Abstract
Introduction
Understanding enzymes and enzyme systems involved in lignocellulosic biomass degradation is the central topic of the OXYMOD project, funded by the Center of Digital Life Norway. Beside enzymes that are active against cellulose, redox enzymes such as laccases and peroxidases contribute to the process of breaking down lignin fraction. From long term bioprospecting activities by SINTEF and NTNU, a unique collection of actinobacteria strains has been established and 1200 strains have been sequenced using Illumina shotgun sequencing. The actinobacteria phylum has been known as a rich source of enzymes for lignin degradation and therefore is an attractive target for in silico mining of novel redox biocatalysts.
Methods
A customized workflow of data mining based on profiles HMM, multiple sequence alignment, and sequence similarity network has been developed and used for mining of new laccases and peroxidases from the database of 1200 actinobacteria genomes. The detected hits were further manually curated to have a shortlist of sequence candidates which were subsequently sent for gene synthesis and subcloned in expression vectors. The candidates were expressed, purified and the activity of the enzymes were tested against model substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS). The laccases and peroxidases with confirmed ABTS activity were subsequently screened against a panel of small phenolic compounds as well as subjected to physiochemical characterization (pH optimum and stability, temperature stability).
Result and discussion
For mining of the new laccases, a custom profile HMM was created based on the sequences of two domain small laccases, SLAC-like family (Hfam39), obtained from Laccase and Multicopper Oxidase Engineering Database (LCCED). Four out of seven new laccase candidates were expressed in the soluble form and showed activity against ABTS after purification. Two enzymes, namely P06-A08 and P20-F12 appeared to be thermostable (activity was maintained even after two hours incubated at 80 °C). The pH optimum of the enzymes was identified in the range of pH 3.8 and 4.2 with the sharp decline of activity from pH 5 and forward. Among compounds tested for substrate scope study, activities against ferulic acid, sinapic acid, catechol, and 3,4-dihydroxybenzoic acid were confirmed.
Most of identified peroxidase hits from the strain collection belong to the Dyp-type family (dye de colourising peroxidase). Like the new laccases, four peroxidases were active against ABTS after purification and have a pH optimum in the limited range of 3.0 and 3.4. The de colourising activity was confirmed using commercial dyes, Remazol Brilliant Blue R and Reactive Blue 5.
Understanding enzymes and enzyme systems involved in lignocellulosic biomass degradation is the central topic of the OXYMOD project, funded by the Center of Digital Life Norway. Beside enzymes that are active against cellulose, redox enzymes such as laccases and peroxidases contribute to the process of breaking down lignin fraction. From long term bioprospecting activities by SINTEF and NTNU, a unique collection of actinobacteria strains has been established and 1200 strains have been sequenced using Illumina shotgun sequencing. The actinobacteria phylum has been known as a rich source of enzymes for lignin degradation and therefore is an attractive target for in silico mining of novel redox biocatalysts.
Methods
A customized workflow of data mining based on profiles HMM, multiple sequence alignment, and sequence similarity network has been developed and used for mining of new laccases and peroxidases from the database of 1200 actinobacteria genomes. The detected hits were further manually curated to have a shortlist of sequence candidates which were subsequently sent for gene synthesis and subcloned in expression vectors. The candidates were expressed, purified and the activity of the enzymes were tested against model substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS). The laccases and peroxidases with confirmed ABTS activity were subsequently screened against a panel of small phenolic compounds as well as subjected to physiochemical characterization (pH optimum and stability, temperature stability).
Result and discussion
For mining of the new laccases, a custom profile HMM was created based on the sequences of two domain small laccases, SLAC-like family (Hfam39), obtained from Laccase and Multicopper Oxidase Engineering Database (LCCED). Four out of seven new laccase candidates were expressed in the soluble form and showed activity against ABTS after purification. Two enzymes, namely P06-A08 and P20-F12 appeared to be thermostable (activity was maintained even after two hours incubated at 80 °C). The pH optimum of the enzymes was identified in the range of pH 3.8 and 4.2 with the sharp decline of activity from pH 5 and forward. Among compounds tested for substrate scope study, activities against ferulic acid, sinapic acid, catechol, and 3,4-dihydroxybenzoic acid were confirmed.
Most of identified peroxidase hits from the strain collection belong to the Dyp-type family (dye de colourising peroxidase). Like the new laccases, four peroxidases were active against ABTS after purification and have a pH optimum in the limited range of 3.0 and 3.4. The de colourising activity was confirmed using commercial dyes, Remazol Brilliant Blue R and Reactive Blue 5.