Sephadex G-100 Gel Filtration Chromatography Research Articles

Purification and characterization of L-asparaginase produced from Bacillus sp.

The objective of this study was to isolate and identify the asparaginase-producing bacteria, then purify and characterize the enzyme in order to investigate their properties in the future. Fifteen local bacterial isolates were isolated from various sites in the city of Baghdad, identified by conventional morphological and biochemical procedures, and confirmed using vitek 2 methods, and submitted to primary screening processes for asparaginase production. For secondary screening, eight isolates with the greatest yellow zone ability on a specific solid medium were chosen. Bacillus sp. was reported to have the highest enzyme production (7.5 U/mg proteins). After 24 hours of incubation, submerged fermentation yielded optimal conditions for the production of L-asparaginase (L-ASNase) by the chosen isolate, with medium (2) serving as the optimal medium for production and fructose serving as the optimal source of carbon. In pH 6 at 40°C, Sephadex G-150 gel filtration chromatography was used to purify the enzyme. The final purification folds were increased by 2.5 times, resulting in an enzyme yield of 93.7%. It also showed the highest purified enzyme activity and stability was at 37°C. Also it revealed the highest activity and stability at pH 7.0 and pH 8.0 respectively. Enzyme lost activity when exposed to several metallic ions at concentrations of 1, 5, and 10 mM.

Characterization of lipases and preliminary purification of tributyrinesterases from lactic acid bacteria strains belonging to the Enterococcus and Lactococcus genera

The objective of this study is to characterize and attempt to purify lipolytic enzymes derived from strains of Enterococcus and Lactococcus genera isolated from various environments. For this purpose, ten strains of lactic acid bacteria were studied for their lipolytic and esterasic activities on an MRS (Man, Rogosa, and Sharpe) medium containing natural and/or artificial lipid substrates. Among them, two strains: Lactococcus lactis subsp lactis and Lactococcus lactis subsp cremoris showed maximum extracellular lipolytic activity in the presence of 1% olive oil. It was observed that both strains exhibited higher lipolytic activity at pH 7 and 8, with an optimal temperature of 30 °C and 37 °C, respectively. Glucose was found to be the best carbon source for both tested strains. The study of growth kinetics and fatty acid production over time revealed that fatty acid production begins during the exponential phase and reaches its maximum during the stationary phase. The purification of tributyrinesterases was carried out on two strains of Enterococcus genus: Enterococcus feacium and Enterococcus durans through ammonium sulfate precipitation and Sephadex G-100 gel filtration chromatography allowed the estimation of the molecular weight of the tributyrinesterases as 32.09 kDa and 38.49 kDa for both strains, respectively.

Bioprospecting of a Thermostable L-Methioninase from Alcaligenes aquatilis BJ-1 in Agro-Industrial Waste

L-methioninase is an enzyme that has recently gained significant interest in the scientific community because of its potential as a targeted therapy for cancer. This study aims to isolate and identify extremophilic bacteria that could produce L-methioninase and to access the enzymatic potential of isolated bacteria under stress conditions, specifically in agro-industrial waste. In this study, a rare marine bacterium, Alcaligenes aquatilis BJ-1, exhibited the highest specific activity of 4.61 U/mg at an optimum pH of 8.3. The L-methioninase was purified 4.3-fold and 7.15-fold by acetone precipitation and Sephadex G-100 gel filtration chromatography, which revealed a molecular weight of 46 kDa. In addition, agriculture waste materials such as cottonseed oil cake had the highest L-methioninase production. Moreover, A. aquatilis BJ-1 can tolerate and produce enzymes in the presence of 10% NaCl, 6% KCl, and 4% MgSO4. Similarly, substrates such as L-asparagine, L-glutamine, L-alanine, and L-tyrosine were found suitable to increase enzyme production. The strain produced L-methioninase in the presence of various heavy metals. Maximum enzyme activity was found in Zn2+ at 0.1% (2.52 U/mL), Li2+ at 0.03% (2.90 U/mL), and Ni2+ at 0.01% (2.78 U/mL), as compared to the control (2.23 U/mL) without metal. Enzyme production was also observed at a high temperature (60 °C), with the produced enzymes possessing antioxidant properties. In addition, no hemolytic activity was observed. The results indicate that A. aquatilis BJ-1 is an appropriate bacterium for metal bioremediation procedures in unfavorable circumstances.

