Matrine-producing endophytic fungus Galactomyces candidum TRP-7: screening, identification, and fermentation conditions optimization for Matrine production.

Matrine (MA) is an alkaloid extracted from the root of genus Sophora with various pharmacological activities. Production of MA by endophytic fungi offers an alternative challenge to reduce the massive consumption to meet the increasing demand of MA. In the current study, the positive strains with MA producing ability were screened from endophytic fungal isolated from the root of Sophora tonkinensis Gagnep. Chromatographic analyses verified the identity of the produced MA. Among these fungi, Galactomyces candidum strain TRP-7 was the most valuable strain for MA production with the initial yield 8.26 mg L-1. The MA production was efficiently maximized up to 17.57 mg L-1 of fermentation broth, after optimization of eight process parameters using Plackett-Burman and Box-Behnken designs. The statistical optimization resulted in a 1.127 times increase in MA production as compared to the initial yield of TRP-7. This is the first report to isolate endophytic fungi with MA-producing activity from S. tonkinensis Gagnep., and to identify an endophytic fungus G. candidum TRP-7 as a new promising start strain for a higher MA yield.

Efficient, fast and robust degradation of chlortetracycline in wastewater catalyzed by recombinant Arthromyces ramosus peroxidase.

Chlortetracycline (CTC), a widely used antibiotic, is recalcitrant and ubiquitous in the environment. Enzymatic degradation of CTC is an economical and efficient bioremediation method. In this work, recombinant Arthromyces ramosus peroxidase (rARP) at a concentration of 3.13 × 10-9 M was used to catalyze rapid degradation of CTC in water. The second-order rate constants of rARP showed up to 62-fold catalytic efficiency of horseradish peroxidase (HRP) toward CTC. The degradation half-life of CTC at the concentrations of 2 and 40 mg L-1 in wastewater under the rARP catalysis was, respectively, 5.3 and 5.7 min at 25 °C, and 2.7 and 3.1 min at 40 °C, which were up to 15-fold and 111-fold faster than HRP and laccase, respectively, but use of 3 % the amount of rARP as HRP. rARP catalyzed degradation of CTC at 2-40 mg L-1 in wastewater completed in 20-24 min, and its catalytic efficiency varied within only 2-fold at 25-40 °C. rARP showed only 2-3-fold discrepancy of catalytic efficiency among pH 5.0, 7.5 and 9.0. CTC under rARP catalysis underwent demethylation and oxidation to form nontoxic N-dedimethyl-9-hydroxy-CTC. The high catalytic efficiency of rARP agreed with a short distance between rARP's δN-His56 and CTC's dimethylamine N as indicated by docking simulation. rARP is a useful enzyme for CTC bioremediation.

Recombinant Arthromyces ramosus Peroxidase Has Similar Substrate Specificity Profiles as, but a Catalytic Efficiency up to 11-Fold Higher than, Horseradish Peroxidase.

Fungal peroxidases are valuable enzymes. Arthromyces ramosus peroxidase (ARP) and horseradish peroxidase (HRP) share a conserved catalytic site. Both native ARP and recombinant ARP (rARP) were not commercially available. The substrate specificity and kinetic parameters of rARP and HRP were not well compared, particularly relevent to structure-activity relationship. In this work, rARP expressed by Komagataella phaffii had a production yield of 6.2 mg/L, up to 155-fold higher than ARP and other recombinant peroxidases, and a specific activity of 3240 units/mg toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), up to 29-fold higher than HRP and other peroxidases. The Michaelis constant (Km) and first-order rate constant (kcat) of rARP showed 10-fold substrate affinity and consequently 6-fold catalytic efficiency of HRP toward ABTS. Under optimal conditions, rARP shared similar substrate specificity profiles as commercial HRP; the second-order rate constants (kapp) of rARP showed 2-11-fold catalytic efficiency of HRP toward well-known peroxidase substrates. rARP's higher catalytic efficiency was also in agreement with the shorter binding distance of H/N-His56 in rARP/substrate in comparison to that of HRP/substrate, as illustrated by docking simulation. The rARP had similar substrate specificity profiles as, but higher specific activity and catalytic efficiency than, HRP, which merits its further structure-functional characterization and applications.