Cohnella Sp Research Articles

A novel thermophilic lysozyme 4356 from Cohnella sp. A01: Cloning, heterologous expression, biochemical and kinetic characterization

Lysozymes have gained attention for their antiseptic properties. In silico studies have shown that the enzyme containing lysM can act as an antibacterial agent. Binding of the lysM motif of rSELys to peptidoglycan and molecular dynamics simulations showed that the protein-ligand binding is very stable. rSELys (2016 bp) is a new recombinant glycoside hydrolase from the thermophilic bacterium Cohnella sp. A01 (PTCC number: 1921). Protein expression and purification, a single band with an apparent molecular weight of ∼74 kDa was observed by SDS-PAGE. The kinetic parameters were Km 1.163 mg/ml, Vmax 670.3 U/mg, kcat 1675.75 (S−1), and kcat/Km 1440.88 (M−1S−1). Its optimum temperature was 55 °C and pH 8. Temperature stability also showed that the temperature of 50–60 °C retained more than half of its activity after 90 min. Based on the results, rSELys demonstrated antibacterial effects on both Gram-positive and Gram-negative strains, with inhibition zones of 11 and 9 mm, respectively. SEM analysis confirmed hydrolysis activity, the MIC was determined to be 31.25 μg/ml and 3.9 μg/ml, and MBC 0.97 μg/ml, respectively. CD and fluorescence studies showed that up to a temperature of 85 °C and a pH value of 8–12 no structural changes occur, and thermal stability protein was confirmed.

Expression optimization and characterization of a novel amylopullulanase from the thermophilic Cohnella sp. A01

Amylopullulanase (EC. 3.2.1.41/1) is an enzyme that hydrolyzes starch and pullulan, capable of breaking (4 → 1)-α and (6 → 1)-α bonds in starch. Here, the Amy1136 gene (2166 base pairs) from the thermophilic bacterium Cohnella sp. A01 was cloned into the expression vector pET-26b(+) and expressed in Escherichia coli BL21. The enzyme was purified using heat shock at 90 °C for 15 min. The expression optimization of Amy1136 was performed using Plackett–Burman and Box–Behnken design as follows: temperature of 26.7 °C, rotational speed of 180 rpm, and bacterial population of 1.25. The Amy1136 displayed the highest activity at a temperature of 50 °C (on pullulan) and a pH of 8.0 (on starch) and, also exhibited stability at high temperatures (90 °C) and over a range of pH values. Ag+ significantly increased enzyme activity, while Co2+ completely inhibited amylase activity. The enzyme was found to be calcium-independent. The kinetic parameters Km, Vmax, kcat, and kcat/Km for amylase activity were 2.4 mg/mL, 38.650 μmol min−1 mg−1, 38.1129 S−1, and 0.09269 S−1mg mL−1, respectively, and for pullulanase activity were 173.1 mg/mL, 59.337 μmol min−1 mg−1, 1.586 S−1, and 1.78338 S−1mg mL−1, respectively. The thermodynamic parameters Kin, t1/2, Ea#, ΔH#, ΔG# and ΔS# were calculated equal to 0.20 × 10−2 (m−1), 462.09 (min), 16.87 (kJ/mol), 14.18 (kJ/mol), 47.34 (kJ/mol) and 102.60 (Jmol K−1), respectively. The stability of Amy1136 under high temperature, acidic and alkaline pH, surfactants, organic solvents, and calcium independence, suggests its suitability for industrial applications.

In vitro and in silico characterization of a novel glutamate carboxypeptidase from Cohnella sp. A01

