Engineering of small laccase (SLAC) from Streptomyces coelicolor for application in biocatalysis and surface functionalisation

Abstract

The small laccase (SLAC) from Streptomyces coelicolor A3(2) is a versatile and industrially relevant biocatalyst mainly because of desirable characteristics such as activity at high temperatures and alkaline pH and relative stability against common laccase inhibitors. However, low yield from natural hosts, low catalytic efficiency and lack of reusability are some of the technological barriers that tend to limit the industrial application of the SLAC. In the present study, strategies have been developed to engineer SLAC for the improvement of catalytic properties and expression of SLAC for potential application in biocatalysis and surface functionalisation. Site directed mutagenesis was used to enhance properties of the enzyme. Four mutant sites (Y229A, Y230A, V290N and M198G) were selected on the basis of some positive results from literature and from the four sites, ten composite mutants including double, triple and quadruple mutants were generated. The produced variant SLAC enzymes were purified to homogeneity by Ni2+ affinity chromatography in a single step. A double mutant (Y230A/V290N) showed a low Km value which was 75% lower than wild type small laccase (WT-SLAC). Double mutations (Y230A/V290N and Y229A/M198G) enhanced the ability of the enzyme to decolourise common industrial dyes. For example, the double mutant Y229A/M198G exhibited a 1.2-fold higher catalytic efficiency (9.12 min-1mM-1) for the oxidation of ABTS than the WT-SLAC (7.46 min-1mM-1) and was able to decolourise 50 mg/L of Methyl Red (MR) completely, whilst only 28% decolourisation was observed with the WT-SLAC. To enhance enzyme production, the SLAC (from S. coelicolor A3(2)) was expressed in the methylotrophic yeast Pichia pastoris. The SLAC gene was cloned under the control of methanol inducible alcohol oxidase 1 (AOX1) promoter. The recombinant P. pastoris yielded high titres of extracellular laccase (500 ± 10 U/L) upon induction with methanol. The extracellular SLAC (~38 kDa) was purified to homogeneity with a specific activity of 8916.66 U/mg. The purified SLAC had an optimum activity at 80 C, but optimum pH varied with substrate used (pH 4 for ABTS and pH 8 for syringaldazine (SGZ) and 2,6-dimethoxy-phenol (2,6-DMP). Km values for ABTS, SGZ and 2,6-DMP were 142.85 μM, 10 μM and 54.55 μM and the corresponding kcat values were 60.6 s−1 , 25.36 s−1 and 27.84 s−1 , respectively. The t1/2 values of the recombinant SLAC (rSLAC) at 60 ºC, 70 ºC, 80 ºC were found to be 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (Ed) was 117.275 kJ/mol while ∆G, ∆H and ∆S for thermal inactivation of the rSLAC were all positive. To further enhance enzyme production, a dual promoter system was investigated using a combination of AOX (inducible promoter) and GAP (constitutive promoter). To this end, a recombinant P. pastoris strain harbouring rSLAC under control of both AOX and GAP promoters, was generated. A production level of 1800 U/L was recorded for rSLAC-GAPAOX which was about 3.6-fold higher than the single promoter system (rSLAC-AOX). Mixed feed strategy (9:1 ratio of methanol/glycerol) led to a positive influence in biomass accumulation and 1.5-fold increase in rSLAC expression, compared with induction of the double promoter system with methanol alone. To facilitate reusability of SLAC, the enzyme was immobilised onto surface of silanized magnetic nanoparticles (Si-MNP’s). Briefly, a silane layer was coated using 3-(aminopropyl) triethoxysilane (APTES) over the surface of MNPs, which was followed by immobilisation of rSLAC via covalent attachment. Field emission-scanning electron microscopy (FE-SEM) images of MNPs revealed that the diameter of the MNPs was in the range of 50-200 nm. Fourier transform-infrared spectroscopy (FT-IR) spectra of MNP, MNP/APTES and MNP/APTES/rSLAC, confirmed the presence of characteristic peaks at 589 cm-1, 2360 cm-1 and 1648 cm-1, respectively. MNP-rSLAC showed remarkable storage stability (retained ≥95% of initial activity after storage in Tris-HCl buffer 20 mM, pH 8 at 4 °C over a period of 30 days), temperature stability, and tolerance towards organic solvents and heavy metals. Repeated usage of MNP-rSLAC showed >73% of its initial activity after 10 catalytic cycles and the enzyme was easily recovered from the reaction mixture by the application of a magnetic field. The potential application of rSLAC was investigated in the degradation of several pollutants and in surface functionalisation. rSLAC efficiently decolourised two synthetic dyes tested belonging to triphenylmethane and azo group of dyes. More than 90% decolourisation was achieved for Brilliant Blue G and Trypan Blue in 6 hours without the assistance of any mediator. Several phenolic pollutants such as phenol, 4-chlorophenol (4-CP) and 4- fluorophenol (4-FP), categorised as “priority pollutants”, were completely degraded within 2 hours using only 2 U of rSLAC. Growth inhibition studies using Escherichia coli showed that rSLAC-mediated treatment of phenolic compounds reduced the toxicity of phenol, 4-CP and 4-FP by 90, 60 and 55%, respectively. In addition, ciprofloxacin and tetracycline, two of the most persistent classes of drugs were also degraded by rSLAC in combination with acetosyringone (AS) as mediator. rSLAC-AS degraded 95% of 5 mg/L ciprofloxacin (CIP) and 100 % of 150 mg/L of tetracycline (TC) within 6 hours. The removal of TC resulted in complete elimination of antibacterial activity while up to 48% reduction in antibacterial activity was observed when CIP was removed. The rSLAC also catalysed the functionalisation of two biological materials (chitosan and coconut fibres) to improve antimicrobial properties. The rSLAC oxidised functional molecules to corresponding radicals which reacted with the lignin moieties and amino groups of the coconut fibres and chitosan, respectively. The appearance of broad absorption band around 380 and 450 nm of the UV/Vis spectra of grafted chitosan films, indicated a reaction between oquinone and amino groups of chitosan. FT-IR spectrum of grafted biomaterials showed new aromatic skeletal vibrations as well as phenolic absorption bands indicating conjugation of allelochemicals onto chitosan and coconut fibres. Antimicrobial activities of grafted biomaterials were found to be up to 60% higher than that of their ungrafted counterparts. In conclusion, the SLAC from S. coelicolor A3(2) was engineered to enhance its catalytic properties and expressed extracellularly in P. pastoris. With combined use of constitutive and inducible promoters, production level reached up to 1800 U/L, which was among the highest yields attained for recombinant bacterial laccases expressed in P. pastoris. This study has shown that a combination of site-directed mutagenesis, secretory expression and immobilisation could lead to the production of a viable rSLAC for application in bioremediation and surface functionalisation.