Purification, application and immunolocalization of thermostable xylanases

2014 ◽  
Author(s):  
◽  
Stephanie Govender
Keyword(s):  
Reducing Sugars ◽  
Animal Feed ◽  
Paper Industry ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Thermomyces Lanuginosus ◽  
Sds Page ◽  
Km Value ◽  
Enzyme Dosage ◽  
Paper And Pulp

Microbial enzymes are gaining worldwide attention due to their potential industrial applications. Microorganisms producing thermostable -xylanase and their associated hemicellulases have significant application in the paper and pulp, food, animal feed, and textile industries. The potential of partially purified xylanase from Thermomyces lanuginosus MC 134, Luminase PB 100, Luminase PB 200 (a commercial xylanase) and T. lanuginosus DSM 5826 (Sigma Aldrich) was evaluated in bleaching of bagasse pulp. The temperature and pH optima for all the enzymes were 60°C and pH 6, respectively. The temperature (50- 80°C) and pH (5-8) stability of the enzymes were also assessed. All the enzymes were relatively stable at 60°C and pH 6 for 180 min. T. lanuginosus MC 134 retained 80% of its activity at 60°C and pH 6 for 180 min and PB 200 retained 75% of its activity at 80°C for 180 min. T. lanuginosus MC 134 also exhibited good alkaline stability at pH 8. The commercial xylanases Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 (Sigma Aldrich) were purified to homogeneity using a gel filtration column packed with sephadex G-100 and characterized for Km and Vmax. However extracellular crude xylanases from T. lanuginosus MC 134 was purified to homogeneity using (N )2S04 precipitation and gel filtration column, packed with sephadex G-100. The purified xylanases exhibited a molecular mass of- 26 to 24 kDa, given range as determined by SDS page. The Km and Vmax values of Luminase PB 100, Luminase PB 200, T. lanuginosus MC 134, and T. lanuginosus DSM 5826, xylanases were determined by the Michaelis-Menten equation using birchwood xylan as the substrate. The Km value for Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 and T. lanuginosus MC 134 were, 8.1 mg/mL, 11.7 mg/mL and 14.3 mg/mL respectively. The Vmax for Luminase PB 100, Luminase PB 200, T lanuginosus DSM 5826 and T lanuginosus MC 134 were 232.6, 454.6 and 74.6 !Jl11ol/min/mg. Biobleaching conditions of the xylanases were also optimised and the release of reducing sugars and lignin derived compounds showed that an enzyme dosage of 50U/g of pulp was ideal for biobleaching at pH 6 and 60°C for 180 min. This brightness for T lanuginosus MC 134, Luminase PB 200, Luminase PB 100 was 45.5 ± 0.11%, 44.1 ± 0.007% and 42.7 ± 0.03% respectively at pH 6, compared to untreated samples. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. All the enzymes analysed exhibited similar trends in the release of lignin derived compounds and reducing sugars which indicated their potential in the pulp and paper industry.

Immobilization of Purified Fungal Laccase on Cost Effective Green Coconut Fiber and Study of its Physical and Kinetic Characteristics in Both Free and Immobilized Form

2019 ◽  
Vol 8 (1) ◽  
pp. 3-14 ◽  
Author(s):  
Priyanka Ghosh ◽  
Uma Ghosh
Keyword(s):  
Activation Energy ◽  
Gel Filtration ◽  
Cost Effective ◽  
Industrial Applications ◽  
Kinetic Properties ◽  
Coconut Fiber ◽  
Immobilized Laccase ◽  
Sds Page ◽  
Glutaraldehyde Solution ◽  
Laccase Enzyme