Enhanced Biosynthesis and Purification of Proteases from Bacillus sp. AI-5 by SmF: A Green Approach for Degradation of Peptide Bonds in Complex Proteins

Proteases are theprotein degrading commercial enzymes with considerable importance for many industrial applications such as paper, leather, food industries as well as used in detergent formulation, toxic waste removal, pharmaceutical and drilling for oil. The focus of the current study was to isolate the novel protease producing bacterial sp. from environment, to optimize the submerged fermentation parameters in order to design the best supportive media for maximum enzyme yield and to purify the enzyme to increase its utilization in various industries, most importantly the pharmaceutical industry.Five pure cultures were isolated from soil of coastal area, Karachi and the bacterial strain that was proved to be the most potent producer of protease was selected for research purposeand named as Bacillus sp AI-5 after series of morphological and biochemical tests. The fermentation conditions and media composition were optimized using the starter medium that was selected among the three previously reported media for protease production. It was found that protease production from Bacillus sp AI-5 reached to maximum when media was supplemented with 0.4 gm % casein as carbon source, the combination of 0.5 gm % yeast extract and 0.5 gm % peptone as nitrogen source and 0.05 gm CaCl₂ as inducer and stabilizer of proteases. The optimum pH and temperature for maximum production of protease were found to be pH 5 and 45 and#176;C respectively after 24 hours of incubation. The protease of the Bacillus sp. AI-5 was purified to homogeneity by salt fractionation method using 60 gm % ammonium sulfate, dialysis and Sephadex G-100 Gel filtration chromatography. The specific activity of purified protease was found to be 322.25 U/mg with 13.80 fold purification as compared to crude enzyme. The purified enzyme gave a single band on Sodiumdodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)corresponding to a molecular weight of 66 kDa.

The Tannase from red yeast Rhodotorula glutinis: purification and characterization

The tannase enzyme was successfully purified to homogeneity from the culture broth of red yeast strain Rhodotorula glutinis DB2 in a three-tandem step involving ultrafiltration (5 kDa and 100 kDa systems), Sephadex G-200 gel filtration chromatography, and DEAE Sepharose CL-4B anion exchange chromatography. The purified tannase appeared to be homogeneous on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The purified tannase had a specific activity of 3.33 U mg−1, with a 1.3% recovery and overall purification of 302-fold. The molecular mass of the tannase estimated by SDS-PAGE was about 73 kDa. The tannase had an optimum pH of 6.0 and an optimum temperature of 40 °C. The most stable pH was 7.0, and the enzyme was stable up to 40 °C. One mmol L−1 of Fe3+, Sr2+, Na+, and Pb2+ were found to promote tannase activity, whilst 1.0 mmol L−1 of Ba2+, Ca2+, Mg2+, Zn2+, Hg+, Ag+, Co2+, Fe2+, Mn2+, Cu2+, Cd2+, Al3+, K+, Ni2+, and Li+ inhibited tannase activity.

Antigenic components, identification, and characterization of whole worm extract of Platynosomum illiciens

The antigenic components of adult Platynosomum illiciens were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cats naturally infected with P. illiciens, Dipylidium caninum, Toxocara cati and uninfected cat sera. The whole worm extract (WWE) of P. illiciens was fractionated by Sephadex G-200 gel filtration chromatography. The results showed that WWE fraction and F2 were highly antigenic as well as F1 and F3, which were moderately antigenic. For SDS-PAGE and immunoblotting, the antigenic molecules of WWE and all three fractions were mostly at molecular weights (MW) ranging from 11 to 150 kDa. Four antigenic proteins of 11, 18, 27 and 75 kDa detected in WWE and F1-F3 were found to give a reaction with sera from P. illiciens infected cats, and these proteins were also identified using liquid chromatography-mass spectrometry (LC-MS/MS). For immunolocalization observation, it was revealed that the P. illiciens antigen was present in high concentration in the cytoplasm of vitelline cells in the vitelline glands, the shell of the eggs and the eggs within the uterus, but not in other organs, i.e., tegument, muscle, parenchymal cells, testes and oral and ventral suckers of adult fluke. This finding indicates that these proteins may be potential antigen candidates for the immunodiagnosis of feline platynosomosis caused by P. illiciens.

