-binding component, and AraH is the membrane-bound component (Schleif, 2010). AraC acts directly as an inducer or an activator of gene expression. The araBAD operon encodes 3 different enzymes required for catabolism of L-arabinose, which are responsible for the conversion of L-arabinose into D-xylulose-5-phosphate. AraA, as an isomerase (L-arabinose isomerase), converts arabinose to L-ribulose; AraB, as a kinase (L-ribulokinase), phosphorylates L-ribulose; and AraD, as an epimerase (L-ribulose-5phosphate 4-epimerase), converts L-ribulose-phosphate
Biological methods of soil and water purification from petroleum are based on activation natural ... more Biological methods of soil and water purification from petroleum are based on activation natural microorganisms with the help of agrotechnical techniques and introducing special selected active soil microorganisms-oil destructors. From active oil-oxidizing psychrophilic bacteria isolated from oil contaminated soils of West Kazakhstan Serratia marcescens Н3К, Rhodococcus erythropolis DN-1, Bacillus amyloliquefaciens I-15) was formulated a bioagent for bioremediation of oil polluted soils at low temperature conditions. The ability of the bioagent to transform various oil was studied: light oil from Zhanatalap oil deposits (Atyrau region, Kazakhstan), crude oil from Karazhanbas oil deposits (Atyrau region, Kazakhstan) and medium density oil from Kumkol (Kyzyl-Orda region, Kazakhstan) oil field. Bioagent was submitted into 100 of liquid mineral medium in cells/at temperature +10 • C. Effectiveness of bioagent was determined from the residual oil content. It was established that bioagent actively decomposed the oil from Zhanatalap and Kumkol oil fields, level of destruction comprised 92% and 88% respectively. Degree of destruction of oil from Karazhanbas oil field comprised 69%. Thus, the bioagent had the greatest destructive activity in relation to light and medium density oil.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Abstract: In this study. using Autographa californica nuclear polyhedrosis virus (AcNPV)-5podopte... more Abstract: In this study. using Autographa californica nuclear polyhedrosis virus (AcNPV)-5podoptera frugiperda (Sf) infection as control. the comparative replication of AcNPV in Anticarsia gemmatalis (Ag) cells was investigated by Iight microscopy. SDS-polyacrilamide gel electrophoresis ( ...
This study aims to determine the diversity of culturable thermophilic bacteria isolated from eigh... more This study aims to determine the diversity of culturable thermophilic bacteria isolated from eight terrestrial hot springs in Northeastern of Algeria using the conventional methods, SDS-PAGE fingerprinting of whole-cell proteins and 16S rRNA gene sequencing. In addition, their hydrolytic enzyme activities were also investigated. A total of 293 strains were isolated from the hot springs' water and sediment using different culture media. Overall, five distinct bacterial groups were characterized by whole-cell protein pattern analysis. Based on the 16S rRNA gene sequencing of 100 selected strains, the isolates were assigned to the following three major phyla: Firmicutes (93%), Deinococcus-Thermus (5%), and Actinobacteria (2%), which included 27 distinct species belonging to 12 different phylotypes, Aeribacillus, Aneurinibacillus, Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, Laceyella, Meiothermus, Saccharomonospora, Thermoactinomyces, Thermobifida, and Thermus. The screening for nine extracellular enzymes showed that 65.87% of the isolates presented at least five types of enzyme activities, and 6.48% of strains combined all tested enzymes (amylase, cellulase, pectinase, esculinase, protease, gelatinase, lipase, lecithinase, and nuclease). It was found that Bacillus, Anoxybacillus, Aeribacillus, and Aneurinibacillus were the genera showing the highest activities. Likewise, the study showed an abundant and diverse thermophilic community with novel taxa presenting a promising source of thermozymes with important biotechnological applications. This study showed that a combined identification method using SDS-PAGE profiles of whole-cell proteins and subsequent 16S rRNA gene sequence analysis could successfully differentiate thermophilic bacteria from Algerian hot springs.
Journal of Enzyme Inhibition and Medicinal Chemistry, Oct 26, 2015
A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter s... more A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co 2+ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the b-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 C and pH of 8.3: k cat of 4.8 Â 10 5 s À1 and k cat /K m of 5.6 Â 10 7 M À1 Â s À1. This activity was potently inhibited by acetazolamide which showed a K I of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO 3 biomineralization processes.
