Annual Meeting Abstract Open Access

Biotechnology Congress 2015 : Activity and thermostabilty improvement of Trametes versicolor IBL-04 laccase by chitosan beads immobilization - Muhammad Asgher - University of Agriculture

Abstract

 The laccase enzyme has got tremendous applications in diverse industrial processes including biomass delignification, biopulping in paper and pulp, as denim stone wash agent, detergents, bioremediation and biosensor development. Monomeric 66 kDa laccase produced by Trametes versicolor IBL-04 in SSF of corn cobs (911 U/mL) was purified through ammonium sulphate precipitation, dialysis, ion-exchange chromatography and gel filtration. The purified laccase was immobilization using chitosan as support material and glutaraldehyde as activator/crosslinking agent. Chitosan concentration of 2.5% was optimum for preparation of most stable 2.0 mm size chitosan beads activated by1.5% gluteraldehyde for best laccase immobilization. Scanning electron microscopy showed that beads with immobilized laccase on the surface were spherical in shape having large surface area. The immobilized laccase was found to be catalytically more vigorous and stable and it worked over a wide range of pH 3-6 and temperature 45-65 oC. Laccase immobilized with chitosan beads had 936 U/mL at pH 6 and 60 °C and improved thermal behavior. The chitosan beads immobilized laccase had higher Km (93 μM) and V-max 944 μM/min values as compared to its soluble counterpart, thus, demonstrating its higher catalytic efficiency. The kinetic and thermostability characteristics of chitosan beads immobilized laccase reflect that the enzyme has potential for use in industrial and environmental biotechnology. The lignin mineralizing compounds arrangement of white decay growths has huge synergist prospect for oxidative bioremediation of various harmful poisons and numerous other mechanical employments. Immobilization empowers the reuse of catalysts and making them modernly significant and conservative biocatalysts. Schizophyllum collective IBL-06 was developed for the emission of lignin peroxidase (LiP) in pre-upgraded strong state maturation mode of corn stover. A high return of lignin peroxidase (1347.3 U/mL) was recorded in rough culture supernatant. LiP was filtered (5.65 folds) by a pre-normalized four stage convention containing ammonium sulfate fractionation, dialysis, DEAE cellulose particle trade and Sephadex G-100 segment chromatography. The 38 kDa single polypeptide S. collective IBL-06 LiP relocating as a solitary clear band on both local and SDS-PAGE gels. The refined compound was then immobilized on chitosan globules actuated with gluteraldehyde (cross-linker).Scanning electron microscopy (SEM) was led for the affirmation of LiP connection on chitosan globules. The most noteworthy material color decolorization (95.45%) potential was seen with chitosan-immobilized chemical at 30°C without hemolytic harmfulness. The chitosan globules LiP held roughly over 70% action after three rehashed runs that step by step diminished to 35 % after seventh pattern of reusability. The immobilized LiP was found to show better color evacuation properties as looked at than free LiP. Higher thermo steadiness, lower Kmand high Vmax highlights of chitosan dots immobilizedLiP proposed its suitibility for variousbiotechnological and modern applictaions. Watchwords: Schizophyllum cooperative IBL-06, lignin peroxidase, chitosan globules immobilization, portrayal, color decolorization. Presentation The catalysts are nitrogenous complex protein particles with explicit synergist capacities which are created by living cells to catalyze biochemical responses required forever (Alam et al., 2009). Compounds have superb properties, as synergist movement, high selectivity and particularity. In spite of all these attributes, they additionally have a few disservices. As dissolvability in response media, shakiness, thermo dependability, so it is hard to recover catalysts from the response blend (Chen et al., 2012). As a result of this issue, a few properties of the proteins ought to be improved prior to their usage at mechanical scale so as to decrease the expense of a concoction procedure. The operational soundness, reusability and recuperation of catalysts applied in the mechanical procedure can be improved by the utilization of mutagenic impacts, hereditary designing, and the immobilization of proteins or procedure changes (Asgher et al., 2013a). Protein attributes, for example, synergist effectiveness, particularity and different uses in modern forms are progressively supplanting the ordinary compound strategies in research facilities