Background: Laccases are important enzymes that have numerous applications in different biotechnological sectors. Objective: The aim was to purify laccase from Aspergillus flavus PUF5, successfully immobilize it on coconut fiber and characterize different physical and kinetic properties under both free and immobilize conditions. Methods: Laccase from A. flavus PUF5 was purified using ammonium sulfate precipitation, followed by DEAE column chromatography and gel filtration using Sephadex G100. The molecular weight was determined through SDS-PAGE (12%). It was immobilized on pretreated coconut fiber through crosslinking by glutaraldehyde (4% v/v). Physical and kinetic parameters like optimum temperature, pH, thermostability, the effect of additives, activation energy, Km and Vmax for free and immobilized laccase were also analyzed. Recycling stability of the immobilized laccase was further determined. Results: The extracellular laccase (65 kDa) was purified up to homogeneity and was immobilized on acid-pretreated coconut fiber by 4% (v/v) glutaraldehyde solution at 30°C, pH 5.0. Activation energy (Ea) of free and immobilized laccase for oxidation of guaiacol was found to be 24.69 and 32.76 kJ mol-1 respectively. Immobilized laccase showed higher melting temperature (Tm) of (82.5°C) than free enzyme (73°C). Km and Vmax for free and immobilized laccase were found to be 0.67 mM, 0.70 mM and 280 U/mg, 336 U/mg respectively when guaiacol was used as substrate. Additionally, in immobilized condition laccase retained ˃80% of its initial activity after use till six repeated cycles. Conclusion: The purified laccase enzyme and the cheap immobilization seem to be a prospective process for different biotechnological and industrial applications.

scholarly journals Peroxidase from Coleus Forskohlii: Purification and Biochemical Characterization

2020 ◽  
Vol 5 (1) ◽  
pp. 9-20
Author(s):  
Yaaser Q. Almulaiky ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Ion Exchange ◽  
Peroxidase Activity ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Coleus Forskohlii ◽  
Sds Page ◽  
Optimum Ph

In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.

Purification and Thermodynamic Characteristics of an Exo-Polygalacturonase from Trichoderma Pseudokoningii

2020 ◽  
Vol 42 (5) ◽  
pp. 767-767
Author(s):  
Wesam H Abdulaal and Yaaser Q Almulaiky Wesam H Abdulaal and Yaaser Q Almulaiky
Keyword(s):  
Food Industry ◽  
Potential Role ◽  
Fruit Juice ◽  
Gel Filtration ◽  
Thermodynamic Characteristics ◽  
Orange Peel ◽  
Sds Page ◽  
Trichoderma Pseudokoningii ◽  
Km Value

Polygalacturonases (PGs) are necessary to degrade the insoluble viscous pectin components during the clarification process of fruit juice and are produced by some plants and various microbes, such as bacteria, yeasts and fungi. In this study, an exo-polygalacturonase (exo-PGP4a) was purified from T. Pseudokoningii using DEAE-Sepharose and Sephacryl S-200 columns. We show that the enzyme produced in this study by solid-state fermentation of citrus Orange peel was purified 20-fold with 12.8% recovery. The apparent molecular mass of the enzyme was determined to be 25 kDa using gel filtration and SDS-PAGE. The optimum temperature and pH of the exo-PGP4a were 45and#176;C and 6, respectively. The exo-PGP4a showed half-lives of 50.95 and 21.32 min at 55 and 75and#176;C, respectively. The activation energy for denaturation (Ea*) was 42.596 kJ/mol. The Km value of the enzyme for PGA hydrolysis was 2 mg/ml, and the Vmax was 3.27 and#181;mol min-1 mg-1. Several metal cations, such as Cu2+and Zn2+, were found to enhance the enzymatic activity of the exo-PGP4a, while Pb2+, Ca2+, Ni2+, Cd2+, Co2+ and Hg2+ ions were found to be inhibitory. In this study, we suggest the exo-polygalacturonase has potential role of the clarification of Orange, Apple, Grape, and Peach juices in the food industry.

scholarly journals Purification and Characterization of Peroxidase From Anthracnose Disease Infected Papaya (Carica papaya L.)

2014 ◽  
Vol 6 (2) ◽  
pp. 49-57 ◽  
Author(s):  
L Bari ◽  
P Hassan ◽  
N Absar ◽  
S Khatun ◽  
MI Hossain
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Potassium Cyanide ◽  
Ammonium Sulphate Precipitation ◽  
Reducing Conditions ◽  
Sds Page ◽  
Peroxidase Enzyme ◽  
Km Value ◽  
Temperature Optima