A glycoprotein from mountain cultivated ginseng: Insights into their chemical characteristics and intracellular antioxidant activity

A glycoprotein (MGP2) from mountain-cultivated ginseng (MCG) was purified by Tris-HCl extraction followed by DEAE-52 ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The approximate molecular weight (27.0 kDa) and monomeric nature were determined by reduced and non-reduced SDS-PAGE. The structure of MGP2 was characterized by a practical and reliable “protein-polysaccharide analyzed by spectroscopy combined with chemical analysis” strategy. The results showed that MGP2 belonged to Arabinogalactan proteins (AGPs) which contained high amount of Glc (35.1 %). The hemagglutination test concluded that MGP2 was not a lectin. In addition, the MGP2 exhibited antioxidant activity by scavenging radical capacity tests and the ability to protect human erythrocytes and RAW264.7 cells from oxidative damage induced by AAPH. Therefore, these results suggested that glycoprotein MGP2 could be used as a natural antioxidant in drug and food industry.

Microbiological degradation of food dyes and amylase production, immobilization by Iraqi isolate Anoxybacillus rupiensis strain Ir3 (JQ912241)

Amylases are employed in the starch processing industries for the hydrolysis of polysaccharides such as starch into simple sugar constituents. With the advent of new frontiers in biotechnology, the spectrum of amylase applications has expanded into many new fields such as clinical, medicinal, and analytical chemistry. Α-Amylase from Anoxybacillus rupiensis strain Ir3 (JQ912241) isolated from hydrocarbon soil of Iraq was purified using ammonium sulphate precipitation and Sephadex G-200 gel filtration chromatography. Partially purified enzyme with 189U/ml activity was used for immobilization study by sodium alginate and Agar, The immobilized enzyme was very effective increase the activity of the enzyme to be (10.7, 14.8 U/ml) respectively for the sodium alginate and agar. This immobilized enzyme can be used commercially as a replacement of free enzyme because it has shown greater operational flexibility and higher enzymatic activity. The A. rupiensis strain Ir3 shows high decolorization (99%) of food dyes in the concentration 0.25 mg/ml for three days while (98%) decolorization the concentration 0.5 mg/ml for ten days.

The mechanism of interaction between lotus rhizome polyphenol oxidase and ascorbic acid: Inhibitory activity, thermodynamics, and conformation analysis.

In this study, the interaction between lotus rhizome polyphenol oxidase (PPO) and ascorbic acid (AA) was discussed from the aspects of inhibitory activity, thermodynamics, and conformation. Results showed that PPO was purified from lotus rhizome by DEAE-52 anion exchange chromatography and Sephadex G-100 gel filtration chromatography, with its optimum substrate being determined as pyrogallic acid. Spectrophotometric and polarographic assays demonstrated that AA exhibited strong inhibitory activity against PPO. Thermodynamics, fluorescence, and circular dichroism spectral analysis showed that hydrophobic interactions caused the formation of AA-PPO complex, leading to the remarkable fluorescence quenching and conformational change of PPO. Atomic force microscopic analysis revealed that binding to AA induced significant changes in the surface morphology and molecular aggregation of PPO molecules. In this study, the interaction mechanism between PPO and AA was proposed for the first time, which provided a theoretical basis for AA to inhibit lotus rhizome browning. PRACTICAL APPLICATIONS: Lotus rhizome, an aquatic vegetable, is prone to enzymatic browning in processing operations, which leads to a decrease in market value and economic loss. At present, ascorbic acid (AA) is widely used in industries as an excellent antioxidant because of its good antibrowning effect and relatively low cost. However, the interaction between the enzymatic browning-related polyphenol oxidase (PPO) from lotus rhizome and ascorbic acid has not been clearly studied. Understanding the mechanism of inhibiting PPO will help to prevent vegetable browning, especially fresh-cut products. The inhibitory effect of AA on PPO in lotus rhizome favors simultaneous use with other types of PPO inhibitors because of their likely synergistic effects.

Purification and characterization of chitinase produced by thermophilic fungi Thermomyces lanuginosus

Background In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed. Result The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of β-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa. Conclusion The characterization study concludes that Cu2+, Hg2+, and EDTA have an inhibitory effect on chitinase activity, whereas β-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste.