Glutamate carboxypeptidase is a bacterial enzyme of metallopeptidase superfamily. This enzyme is an exo-peptidase that catalyzes the hydrolysis of glutamate residues at the C-terminus of folic acid. The rCP302 is a novel zinc ion-dependent recombinant glutamate carboxypeptidase derived from a thermophilic bacterium, Cohnella sp. A01 (PTCC No: 1921). By simulating the structure of rCP302, analyzing its activity in various environmental settings, and contrasting it with that of related enzymes, we wanted to evaluate the heterologous production, purification, and characterization of this enzyme. The bioinformatics study showed that rCP302 had maximum similarity to M20 family of metallopeptidases. The purified rCP302 molecular weight was about 41.6 kDa. The optimum temperature and pH for the catalytic activity of rCP302 were 50 °C and 7.2, respectively. Fluorescence spectroscopy data elucidated the secondary structure of rCP302 and determined conformational changes caused by alterations in ambient conditions. Using folate as a substrate, Km and specific activity values were calculated as 0.108 μM and 687 μmol/min/mg, respectively. The enzyme activity was strongly inhibited when EDTA sequestered zinc ions. The half-life of this enzyme at 30 °C was 2012 min. Regarding the ability of rCP302 to degrade folic acid, and its long half-life at 37 °C, the normal temperature of many mammals, this enzyme can be introduced for further study for use in the pharmaceutical industry.

A genome-proteome-based approach for xylan degradation by Cohnella sp. AR92.

Several members of Cohnella genus have been reported as xylanolytic bacteria with significant capacity as carbohydrate-active enzyme producers (CAZymes), whose mechanisms involving xylan degradation are a key goal for suitable applications in bio-based industries. Using Cohnella sp. AR92 bacterium, we ensembled a genomic-proteomic approach to assess plant biomass conversion targeting its xylanolytic set of enzymes. Also, the genomic traits of the strain AR92 were compared to other Cohnella spp., showing a significant variability in terms of genome sizes and content of genes that code CAZymes. The AR92 straingenome harbours 209 CAZymes encoding sequences active on different polysaccharides, particularly directed towards xylans. Concurrent proteomic data recovered from cultures containing three kinds of lignocellulosic-derived substrates showed a broad set of xylan-degrading enzymes. The most abundant CAZymes expressed in the different conditions assayed were endo-β-1,4-xylanases belonging to the GH11 and GH10 families, enzymes that were previously proved to be useful in the biotransformation of lignocellulosic biomass derived from sugarcane as well as onto xylan-enriched substrates. Therefore, considering the large reserve of CAZymes of Cohnella sp. AR92, a xylan processing model for AR92 strain is proposed.

Expression, characterization, and activity optimization of a novel cellulase from the thermophilic bacteria Cohnella sp. A01

Cellulases are hydrolytic enzymes with wide scientific and industrial applications. We described a novel cellulase, CelC307, from the thermophilic indigenous Cohnella sp. A01. The 3-D structure of the CelC307 was predicted by comparative modeling. Docking of CelC307 with specific inhibitors and molecular dynamic (MD) simulation revealed that these ligands bound in a non-competitive manner. The CelC307 protein was purified and characterized after recombinant expression in Escherichia coli (E. coli) BL21. Using CMC 1% as the substrate, the thermodynamic values were determined as Km 0.46 mM, kcat 104.30 × 10–3 (S−1), and kcat/Km 226.73 (M−1 S−1). The CelC307 was optimally active at 40 °C and pH 7.0. The culture condition was optimized for improved CelC307 expression using Plackett–Burman and Box–Behnken design as follows: temperature 20 °C, pH 7.5, and inoculation concentration with an OD600 = 1. The endoglucanase activity was positively modulated in the presence of Na+, Li+, Ca2+, 2-mercaptoethanol (2-ME), and glycerol. The thermodynamic parameters calculated for CelC307 confirmed its inherent thermostability. The characterized CelC307 may be a suitable candidate for various biotechnological applications.

Characterization of Agarolytic Pathway in a Terrestrial Bacterium Cohnella sp. LGH.