just as in enterprises. In any case, the advertising of proteins is at a more slow pace because of their significant expense and issues of capacity (Asgher et al., 2013a; Shi et al., 2003). Immobilization techniques help to create steady, recoverable and reusable chemicals for multi-reason mechanical and ecological applications bringing about critical money related advantages (Asgher et al., 2008b). Immobilized catalyst details will in general be affected by the basic properties of compound just as the help lattice (Wang et al., 2012). The particular connections between bearer material and the catalyst give altered chemicals unique biochemical, dynamic, mechanical and compound properties. Most much of the time utilized protein (compound) immobilization strategies are surface official, adsorption what's more, entanglement (Thakur et al., 2015). While, entanglement or epitome is chosen over surface restricting on the grounds that it is simpler, less expensive, and helpful and the structure of catalyst stays secured (Asgher et al., 2012c). Various strategies have been accounted for the treatment of material modern effluents and color corruption which are not financially and earth appropriate. These realities surely require the improvement of a successful, cost-proficient and earth benevolent innovation for the detoxification and corruption of mechanical effluents containing colors. At present the essential strategies applied The Journal of Animal and Plant Sciences, 26(5): 2016, Page: 1451-1463 ISSN: 1018-7081 Parveen et al., The J. Anim. Plant Sci. 26(5):2016 1452 for the treatment of material effluents dependent on physical or concoction methods, which are unfathomably costly and the collection of packed slop results in ecological fiascos (Alam et al., 2009; Parshetti et al., 2012). On the other hand, bio-sensible procedures offer reasonable and eco-accommodating remediation innovations and they simply don't create huge amounts of slop (Bilal et al., 2015).The interest in new biocatalyst practice has expanded during the most recent two decades in view of the expanding uses of xenobiotics, the corruption of these synthetic concoctions are not compelling and capable with customary compound procedures (Missau et al., 2014).The basidiomycetes; white decay growths (WRF) are the small scale living beings which broadly read for modern color corruption. With no affirm to fulfillment, the ligninolytic chemicals (LiP, MnP, Lac) of WRF included during the time spent color decolorization (Cheng et al., 2007; Maciel et al., 2010). LiP has incredible Redox potential and wide scope of utilizations in different mechanical methods. Refined LiP from Kocuria rosea uncovered a higher synergist action to decolorize responsive colors through different gatherings, demonstrating that it is an amazingly flexible peroxidase (Qiu et al., 2009). Biography Muhammad Asgher is the Chairman, Department of Biochemistry, University of Agriculture, Faisalabad. He graduated in 1985 from Gujranwala and got his MSc degree in 1988, Mphil in 1993 and PhD in 1998 in the subject of Biochemistry from University of Agriculture, Faisalabad, Pakistan. In 2002, he was awarded UNESCO Fellowship in Biotechnology and in 2004, a one year HEC Post-Doctoral Fellowship to work on production, purification and immobilization of industrial enzymes in University of Waterloo, Canada. In 2009, HEC awarded him Sabbatical Leave Fellowship to learn advanced enzyme immobilization techniques at University of Waterloo, Canada. After another 5 years he was awarded Sabbatical Research Fellowship by Biotechnology Lab. University of Waterloo, Canada for collaborative research and training on production and characterization of Biocomposites. He also has 2 national and 5 international Post-Graduate Research Training Courses to his credit. He joined the University of Agriculture, Faisalabad, Pakistan as a Lecturer of Biochemistry in 1990. Based on his outstanding academic performance, he was elevated as Assistant Professor in 1999, Associate Professor in 2004, Professor in 2007 and Professor TTS in 2010. He has been guiding the Postgraduate students in research and has so far produced 124 research students as Major Supervisor, including 10 PhD, 36 MPhil and 78 MSc students to provide trained manpower to industry and academia. As Researcher, he focused his investigations on indigenous production, purification, characterization and immobilization of microbial enzymes and their industrial applications. He has won and executed a number of research projects as PI and Co-PI.

Muhammad Asgher and Sadia Noreen

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