16SrRNA analyses of the clones obtained from a composite water sample, the bacterial community wa... more 16SrRNA analyses of the clones obtained from a composite water sample, the bacterial community was determined to consist of Acidithiobacillus ferrivorans, Ferrovum myxofaciens, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans as iron-oxidising bacteria, Acidocella facilis, Acidocella aluminiidurans, Acidiphilium cryptum and Acidiphilium multivorum as iron-reducing bacteria, and Acidithiobacillus ferrivorans, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidiphilium cryptum as sulphur-oxidising bacteria. This association of bacteria with varying roles was interpreted as evidence of a concomitant occurrence of sulphur and iron cycles during the generation of AMD along the Acısu effluent draining the Karaerik mine. Keywords Acid mine drainage • 16S rRNA • Acidithiobacillus ferrivorans • Leptospirillum ferrooxidans • Acidiphilium cryptum • Acidocella facilis • Ferrovum myxofaciens Communicated by H. Atomi.
Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization d... more Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization due to its heterogeneous structure. Degradation of lignin can be achieved by the use of potential lignin-degrading bacteria. The current study was designed to evaluate the degradation efficiency of newly isolated Bacillus altitudinis SL7 from pulp and paper mill effluent. The degradation efficiency of B. altitudinis SL7 was determined by color reduction, lignin content, and ligninolytic activity from degradation medium supplemented with alkali lignin (3 g/L). B. altitudinis SL7 reduced color and lignin content by 26 and 44%, respectively, on the 5th day of incubation, as evident from the maximum laccase activity. Optimum degradation was observed at 40°C and pH 8.0. FT-IR spectroscopy and GC-MS analysis confirmed lignin degradation by emergence of the new peaks and identification of low-molecular-weight compounds in treated samples. The identified compounds such as vanillin, 2-methyoxyhenol, 3-methyl phenol, oxalic acid and ferulic acid suggested the degradation of coniferyl and sinapyl groups of lignin. Degradation efficiency of B. altitudinis SL7 towards high lignin concentration under alkaline pH indicated the potential application of this isolate in biological treatment of the lignin-containing effluents.
Sakarya University Journal of Science, Oct 20, 2022
In this study, esterase of Aneurinibacillus sp. PDF24 strain, a thermophilic bacteria, was purifi... more In this study, esterase of Aneurinibacillus sp. PDF24 strain, a thermophilic bacteria, was purified to homogenity (5.25 fold purification) by column chromotography, and characterized. The molecular weight of Aneurinibacillus sp. PDF24 esterase was determined about 40 kDa. The maximum activity of the purified esterase was analyzed at 55°C, pH 8.5. The esterase was found to be stable at 40ºC, 50ºC and 60ºC for 1 hour. Km and Vmax values for p-nitrophenyl butyrate were determined as 0.120 mM and 3164.8 U/mg, respectively. Considering Km values in the literature, Aneurinibacillus sp. PDF24 esterase was found to have a good Km value compared to other esterases. In the presence of 1 mM and 5 mM metal salts of Mg 2+ , Li + , Ca 2+ , K + , no significant change occured in enzyme activity. The activity of Aneurinibacillus sp PDF24 esterase was found to be stable also in the presence of ethanol, DMSO, EDTA, DTT and ß-mercaptoethanol. The data obtained suggest that the enzyme is a serine esterase, not a metalloprotein, and that disulfide bonds are not required to maintain enzyme conformation, and therefore, depending on its features, this esterase may be a suitable candidate for industrial applications.
Bilim adamları, dünyada hızla artan hastalıklara karşı kullanılacak ilaçların bulunması ve gelişt... more Bilim adamları, dünyada hızla artan hastalıklara karşı kullanılacak ilaçların bulunması ve geliştirilmesinde, besin maddesi olarak ihtiyaç duyulan bazı proteinlerin üretilmesinde ve çe
A cryptic plasmid pHIG22 from Thermus scotoductus sp. K6, an isolate from the Alangullu Hot Sprin... more 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 commiting 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 ± 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.