Previously, we have reported that an endo-type β-agarase AgaW was responsible for the hydrolysis of agarose into the major product neoagarotetraose in a terrestrial agar-degrading bacterium Cohnella sp. LGH. Here, we identify and characterize the following depolymerization pathway in strain LGH through the genomic and enzymatic analysis. In the pathway, neoagarotetraose was depolymerized by a novel α-neoagarooligosaccharide (NAOS) hydrolase CL5012 into 3,6-anhydro-α-L-galactose (L-AHG) and agarotriose; Agarotriose was further depolymerized by a novel agarolytic β-galactosidase CL4994 into D-galactose and neoagarobiose; Neoagarobiose was finally depolymerized by CL5012 into L-AHG and D-galactose. Although α-agarase has not been identified in strain LGH, the combined action of CL5012 and CL4994 unexpectedly plays a critical role in the depolymerization of agarotetraose, one theoretical product of α-agarase hydrolysis of agarose. In this pathway, agarotetraose was depolymerized by CL4994 into D-galactose and neoagarotriose; Neoagarotriose was then depolymerized by CL5012 into L-AHG and agarobiose. Furthermore, another novel endo-type β-agarase CL5055 was identified as an isozyme of AgaW with different pH preference in the hydrolysis of agarose into α-NAOSs. Strain LGH seemed to lack a common exo-type β-agarase responsible for the direct depolymerization of agarose or neoagarooligosaccharide into neoagarobiose. These results highlight the diversity of agarolytic manner in bacteria and provide a novel insight on the diversity of agarolytic pathways.

Characterization of Thermotolerant Chitinase from the Strain Cohnella sp. IB P-192 and Its Application for the Production of Bioactive Chitosan Oligomers

Characterization of Thermotolerant Chitinase from the Strain Cohnella sp. IB P-192 and Its Application for the Production of Bioactive Chitosan Oligomers

Endo-xylanases from Cohnella sp. AR92 aimed at xylan and arabinoxylan conversion into value-added products.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-β-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4°C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

Thermophilic iron containing type superoxide dismutase from Cohnella sp. A01

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol−1, −76.8 kJ.mol−1.°K−1, and 84.1 kJ.mol−1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0–10.0) and temperatures (35–75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

Enhancement of soil health, germination and crop productivity in Andrographis paniculata (Burm.f.) Nees, an important medicinal crop by using a composite bio inoculant

The untapped microbial communities in medicinal plants can have a genetically varied population with multi-functional plant growth promoting characters. An attempt was made to investigate the effect of bioinoculant, Cohnella sp., Chryseobacterium taklimakanense, Lysobacter soli and Paenibacillus glycanilyticus isolated from the medicinal plant (Hemidesmus indicus) rhizosphere on the growth parameters of Kalmegh (Andrographis paniculata) without chemical fertilizers. A vermicompost carrier based single, dual and multiple bio-inoculant formulation was developed and tested for the survival of individual strains, showed a maximum population of 1 × 106 cells g−1 after 90 days of storage at 28 ± 2 °C invariably in all formulations. The inoculums’ efficiency on Kalmegh under greenhouse conditions resulted in boosted growth with the maximum plant height (95.8 cm) in Cohnella sp. application, followed by consortium of all strains recorded 91.5 cm. Flower initiation occurred sooner in plants inoculated with bacterial consortium of all as well in Cohnella sp. alone, consecutively resulted in highest Andrographolide content of 3.06% and 3.50%, respectively. Maximum fresh weight herbage yield of 39.5% and 27.5% (dry weight) was recorded in plants treated with bacterial consortium (Cohnella sp, C. taklimakanense, L. soli and P. glycanilyticus) over non-inoculated control as well the available nitrogen, phosphorus and potassium content. Germination of seed experiment revealed the synergism of bacterial isolates in consortium for promoting plant growth. Further, the quanta of inoculums is reduced by 25 percent in composite inoculation, in turn reduces the fertilizers expenditure and persist until the harvesting stage of the crop with the need to apply once.

Structural and biochemical characterization of a novel thermophilic Coh01147 protease.

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

L-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). Km, Vmax, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg2+, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.

