A thermostable laccase from Thermus sp. 2.9 and its potential for delignification of Eucalyptus biomass

Laura E Navas,María E Taverna,Eleonora Campos,Verónica Nicolau,Marcelo F Berretta,Lindsay D Eltis,Morgan M Fetherolf,Graciela B Benintende,Fernando D Martínez,Diana Estenoz

doi:10.1186/s13568-019-0748-y

Abstract

Laccases are multicopper oxidases that are being studied for their potential application in pretreatment strategies of lignocellulosic feedstocks for bioethanol production. Here, we report the expression and characterization of a predicted laccase (LAC_2.9) from the thermophilic bacterial strain Thermus sp. 2.9 and investigate its capacity to delignify lignocellulosic biomass. The purified enzyme displayed a blue color typical of laccases, showed strict copper dependence and retained 80% of its activity after 16 h at 70 °C. At 60 °C, the enzyme oxidized 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) at optimal pH of 5 and 6, respectively. LAC_2.9 had higher substrate specificity (kcat/KM) for DMP with a calculated value that accounts for one of the highest reported for laccases. Further, the enzyme oxidized a phenolic lignin model dimer. The incubation of steam-exploded eucalyptus biomass with LAC_2.9 and 1-hydroxybenzotriazole (HBT) as mediator changed the structural properties of the lignocellulose as evidenced by Fourier transform infrared (FTIR) spectroscopy and thermo-gravimetric analysis (TGA). However, this did not increase the yield of sugars released by enzymatic saccharification. In conclusion, LAC_2.9 is a thermostable laccase with potential application in the delignification of lignocellulosic biomass.

Highlights

Laccases (EC 1.10.3.2, benzenediol:oxygen oxidoreductase) are multicopper oxidases (MCO) that catalyze the oxidation of a variety of phenolic and non-phenolic compounds with the concomitant reduction of molecular oxygen to water (Thurston 2019)
Bacterial laccases possess a variety of attractive characteristics as compared to fungal laccases, especially with respect to pH-range of activity, thermal stability and resistance to chlorides (Chauhan et al 2017)
The DNA encoding the mature LAC_2.9 protein with an N-terminal 6xHis tag was amplified from Thermus sp. 2.9 genomic DNA by PCR using primers: 5′-aggccttcatatgcatcatcaccatcaccaccaggccccctttcccga-3′ and 5′-tggtggt-gggtctagattagctcacctccagaat-3′ (NdeI and XbaI restriction sites are underlined)

Summary

Introduction

Laccases (EC 1.10.3.2, benzenediol:oxygen oxidoreductase) are multicopper oxidases (MCO) that catalyze the oxidation of a variety of phenolic and non-phenolic compounds with the concomitant reduction of molecular oxygen to water (Thurston 2019). These enzymes are widely distributed in nature, occurring in plants, insects, fungi and bacteria (Bertrand et al 2017). Laccases typically comprise three domains and contain four copper ions arranged in mononuclear and trinuclear clusters: substrate oxidation at the mononuclear site generates electrons that are transferred to the trinuclear site where O2 is reduced (Thurston 2019). Among 2719 proteincoding genes predicted from the bacterium’s genome sequence (Navas et al 2015), we identified a gene designated lac_2.9 that encodes a protein with amino acid sequence similarity to laccases

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Conclusion