In order to characterize two α-Ð-arabinofuranosidases (α-Ð-AFases), Abf1Geo12 and Abf2Geo12, prod... more In order to characterize two α-Ð-arabinofuranosidases (α-Ð-AFases), Abf1Geo12 and Abf2Geo12, produced by Geobacillus stearothermophilus strain 12, the genes (abf 1 and abf 2) coding for these enzymes were cloned and sequenced. Based on the protein sequence similarities, approximately 57 kDa two α-Ð-AFases were assigned to the glycoside hydrolase family 51. To obtain pure enzymes, the abf 1 and abf 2 genes were cloned into pET28a+ expression vector and recombinant α-Ð-AFases were produced in E.coli BL21(DE3): pLysS. Characterization of recombinant α-Ð-AFases revealed that Abf1Geo12 and Abf2Geo12 were active in a broad temperature range from 50 to 85 • C and from 40 to 80 • C, respectively. Also, the Abf1Geo12 was active in a broad pH range from 5.0 to 9.0. The optimum pH and temperature for Abf1Geo12 were determined as pH 6.0 and 65 • C, respectively, whereas the optimum pH and temperature for Abf2Geo12 were determined as pH 5.5 and 60 • C, respectively. Based on characterization studies, it was determined that the Abf1Geo12 was more stable than Abf2Geo12 and previously identified α-Ð-AFases from G. stearothermophilus. Using p-nitrophenyl α-Ð-arabinofuranoside as a substrate, the Km and Vmax values for Abf1Geo12 and Abf2Geo12 were determined as 0.31 mM and 290 U/mg for the former enzyme and 0.19 mM and 213.2 U/mg for the latter enzyme, respectively. The activities of Abf1Geo12 and Abf2Geo12 were strongly inhibited by 1 mM Hg 2+. Interestingly, Cu 2+ and Co 2+ stimulated the activity of Abf1Geo12, but they reduced the activity of Abf2Geo12. The recombinant enzymes released Ð-arabinose from sugar beet arabinan, arabinobiose, arabinotriose, arabinotetraose and arabinopentaose. Consequently, these characterized two enzymes may be used in industrial fields since they are stable at high temperatures.
Highlights Purification of endo-chitinase (ChiA-Hh59) from a H. hirschii KB-DZ44 was carried ou... more Highlights Purification of endo-chitinase (ChiA-Hh59) from a H. hirschii KB-DZ44 was carried out. The molecular weight and the NH2-terminal sequence of the chitinase was determined. Optimum pH and temperature values for activity were pH 5.0 and 85 °C, respectively. The kinetic parameters and the TLC of hydrolysis products of the enzyme were studied. ChiA-Hh59 may be used as potential candidate for the enzymatic degradation of chitin.
Applied Biochemistry and Biotechnology, Sep 3, 2019
In order to bleach the eucalyptus kraft pulp, two enzyme treatments involving feruloyl esterase a... more In order to bleach the eucalyptus kraft pulp, two enzyme treatments involving feruloyl esterase and laccase were used in the TCF sequence. Hydroxycinnamic acids, which were released from lignin subunits by the activity of feruloyl esterase, were used as a natural mediator of laccase. The use of sequentially feruloyl esterase and laccase has much higher pulp bleaching effects than the individual enzymes. GthFAE, BmegLac and GthFAE+BmegLac treatments (X) reduced the kappa number of eucalyptus kraft pulps by indicating 9%, 18%, and 30% delignification rates, respectively. Just like in delignification rates, the highest brightness improvement was achieved from the GthFAE+BmegLac combination. The results of the present study indicated that the natural mediators, which are presented in the structure of lignin, could be used as laccase-mediators for pulp bleaching more efficiently and costeffectively.
Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potenti... more Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potential in paper pulp and bleaching processes. Here, we report rapid, cost-effective, and result-oriented combinatorial potential of in silico DNA swapping strategy to engineer the pH optimum of industrially crucial enzymes, particularly engineering of Geobacillus sp. TF16 endoxylanase for alkaline environments. The 3D structures of Geobacillus sp. TF16 and donor Bacillus halodurans C-125 endoxylanases were firstly predicted, analyzed, and compared for their similarities before any in silico design of mutants. Reasonably, to improve its alkaline pH tolerance, the corresponding regions in Geobacillus sp.TF16 endoxylanase were further engineered by swapping with negatively-charged amino acid-rich regions from B. halodurans C-125 endoxylanase. Through only two of four in silico-designed mutants, the optimum pH of GeoTF16 endoxylanase was improved from 8.5 to 10.0. Moreover, as compared to GeoTF16 parental enzyme, both GeoInt3 and GeoInt4 mutants revealed (i) enhanced biobleaching performance, (ii) improved adaptability to alkaline conditions, and (iii) better activity for broader pH range. Unlike GeoTF16 losing activity at pH 11.0 completely, GeoInt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, GeoInt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications. The current study represents an efficient protein engineering strategy to adapt industrial catalysts to diverse processing conditions. Further comprehensive and fine-tuned research efforts may result in biotechnologically more promising outcomes.
A newly identified ligninolytic Rhodococcus strain (Rhodococcus sp. T1) was isolated from forestr... more A newly identified ligninolytic Rhodococcus strain (Rhodococcus sp. T1) was isolated from forestry wastes (Trabzon/Turkey). The DyP type peroxidase of Rhodococcus sp. T1 (DyPT1) was cloned, characterized and paper treated for industrial applications. Molecular weight of the protein was about 38 kDa. The kinetic parameters were 0.94 mM and 1417.53 µmol/min/mg for Km and Vmax, respectively. The enzyme was active at the temperature range of 25-65 °C and optimum temperature was 35 °C, enzyme was stable up to 6 days at room temperature. Optimum pH of the DyPT1 was 4.0 and it was stable between pH 4.0-6.0 up to 8 days at room temperature. Effects of some metal ions, Hemin, and some chemical agents on DyPT1 were determined. Hemin has implemented protective effects on the stability and the activity of the enzyme in long time periods when added into growing medium. DyPT1 was applied to eucalyptus kraft pulp for analyzing the bleaching efficiency, physical and optical tests of the manufuctared paper were carried out. Application of lignin peroxidase to kraft pulp caused a decrease of 5.2 units for kappa number and an increase from 52.05 to 64.18% in the delignification rate.
