Description
An economical method of producing recombinant Arthromyces ramosus peroxidase with high yield, purity, stability and robustness
Background
Peroxidases are valuable heme-containing enzymes that catalyze the oxidation of various phenolic and non-phenolic substrates by hydrogen peroxide. They have important applications in biomedicine, agriculture, industry and the environment. Horseradish peroxides (HRP) from the root of horseradish as a commercially important enzyme has been studied deeply and utilized widely for more than a century due to its high catalytic efficiency and broad substrate specificity. HRP is frequently used in biosensors, bioassays, enzyme-linked immunosorbent assays, bioremediation, and pharmaceutical synthesis. The wide popularity of HRP has led to the neglect of alternative peroxidases with potentially greater characteristics.
The fungus Arthromyces ramosus peroxidase (ARP) has a broad substrate specificity and higher catalytic efficiency than HRP. Furthermore, ARP and HRP share a conserved catalytic site. ARP was once commercially available from Sigma. Attempts have been made to express recombinant ARP (rARP). However, these efforts were hindered by low yield leading to a need for an economical and effective expression system to produce rARP.
Technology Overview
Researchers at the University of Hawaii have developed an economical method of producing rARP with high yield and purity. rARP has similar substrate specificity profiles as HRP, but a catalytic efficiency up to 11-fold higher than HRP.
The gene sequence of rARP from the mature protein of A. ramosus peroxidase was codon optimized and expressed using the methylotrophic Komagataella phaffii (formerly named Pichia pastoris) to obtain the high yields required. rARP has advantages of easy purification, good yield, and high specific activity. The specific activity of purified rARP has up to 29-fold higher than HRP and other recombinant peroxidases. The Michaelis constant (Km) value and first-order rate constant (kcat) value of rARP showed 10-fold substrate affinity and consequently 6-fold catalytic efficiency of HRP toward the substrate ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]).
Application value of rARP is exemplified with efficient, fast and robust degradation of the antibiotic chlortetracycline in wastewater. Chlortetracycline has been extensively used as a veterinary medicine and as a human drug. It is recalcitrant and ubiquitous in the environment. The degradation half-life of chlortetracycline in wastewater under the rARP catalysis was less than 6 min at 25 and 40 °C in a range of pH from 5.0 to 9.0, which were up to 15-fold and 111-fold faster than HRP and laccase, respectively, but use of 3% amount of rARP as HRP. The degradation of chlortetracycline is complete; and the metabolites are nontoxic. The high catalytic efficiency and robustness are related to unique O-glycosylation of rARP.
Benefits
- High catalytic efficiency
- High specific activity
- Similar substrate specificity as HRP
- High stability and robustness
- Inexpensive to produce
- Ease of purification
- High yield
Applications
- HRP replacement
- Agriculture runoff treatment
- Biomedicine
- Environmental remediation
- Pharmaceutical synthesis