Characterization of a mildly alkalophilic and thermostable recombinant Thermus thermophilus laccase with applications in decolourization of dyes
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To examine the potential for applications of TthLAC, a monomeric (~ 53 kDa) laccase encoded by the genome of Thermus thermophilus (strain HB 27) which can be produced at low cost in Escherichia coli.
Functional, thermostable and mildly alkalophilic TthLAC of high purity (> 90%) was produced through simple heating of suspended (TthLAC overexpressing) E.coli cells at 65 °C. For reactions of short duration (< 1 h) the temperature for optimal activity is ~ 90 °C. However, TthLAC undergoes slow partial unfolding and thermal inactivation above 65 °C, making it unsuitable for long incubations above this temperature. With different substrates, optimal function was observed from pH 6 to 8. With the substrate, ABTS, catalytic efficiency (K m) and maximum velocity (Vmax) at 60 °C and pH 6.0 were determined to be 2.4 × 103 µM and 0.04 × 103 µM/min respectively. Ultra-pure, affinity-purified TthLAC was used to confirm and characterize the enzyme’s ability to oxidize known (laccase) substrates such as ABTS, syringaldazine and 4-fluoro-2-methylphenol. TthLAC decoloured up to six different industrial dyes, with or without the use of redox mediators such as ABTS.
Unlike versatile laccases from most other sources, which tend to be thermolabile as well as acidophilic, TthLAC is a versatile, thermostable, mildly alkalophilic laccase which can be produced at low cost in E.coli for various redox applications.
KeywordsDecolourization Delignification Oxidation Thermus thermophilus laccase Thermostable enzyme
AK and NK would like to thank the Centre of Excellence in Protein Science, Design and Engineering (CPSDE) funded by the Ministry of Human Resource Development (MHRD), Government of India, for financial support.
Supplementary Figure 1—(A) Amplification of the gene encoding TthLAC along with restriction sites and 6xHis affinity tag (1.3 kb). (B) Restriction-digestion based verification of the presence of the clone (gene) in the pET 28a Vector (size 5369 bp) through release of the insert of 1.3 kb upon digestion with NdeI and EcoRI.
Supplementary Figure 2—Fragmentation of a tryptic peptide of mass 1936 Da using ESI-TOF MS-MS mass spectrometery and analysis by the Biolynx tool of the Mass Lynx software of the WATERS G2SHDMS.
Supplementary Figure 3—(A) Far-UV circular dichroic (CD) spectrum of TthLAC. (B) Chromatographic profile of of TthLAC eluting from a Superdex-200 gel filtration column. (C) SDS-PAGE showing TthLAC incubated with glutaraldehyde. (D) Dynamic light scattering-derived hydrodynamic diameter data for TthLAC.
Supplementary Figure 4—Tmid values for the partial unfolding transitions of TthLAC.
Supplementary Figure 5—Denaturant-induced unfolding of TthLAC.
Supplementary Figure 6—(A) Oxidization of ABTS into a green product by TthLAC. (B) Variation of TthLAC activity with pH. (C) Absorption spectrum of ABTS after enzymatic treatment at pH 6.
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