Production, Optimization, Purification, Kinetic analysis and applications of alkaline proteases produced from Bacillus subtilis through solid state fermentation of agricultural byproducts

Proteases have gained more commercial value to date due to multiple applications in different industrial sectors. Current research was aimed to use the cheaper agricultural waste for optimal protease production. Maximum level of protease production was achieved at 37 °C, incubation period of 24 h, pH 9.0, inoculum size 3%, 1.5 g sucrose as a carbon source and 30% moisture content by using solid-state fermentation. Among the various inorganic and organic nitrogen sources, ammonium nitrate and yeast extract tremendously increased the production of protease. Among metal ions and surfactants tested, Ca2+ and Tween 40 showed the optimal protease production. The purification of protease was carried out by ammonium salt precipitation followed by sephadex G-100 gel filtration chromatography. The purification resulted in 1.3 fold of purified protease with a specific activity of 51.5 and a total yield of 37.5 %. Molecular weight of purified protease was predicted upon SDS-PAGE with a single band of ~36 kDa. The protease was quite stable over a temperature range of 35-45 °C and pH 7-9 with maximal activity at 40 °C and pH 9. The kinetic parameters Vmax (maximum rate) and Km (Michaelis-Menten constant) were calculated as 0.307 U/g and 11.2 mg/mL, respectively. The alkaline protease significantly de-hair the goat skin and successfully removed the animal blood stain from cotton cloth.

IN VITRO ANTIOXIDANT ACTIVITY OF C-PHYCOCYANIN PURIFIED FROM Spirulina platensis DRY BIOMASS

C-phycocyanin (C-PC) was a phycobiliprotein found exclusively in cyanobacteria as Spirulina and exhibited a variety of pharmacological properties, which were not yet studied for any purpose in commercial applications in the pharmaceutical and cosmetic industry in Vietnam. At present, there are no other sources of natural phycocyanin than Spirulina, it is the sole efficient source of natural phycocyanin production under photoautotrophic conditions. The present research was conducted to determine an efficient extraction method from C-PC from Spirulina platensis dry biomass. Antioxidant activity of purified C-PC was investigated. Extractions were carried out using distilled water and Na-phosphate buffer pH 7.0 as solvents and freeze\thaw and ultrasonication techniques were applied to optimize the extraction process. Extraction using the freeze\thaw method proved to be the most efficient method. Phycocyanin purification from crude extracts was carried out by a combination of methods such as ammonium sulfate (NH4)2SO4 65% precipitation, dialysis filter, and Sephadex G-100 gel filtration chromatography, to give 121.63 ± 0.03 mg\g C-PC content and 3.52 ± 0.04 purity. Antioxidant activity in vitro was determined by nitric oxide (NO) scavenging activity, percentage inhibition of NO by the C-PC in concentration 10 μg\mL to 100 μg\mL which significantly scavenged 55.89% to 91.54% of NO radicals, respectively. They exhibited strong NO radical scavenging capacity, the C-PC and vitamin C were able to reduce the stable NO radical to 50% reduction with EC50 of 4.53 and 3.66 µg\mL. Research results showed that S. platenis dry biomass provided by Dalitra was potential for obtaining C-phycocyanin (12.16% of dry biomass) and it was evaluated as an antioxidant in vitro able to scavenge nitric oxide. C-PC (12.16% of dry biomass) and was evaluated as an antioxidant in vitro able to scavenge nitric oxide.

Purification, Characterization and Immunomodulatory Activities of Polysaccharides fromMulberryLeaf Fermented withPhellinus igniarius

Phellinus igniariusis a rare and precious medicinal fungus, displaying an outstanding physiological effect, especially the immunomodulatory effects. Previous studies indicated that water-soluble crude polysaccharide (MPFP) was obtained from mulberry leaf fermented withPhellinus igniarius. In vitrocell assay revealed that MPFP showed higher immunomodulatory activity than that of mulberry leaves polysaccharide (MP) andPhellinus igniariusmycelial polysaccharide (PP). Therefore, in this study, structure and immunomodulatory activity of MPFP were measured, a novel polysaccharide named MPFP2-1 was separated through DEAE-52 cellulose column and SephadexG-100 gel-filtration chromatography. Monosaccharide composition analysis showed that MPFP2-1 was mainly composed of L-rhamnose and D-glucose with the molar ratio of 1.0:5.4. The average molecular weight was 50.3 kDa by high performance gel permeation chromatography (HPGPC). FT-IR spectrum showed that MPFP2-1 contained a characteristic absorption peak of polysaccharide. The NMR spectrum indicated MPFP2-1 contained 1 → 6 glucosidic bond.In vitroimmunomodulatory assay revealed that MPFP2-1 significantly enhanced the macrophages proliferation, stimulated the macrophages phagocytic capacity, as well as induced NO and TNF-a generation. We further discovered that MPFP2-1 stimulated iNOS and TNF-αprotein expression in RAW264.7 cells by western blotting. The results are in agreement with ELISA. All the results suggest that MPFP2-1 possesses potent immunomodulatory activity and could be taken forward as new products for medicines.