Introduction Anoxybacillus is a relatively new genus compared to the well-studied genera Geobacil... more Introduction Anoxybacillus is a relatively new genus compared to the well-studied genera Geobacillus or Bacillus. The genus Anoxybacillus represents aerobic or facultatively anaerobic, neutrophilic, obligately thermophilic, endospore-forming bacteria (İnan et al., 2011). Most of the reported data have revealed that the members of this genus produce interesting enzymes that are thermostable and tolerant to alkaline pH. Some of the well-studied enzymes were discovered through partnerships with industry; for example, the raw starch-degrading amylase was discovered by a Novozyme team (Viksø-Nielsen et al., 2006), and the BfιI RE was discovered by New England Biolabs (D'Souza et al., 2004; Goh et al., 2013). L-ribulokinase (RK; EC 2.7.1.16) is 1 of 3 major enzymes of the arabinose catabolic pathway. L-arabinose is 1 of the major polysaccharide components in plant cell walls and among the most abundant monosaccharides in nature. Furthermore, its utilization pathway in bacteria has been investigated extensively (Zhang et al., 2012). The arabinose regulon is 1 of many gene systems in Escherichia coli and the regulon consists of 4 operons, araBAD, araC, araE, and araFGH, which are responsible for L-arabinose catabolism, gene regulation, low-affinity transport, and high-affinity transport, respectively (Englesberg and Wilcox, 1974; Lichenstein et al., 1987). In the lowaffinity transport system, the transporter, the araE gene product, is bound to the inner membrane and utilizes the electrochemical potential to transport arabinose. The araFGH genes encode arabinose-specific components of a high-affinity transport system, ABC transporters. These are 3 proteins of the ATP-binding cassette transporter family. AraF is the periplasmic arabinose-binding protein, AraG is the ATP-binding component, and AraH is the membrane-bound component (Schleif, 2010). AraC acts directly as an inducer or an activator of gene expression. The araBAD operon encodes 3 different enzymes required for catabolism of L-arabinose, which are responsible for the conversion of L-arabinose into D-xylulose-5-phosphate. AraA, as an isomerase (L-arabinose isomerase), converts arabinose to L-ribulose; AraB, as a kinase (L-ribulokinase), phosphorylates L-ribulose; and AraD, as an epimerase (L-ribulose-5phosphate 4-epimerase), converts L-ribulose-phosphate
Biological methods of soil and water purification from petroleum are based on activation natural ... more Biological methods of soil and water purification from petroleum are based on activation natural microorganisms with the help of agrotechnical techniques and introducing special selected active soil microorganisms-oil destructors. From active oil-oxidizing psychrophilic bacteria isolated from oil contaminated soils of West Kazakhstan Serratia marcescens Н3К, Rhodococcus erythropolis DN-1, Bacillus amyloliquefaciens I-15) was formulated a bioagent for bioremediation of oil polluted soils at low temperature conditions. The ability of the bioagent to transform various oil was studied: light oil from Zhanatalap oil deposits (Atyrau region, Kazakhstan), crude oil from Karazhanbas oil deposits (Atyrau region, Kazakhstan) and medium density oil from Kumkol (Kyzyl-Orda region, Kazakhstan) oil field. Bioagent was submitted into 100 of liquid mineral medium in cells/at temperature +10 • C. Effectiveness of bioagent was determined from the residual oil content. It was established that bioagent actively decomposed the oil from Zhanatalap and Kumkol oil fields, level of destruction comprised 92% and 88% respectively. Degree of destruction of oil from Karazhanbas oil field comprised 69%. Thus, the bioagent had the greatest destructive activity in relation to light and medium density oil.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Abstract: In this study. using Autographa californica nuclear polyhedrosis virus (AcNPV)-5podopte... more Abstract: In this study. using Autographa californica nuclear polyhedrosis virus (AcNPV)-5podoptera frugiperda (Sf) infection as control. the comparative replication of AcNPV in Anticarsia gemmatalis (Ag) cells was investigated by Iight microscopy. SDS-polyacrilamide gel electrophoresis ( ...
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