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Development of magnetic cross-linked laccase aggregates and determination of laccase activity on ABTS and vanillin using raman spectroscopy

Perzon, Alixander LU (2016) KBK820 20141
Pure and Applied Biochemistry
Abstract
In order to broaden the use of laccase and increase its economic practicability for industrial oxidative processes, we have immobilized the enzyme in cross-linked enzyme aggregates (CLEA). The immobilization was carried out in two steps consisting of 1) precipitation- and 2) cross-linking of the enzymes. A factorial design was used in order to reveal the importance of different process parameters and their interplay for obtaining active CLEAs. The results show that the activity of the CLEA is increasing with the cross-linker concentration, and that co-immobilization of BSA increases the effect of the cross-linker. In addition, we found that the precipitating agent (ammonium sulphate) reacts with the cross-linker (glutaraldehyde), which... (More)
In order to broaden the use of laccase and increase its economic practicability for industrial oxidative processes, we have immobilized the enzyme in cross-linked enzyme aggregates (CLEA). The immobilization was carried out in two steps consisting of 1) precipitation- and 2) cross-linking of the enzymes. A factorial design was used in order to reveal the importance of different process parameters and their interplay for obtaining active CLEAs. The results show that the activity of the CLEA is increasing with the cross-linker concentration, and that co-immobilization of BSA increases the effect of the cross-linker. In addition, we found that the precipitating agent (ammonium sulphate) reacts with the cross-linker (glutaraldehyde), which added another parameter to the immobilization procedure. To enable magnetic re-use of the CLEA, iron oxide was synthesized and co-immobilized with laccase, which enabled the enzyme to be re-used at least two times. In addition, Raman spectroscopy was introduced as a more elaborate tool to monitor the catalytic activity of laccase. In order to validate the new method, the activity of laccase on ABTS was traced both using the standard colorimetric method, as well as with the Raman scattering technique. The results suggest that the KM value for laccase obtained with Raman scattering (24 μM) is different from the KM value obtained with the colorimetric method (59 μM). Laccase activity on vanillin was also traced with the Raman scattering technique, demonstrating its application on a substrate-to-product conversion that cannot be detected easily using standard absorption spectrophotometry. (Less)
Popular Abstract (Swedish)
Enzymer är biologiskt aktiva molekyler med en katalytisk förmåga. Detta innebär enzym kan medföra en omvandling av ett ämne i en reaktion som normalt inte sker spontant. Laccas är ett enzym som kan bryta ner (oxidera) fenolföreningar, och i naturen förekommer det i växter, svampar och bakterier där det fyller olika funktioner.
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author
Perzon, Alixander LU
supervisor
organization
course
KBK820 20141
year
type
H2 - Master's Degree (Two Years)
subject
keywords
magnetic nano-particle, enzymatic process, biocatalysis, clea, Laccase, cross-linked enzyme aggregates, raman spectroscopy, ABTS, vanillin, applied biochemistry, tillämpad biokemi
language
English
id
8775803
date added to LUP
2016-04-07 09:07:54
date last changed
2016-04-07 09:07:54
@misc{8775803,
  abstract     = {In order to broaden the use of laccase and increase its economic practicability for industrial oxidative processes, we have immobilized the enzyme in cross-linked enzyme aggregates (CLEA). The immobilization was carried out in two steps consisting of 1) precipitation- and 2) cross-linking of the enzymes. A factorial design was used in order to reveal the importance of different process parameters and their interplay for obtaining active CLEAs. The results show that the activity of the CLEA is increasing with the cross-linker concentration, and that co-immobilization of BSA increases the effect of the cross-linker. In addition, we found that the precipitating agent (ammonium sulphate) reacts with the cross-linker (glutaraldehyde), which added another parameter to the immobilization procedure. To enable magnetic re-use of the CLEA, iron oxide was synthesized and co-immobilized with laccase, which enabled the enzyme to be re-used at least two times. In addition, Raman spectroscopy was introduced as a more elaborate tool to monitor the catalytic activity of laccase. In order to validate the new method, the activity of laccase on ABTS was traced both using the standard colorimetric method, as well as with the Raman scattering technique. The results suggest that the KM value for laccase obtained with Raman scattering (24 μM) is different from the KM value obtained with the colorimetric method (59 μM). Laccase activity on vanillin was also traced with the Raman scattering technique, demonstrating its application on a substrate-to-product conversion that cannot be detected easily using standard absorption spectrophotometry.},
  author       = {Perzon, Alixander},
  keyword      = {magnetic nano-particle,enzymatic process,biocatalysis,clea,Laccase,cross-linked enzyme aggregates,raman spectroscopy,ABTS,vanillin,applied biochemistry,tillämpad biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development of magnetic cross-linked laccase aggregates and determination of laccase activity on ABTS and vanillin using raman spectroscopy},
  year         = {2016},
}