Development of a downstream process for purification and purity analysis of glutaminase free L-asparaginase using UPLC, DLS-ZP and DSC-TGA

Several bacterial strains were isolated from soil and water in the current work and screened for glutaminase-free L-asparaginase activity. The selected strain was subjected to the production of glutaminase-free L-asparaginase and thereby purification. Before purification, pH scouting was performed, and 8.6 pH was the optimum working pH to carry out purification. The enzyme was purified using Sephadex G-200 gel filtration chromatography and up to 4.55-folds and its purity percentage was determined using UPLC. The purity of L-asparaginase was determined to be 99.2%. The enzyme was characterized using UV-Visible Spectrophotometer, DLS, ZP, DSC, TGA. The loss of the enzyme was 65% after sublimation, indicating its purity compared to the standard drug. The results report a developed process for the purification of L-asparaginase and its characterization, which can prove instrumental in the purification process of naturally occurring enzymes because today, most developed processes deal with recombinant enzymes.

A halotolerant hyaluronidase from newly isolated Brevibacterium halotolerans DC1: Purification and characterization

An enzyme hyaluronidase (hyase) producing halotolerant bacterium was isolated from dental caries and identified as Brevibacterium halotolerans DC1. Higher growth and hyase production were observed in nutrient broth fortified with hyaluronic acid at pH 7.0, temperature 37 °C, 120 rpm upon 48 h of incubation. Hyase was purified using salt precipitation, DEAE cellulose ion exchange, and Sephadex G-100 gel filtration chromatography. The enzyme was purified to 13-fold with 67.19% recovery of activity and 26.37 U/mg of specific activity. SDS-PAGE and zymography revealed it to be near to homogeneity showing a relative molecular weight of about 43 kDa that was confirmed by MALDI-TOF MS. This hyase was very active and stable at pH 7.0 and temperature 40 °C. The presence of metal ions Ca2+ and Mg2+ increased its activity while Zn2+ and Cu2+ severely inhibited it. Being stable at 2 M NaCl, hyase exhibited its halotolerant nature. This enzyme showed wide substrate specificity where hyaluronic acid (HA) was the best substrate. The kinetic studies revealed that Km and Vmax were 91.3 μg/mL and 306.2 μg/mL/min respectively. This is the first report of hyaluronidase from a halotolerant Brevibacterium spp. which can find applications under high salinity.

Studies on the extremo-lipase produced by the halotolerant Oceanobacillus iheyensis strain QCS

In the current work, different studies were carried out on the lipase enzyme produced by the halotolerant Oceanobacillus iheyensis strain QCS, with an expectation to be an important candidate in the industrial applications. Lipase of strain QCS was halo-alkali-thermo-detergent-solvent stable. Maximum production of lipase was obtained after 72 h of incubation, at 40oC and pH 8 and 9 in a medium containing 25 % (w/v) NaCl and 1% (v/v) olive oil as a lipid substrate. This lipase was partially purified, highest lipase activity was obtained in 80% ammonium sulfate saturation, and in fraction eight of the Sephadex G-200 gel filtration chromatography. Lipase displayed wide spectrum of activity within a broad range of conditions including salinity, temperature and pH, it was optimally active at 25% (w/v) NaCl, 40°C and pH 8 and 9, respectively. The effect of many metal ions, detergents and organic solvents on the activity of lipase was evaluated. Interestingly, lipase was able to retain the majority of its activity in the presence of Ni2+, Mg2+, Oxi and Fairy detergents, ethyl acetate, dimethyl formamide and toluene, respectively. Overall, as the lipase from O. iheyensis strain QCS has a number of interesting properties especially its stability at extreme conditions; it could be used as a potential promising candidate for detergents industry, and as a biocatalyst in low water enzymatic processes.