Cohnella sp. A01 laccase: thermostable, detergent resistant, anti-environmental and industrial pollutants enzyme

Laccase (EC 1.10.3.2; benzenediol; oxygen oxidoreductases) is a multi-copper oxidase that catalyzes the oxidation of phenols, polyphenols, aromatic amines, and different non-phenolic substrates with concomitant reduction of O2 to H2O. Enzymatic oxidation techniques have the potential of implementation in different areas of industrial fields. In this study, the Cohnella sp. A01 laccase gene was cloned into pET-26 (b+) vector and was transformed to E. coli BL21. Then it was purified using His tag affinity (Ni sepharose resin) chromatography. The estimated molecular weight was approximately 60 kDa using SDS-PAGE. The highest enzyme activity and best pH for 2,6-dimethoxyphenol (DMP) oxidation were recorded as 8 at 90 °C respectively. The calculated half-life and kinetic values including Km, Vmax, turn over number (kcat), and catalytic efficiency (kcat/Km) of the enzyme were 106 min at 90 °C and 686 μM, 10.69 U/ml, 20.3 S−, and 0.029 s−1 μM−1, respectively. The DMP was available as the substrate in all the calculations. Enzyme activity enhanced in the presence of Cu2+, NaCl, SDS, n-hexane, Triton X-100, tween 20, and tween 80, significantly. The binding residues were predicted and mapped upon the modeled tertiary structure of identified laccase. The remaining activity and structural properties of Cohnella sp. A01 laccase in extreme conditions such as high temperatures and presence of metals, detergents, and organic solvents suggest the potential of this enzyme in biotechnological and industrial applications. This process has been patented in Iranian Intellectual Property Centre under License No: 91325.

Isolation, characterization and effect of plant-growth-promoting rhizobacteria on pine seedlings (Pinus pseudostrobus Lindl.)

In this study, 10 bacterial strains were isolated from the rhizosphere of coniferous trees on Mount Tlaloc in Mexico. The strains were characterized by their capacity to produce auxins, solubilize phosphates and stimulate mycelial growth of the ectomycorrhizal fungus Suillus sp. All isolates were identified at the molecular level. Moreover, an experiment was established to evaluate the response of Pinus pseudostrobus seedlings to inoculation with the rhizobacteria strains. The isolated strains belonged to the species Cupriavidus basilensis, Rhodococcus qingshengii, R. erythropolis, Pseudomonas spp., P. gessardii, Stenotrophomonas rhizophila and Cohnella sp. All of the strains produced auxins; the best producer was R. erythropolis CPT9 (76.4 µg mL−1). P. gessardii CPT6 solubilized phosphate at a significant level (443 µg mL−1). The strain S. rhizophila CPT8 significantly increased the radial growth of the ectomycorrhizal fungus Suillus sp. by 18.8%. Five strains increased the dry mass of the shoots; R. qingshengii CPT4 and R. erythropolis CPT9 increased growth the most, by more than 20%. Inoculation with plant-growth-promoting rhizobacteria can be a very useful practice in a forest nursery to produce healthy, vigorous plants.

Designing cross-linked xylanase aggregates for bioconversion of agroindustrial waste biomass towards potential production of nutraceuticals

Immobilized biocatalysts design has the potential to efficiently produce valuable bioproducts from lignocellulosic biomass. Among them, the carrier-free immobilization through the cross-linked enzyme aggregates technology is a simple and low-cost alternative. A two steps statistical approach was utilized to evaluate the synthesis of a cross-linked enzyme aggregate from a xylanolytic preparation, which was produced by Cohnella sp. AR92 grown in a peptone-based culture medium.The resulting immobilized biocatalyst, Xyl-CLEA, was significate more stable (25 to 45%) towards temperatures up to 50°C with respect to the free enzyme, and retained over 50% of its initial activity after 5 consecutive cycles of reuse. By means of infrared spectroscopy and electron microscopy, the Xyl-CLEA showed architectural features described as signature of type I and type II of protein aggregates. These, were the result of the simultaneous aggregation of a multiplicity of proteins from the crude enzymatic extract.The enzymatic activity was assessed using alkali pretreated sugar cane bagasse as substrate. Whereas the free enzymatic preparation released xylose as the main product, the immobilized xylanase produced xylooligosaccharides, thus showing that the immobilization procedure modified the potential application of the extracellular xylanase from Conhella sp. AR92.