Peroxidase enzyme was isolated and purified from the pulp of disease infected ripen papaya of local variety by 90% ammonium sulphate precipitation, chromatography on DEAEcellulose followed by hydrophobic chromatography on Phenyl Sepharose CL-4B and the purifications achieved was about 7.2 fold with 2.5% recovery. The purified enzyme was homogeneous as judged by polyacrylamide slab gel electrophoresis. The purified enzyme had a Mr of about 55,000 and 50 000 as determined by gel filtration on Sephadex G-100 and SDS-PAGE, respectively. The molecular mass of the enzyme was found to be very similar under both reducing and non-reducing conditions indicating that the enzyme contains no subunit. The enzyme has the following characteristics: pH optima at 6.0, temperature optima around 38°C, enzyme activity was found to be strongly inhibited in the presence of potassium cyanide and Fe+2 while the activity was found to be remarkably increased in the presence of ammonium sulphate. The Km value for the peroxidase obtained with pyrogallol as substrate was 0.027 mM. DOI: http://dx.doi.org/10.3329/bjmb.v6i2.17643 Bangladesh J Med Biochem 2013; 6(2): 49-57

scholarly journals Laccase Production by Trameteshirsuta, Characterization, and Its Capability of Decoloring Chlorophyll

2014 ◽  
Vol 63 (3) ◽  
pp. 323-333 ◽  
Author(s):  
JIAYANG LIU ◽  
WENHUA LIU ◽  
YUJIE CAI ◽  
XIANGRU LIAO ◽  
QINGGUO HUANG ◽  
...  
Keyword(s):  
Water Hyacinth ◽  
Room Temperature ◽  
Carbon Sources ◽  
Industrial Applications ◽  
Laccase Production ◽  
Fungal Laccases ◽  
Trametes Hirsuta ◽  
Sds Page ◽  
Extracellular Laccase ◽  
Enzyme Dosage

The present study focused on laccase production, characterization, and its involvement in chlorophyll decolorization. Extracellular laccase, with the highest activity of 11 U/ml on day 8, was efficiently produced from Trametes hirsuta in 5 l bioreactor with optimized media comprising dual carbon sources, glucose and water hyacinth. A laccase was then purified from the supernatant to homogeneity with purification fold of 9.51 and recovery of 39.8% and an estimated molecular mass of 62 kDa by SDS-PAGE. The laccase showed activity at pH 2-6 and temperature 30-80°C and was relatively thermally stable at below 70°C and neutral pH. The laccase was applied to decolorize chlorophyll under different factors: temperature, pH, mediator, metal ions, and enzyme dosage. Other fungal laccases were also found to be able to degrade chlorophyll with rating from 52% to 88% following 1 h treatment with two laccase dosages (5 or 10 U/ml) in the absence of any other mediators at room temperature. These findings may be an important step in developing new, important, and commercially viable industrial applications for laccase enzymes.

scholarly journals Cloning and expression of xylanase variants in Pichia pastoris

2017 ◽  
Author(s):  
◽  
Natasha Govindarajulu
Keyword(s):  
Pichia Pastoris ◽  
Animal Feed ◽  
Negative Impact ◽  
Paper Industry ◽  
Xylanase Activity ◽  
Restriction Enzymes ◽  
Cloning And Expression ◽  
Thermomyces Lanuginosus ◽  
High Yield ◽  
Heterologous Proteins

Microbial xylanases have attracted considerable research interest because of their various applications in biotechnology including the biobleaching of kraft pulp, to increase the nutritional value of foods and animal feed as well as for their potential use in the production of ethanol and methane. In the paper and pulp industry, the bleaching process involves the use of toxic chemicals and in the interim produces harmful gases that have a negative impact on the environment. The application of enzymes for this process will potentially reduce the environmental pollution by this industry. In addition, using an enzyme that is thermostable and alkali tolerant means that they will remain active under the required processing conditions. The xylanase gene, xynA derived from Thermomyces lanuginosus DSM 5826, was previously evolved to produce a number of xylanase variants, which were further enhanced for increased thermostability and alkalinity. In this study, these variants were cloned in Pichia pastoris using the pBGP1 vector to achieve extracellular production of the recombinant proteins. The xylanase genes were isolated using PCR. Both vector and DNA inserts were linearized with restriction enzymes EcoRI and XbaI and ligated. Electroporation was employed to transform the yeast with the recombinant plasmids. This was followed by the expression of the enzymes in P. pastoris grown in yeast peptone glucose (YPD) medium. Enzyme activity was thereafter assessed and the yeast was found to produce 164, 78, 96 and 142 IU/ml of S325, S340, G41 and G53 xylanase respectively, higher levels than bacterial hosts. The enzymes were then characterized and it was established that the optimum temperatures and pH for maximum xylanase activity were, 60°C, pH 6 for S325; 40°C, pH 5 for S340; 60°C, pH 6 for G41 and 60°C, pH 7 for G53. i The pH and temperature stabilities of the respective enzymes were investigated, the S325 variant was exceptionally stable at a pH between 5 and 7 and temperature range of 40-80°C and retained a minimum of 40% of activity at higher pH and temperature after an incubation period of 90 min. The S340 variant was the least thermostable and alkali stable from all four variants, it however retained 40% of activity when subjected to conditions of pH 9, 80°C after 90 min. The G41 and G53 were highly stable under the pH and temperature conditions that they were subjected to. Thus being suitable for potential application in the pulp and paper industry. The enzymes were able to retain 80% of activity at pH 9, 80°C after 120 min. P. pastoris has been proven to be a more suitable protein expression vector than E. coli for a number of reasons, including; the ability to perform complex post-translational modifications and grow to high densities in minimal media resulting in the production of a high yield of heterologous proteins.