Purification and biochemical characterization of a novel secretory dipeptidyl peptidase IV from porcine serum.

Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtrationfollowed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160kDa. Distinct single band was observedafter PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37°C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC50 value of 8.473µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia, ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25µg.

Characterization of Extracellular Protease from the Haloarcheon Halococcus sp. Strain GUGFAWS-3 (MF425611).

Halococcus agarilyticus GUGFAWS-3 (MF425611) was isolated from a marine white sponge of Haliclona sp., inhabiting the rocks in the intertidal region of Anjuna, Goa, India. Uniquely, the microbe simultaneously produces two halo-extremozymes in 25% NaCl, namely protease and lipase at 49.5 ± 0.4 and 3.67 ± 0.02 (UmL-1), respectively. The protease is constitutively produced in starch mineral salts medium with consistent 4 ± 1.0mm zone of enzyme production, regardless of the non-availability of protein as substrate. The ethanol precipitated enzyme on dialysis and Sephadex G-200 gel filtration chromatography was partially purified to 12.26-fold and was active between 20 and 80°C, 0-5M NaCl, and pH 3-13. Optimum activity, however, was at 70°C, 3M NaCl, and pH 7. The enzyme was thermo stable at 70°C with 50.26 ± 2.40% of relative enzyme activity at 75min. Furthermore, it was stable in the presence of polar and non-polar organic solvents, detergents, and hydrocarbons. Several metal cations enhanced its activity in the order of Ca2+ > Ni2+ > Fe3+ > Co2+ > Mg2+ > Cu2+ > Mn2+. Dependence of enzyme on cysteine; serine, and metal ions was confirmed by β-mercaptoethanol; PMSF and EDTA, respectively which induced its partial inhibition. Additionally, protease inhibited in vitro biofilm formation in Staphylococcus aureus. Conclusively, the production of a neutral halo-thermophilic protease is reported for the first time in the genus Halococcus.

Purification and characterization of low molecular weight alkali stable xylanase from Neosartorya spinosa UZ-2-11

Abstract Alkaliphilic xylanase from Neosartorya spinosa UZ-2-11 was purified using a three-step of purification scheme of ammonium sulphate precipitation followed by Sephadex G-100 gel filtration and DEAE-cellulose ion-exchange chromatography, and compared its properties with N. tatenoi KKU-CLB-3-2-4-1 of our previous report. The purified xylanase from N. spinosa UZ-2-11 exhibited maximum activity at pH 9.0 and 45 °C which was similar to endo-xylanase from N. tatenoi KKU-CLB-3-2-4-1. However, this enzyme was stable in a range of pH 6.0–11.0. It was also more stable at a high temperature of 50 °C where the activity was still up to 50% after heating for 120 min. The xylanase was purified 7.89-fold with 3.0% of yield to obtain a specific activity of 11.88 U/mg. The molecular weight of xylanase from this fungus was 27.68 kDa. The Km and Vmax values of the purified xylanase were 0.24 mg/mL and 15.85 μmol/min/mg, respectively. The xylanase activity was moderately inhibited by Hg2+ at a concentration of 10 mM, which was different to the case of N. tatenoi KKU-CLB-3-2-4-1 where Hg2+ was a strong inhibitor. In addition, the hydrolysed birchwood xylan was obtained mailnly xylobiose, xylotriose, xylotetraose and xylopentaose as end products, suggesting that it was an endo-xylanase.

Purification, characterization, and functional properties of a novel glycoprotein from tartary buckwheat (Fagopyrum tartaricum) seed

A pure glycoprotein (BGP4-I) was obtained from tartary buckwheat seeds by aqueous extraction followed by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The average molecular weight of BGP4-I, as determined by high performance gel permeation chromatography, was 123.43 kDa. The structure of BGP4-I was characterized based on Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy, etc. Based on the nano-liquid chromatography-coupled electrospray ionization mass spectrometry analysis of the amino acid sequence of BGP4-I, belongs unequivocally to the glycosyl hydrolase family 1 in the Carbohydrate Active Enzymes database by alignment studies. The specific activity of BGP4-I was 18.44 μmol/min/mg on the substrate p-nitrophenyl-β-d-glucopyranoside. Furthermore, BGP4-I is unique in its specificity for some substrates. These results suggest that the BGP4-I from tartary buckwheat seeds is a novel specific β-glucosidase setting the foundation for potential applications in the food industry.