Agrowastes as Feedstock for the Production of Endo-β-Xylanase from Cohnella sp. Strain AR92

Members of Cohnella sp. isolated from a variety of environments have been shown to be glycoside hydrolase producers. Nevertheless, most evaluations of members of this genus are limited to their taxonomic description. The strain AR92, previously identified as belonging to the genus Cohnella, formed a well-supported cluster with C. thailandensis and C. formosensis (>80% bootstrap confidence). Its growth and xylanase production were approached by using a mineral-based medium containing alkali-pretreated sugarcane bagasse as the main carbon source, which was assayed as a convenient source to produce biocatalysts potentially fitting its degradation. By means of a two-step statistical approach, the production of endoxylanase was moderately improved (20%). However, a far more significant improvement was observed (145%), by increasing the inoculum size and lowering the fermentation temperature to 25°C, which is below the optimal growth temperature of the strain AR92 (37°C). The xylanolytic preparation produced by Cohnella sp. AR92 contained mild temperature-active endoxylanase (identified as redundant GH10 family) for the main activity which resulted in xylobiose and xylo-oligosaccharides as the main products from birchwood xylan.

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization.

Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and Vmax values of chitinase to be 5.6 mg/mL and 0.87 μmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH.

An agar-degrading bacterium, strain LGH, was isolated and identified as Cohnella sp. This strain had a capability of utilizing agar as a sole carbon source for growth and showed a strong agarolytic activity. A novel endo-type β-agarase gene agaW, encoding a primary translation product of 891 amino acids, including a 26 amino acid signal peptide, was cloned and identified from a genomic library of strain LGH. The AgaW belonged to the glycoside hydrolase (GH) GH50 family, with less than 39% amino acid sequence similarity with any known protein, and hydrolyzed agarose into neoagarotetraose as the major end product and neoagarobiose as the minor end product through other neoagarooligosaccharide intermediates, such as neoagarohexaose.

Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01

Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5kDa on SDS–PAGE. The values of Km and Vmax, calculated by the Michaelis–Menten equation, were 1077μM and 61.94U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70°C and pH 8.5. Activities at 50, 55 and 60°C for 120min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5–10.0 for 180min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% α-helix, 12.8% β-sheet, 22.7% β-turn and 27% random coil.

Trichloroacetic acid precipitation by ultracentrifugation to concentrate dilute protein in viscous solution.

BioTechniquesVol. 20, No. 6 BenchmarksOpen AccessTrichloroacetic Acid Precipitation by Ultracentrifugation to Concentrate Dilute Protein in Viscous SolutionByung Joon Hwang & Gilbert ChuByung Joon HwangStanford University Medical Center, Stanford, CA, USASearch for more papers by this author & Gilbert Chu*Address correspondence to Gilbert Chu, Department of Medicine, Stanford University Medical Center, Stanford, CA 94305, USA.Stanford University Medical Center, Stanford, CA, USASearch for more papers by this authorPublished Online:2 Aug 2018https://doi.org/10.2144/96206bm09AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail "Trichloroacetic Acid Precipitation by Ultracentrifugation to Concentrate Dilute Protein in Viscous Solution." , 20(6), pp. 982–984FiguresReferencesRelatedDetailsCited ByThermophilic iron containing type superoxide dismutase from Cohnella sp. A01International Journal of Biological Macromolecules, Vol. 187A rapid competitive method for bacteriophage genomic DNA extractionJournal of Virological Methods, Vol. 293Development of a novel S. Typhi and Paratyphi A outer membrane vesicles based bivalent vaccine against enteric fever14 September 2018 | PLOS ONE, Vol. 13, No. 9Identification of Effectors: Precipitation of Supernatant MaterialSimple and Reliable Method To Precipitate Proteins from Bacterial Culture SupernatantApplied and Environmental Microbiology, Vol. 70, No. 1Internal sequence analysis of proteins eluted from polyacrylamide gelsJournal of Chromatography B: Biomedical Sciences and Applications, Vol. 686, No. 2 Vol. 20, No. 6 Follow us on social media for the latest updates Metrics Downloaded 557 times History Published online 2 August 2018 Published in print June 1996 Information© 2018 Author(s)PDF download