scholarly journals Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

1996 ◽  
Vol 317 (1) ◽  
pp. 135-140 ◽  
Author(s):  
Gabriele NIEMANN ◽  
Hans von BESSER ◽  
Rolf D. WALTER
Keyword(s):  
Ornithine Decarboxylase ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Single Copy ◽  
Ecori Fragment ◽  
Nucleotide Sequence Analysis ◽  
Panagrellus Redivivus ◽  
Sds Page ◽  
Km Value

A Southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5´ non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni2+-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 μmol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for l-ornithine was determined as 44 μM. The Ki values for putrescine, α-difluoromethylornithine, α-hydrazino-ornithine and α-methylornithine were calculated as 51, 34, 0.34 and 42 μM respectively.

scholarly journals Improved xylanase Characteristics upon enzyme entrapment Isolated from Thermomyces lanuginosus C9

2021 ◽  
Author(s):  
Muhammad Irfan ◽  
Jawairia Kiran ◽  
Salah Ud Din ◽  
Ameen ullah ◽  
Qurrat Ul Ain Rana ◽  
...  
Keyword(s):  
Calcium Alginate ◽  
Incubation Temperature ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Thermomyces Lanuginosus ◽  
Ph Profile ◽  
Immobilized Catalyst ◽  
Km Value ◽  
Catalyst Reusability ◽  
Enzyme Entrapment

Abstract Xylanases from microbial sources assume basic jobs in an assortment of industrial applications as a biocatalyst, and its applications generally require immobilization on supports to upgrade their stability. Enzyme immobilization is a thrilling decision to show signs of improved strength of enzymatic procedures. In this work, two sorts of polymeric backings (agar-agar and calcium alginate) are utilized to immobilize β-1,4-xylanase from Thermomyces lanuginosus C9 by entrapment, and afterward, biochemical properties of the entangled enzymes were performed. To create immobilized catalyst beads centralization of 4% agar while mix of sodium alginate 5% and calcium chloride 0.4 M was seen as ideal. Ideal reaction time for agar and calcium alginate immobilized protein increments from 10 to 25 and 30 min, separately. The incubation temperature expanded from 70°C to 75°C for agar however stayed unaltered for calcium alginate. The pH profile of free and immobilized xylanase was generally equal in both cases. Be that as it may, both the strategies changed the active boundaries of immobilized β-1,4-xylanase rather than free protein. High sub-atomic load of xylan limits dispersion which brings down the Vmax estimation of immobilized protein while Km value expanded. In contrast with agar-agar, protein immobilized inside calcium alginate display wide thermal stability and kept up 86.6% of its underlying activity at 80°C up to 150 min. Be that as it may, biotechnological portrayal demonstrated that the catalyst reusability was the most surprising discovery, predominantly of agar-agar immobilized xylanase, which held 31% activity after 7 cycles. These outcomes prove the biotechnical and monetary advantages of immobilization which help in an assortment of industrial applications.

scholarly journals Improvement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)

2007 ◽  
Author(s):  
◽  
Sarveshni Pillay
Keyword(s):  
Recombinant Dna ◽  
Paper Industry ◽  
Residual Activity ◽  
Industrial Applications ◽  
Thermomyces Lanuginosus ◽  
Single Amino Acid ◽  
Wild Type ◽  
Single Amino Acid Residue ◽  
Recombinant Dna Techniques

Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

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