High level production of tyrosinase in recombinant Escherichia coli

Abstract

BackgroundTyrosinase is a bifunctional enzyme that catalyzes both the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of the diphenols to o-quinones (diphenolase activity). Due to the potential applications of tyrosinase in biotechnology, in particular in biocatalysis and for biosensors, it is desirable to develop a suitable low-cost process for efficient production of this enzyme. So far, the best production yield reported for tyrosinase was about 1 g L-1, which was achieved by cultivating the filamentous fungus Trichoderma reesei for 6 days.ResultsIn this work, tyrosinase from Verrucomicrobium spinosum was expressed in Escherichia coli and its production was studied in both batch and fed-batch cultivations. Effects of various key cultivation parameters on tyrosinase production were first examined in batch cultures to identify optimal conditions. It was found that a culture temperature of 32 °C and induction at the late growth stage were favorable, leading to a highest tyrosinase activity of 0.76 U mL-1. The fed-batch process was performed by using an exponential feeding strategy to achieve high cell density. With the fed-batch process, a final biomass concentration of 37 g L-1 (based on optical density) and a tyrosinase activity of 13 U mL-1 were obtained in 28 hours, leading to a yield of active tyrosinase of about 3 g L-1. The highest overall volumetric productivity of 103 mg of active tyrosinase per liter and hour (corresponding to 464 mU L-1 h-1) was determined, which is approximately 15 times higher than that obtained in batch cultures.ConclusionsWe have successfully expressed and produced gram quantities per liter of active tyrosinase in recombinant E. coli by optimizing the expression conditions and fed-batch cultivation strategy. Exponential feed of substrate helped to prolong the exponential phase of growth, to reduce the fermentation time and thus the cost. A specific tyrosinase production rate of 103 mg L−1 h−1 and a maximum volumetric activity of 464 mU L−1 h-1 were achieved in this study. These levels have not been reported previously.

Enterobacter sp. LU1 as a novel succinic acid producer – co‐utilization of glycerol and lactose

“…Although the capability of environmental strains of Enterobacter to produce succinic acid as the main product during anaerobic conditions has not been described in the literature, the results obtained in this study are not competitive with those for other thoroughly described microbial producers of succinic acid (Beauprez et al ., ). One way to increase bioproduct concentration during fermentation is by precisely controlling fermentation conditions, which can be achieved using different bioreactors (Ren et al ., ; Yan et al ., ). This study was initially unable to achieve succinic acid concentrations higher than 13 g l −1 .…”

Tyrosinase mediated humic substances synthesis by Bacillus aryabhattai TFG5

“…Sambasiva Rao[31]optimized the production conditions of Streptomyces antibiotics tyrosinase and showed maximum tyrosinase activity of 4.62 U.ml −1 . In another study recombinant E.coli produced tyrosinase to a high level of 13U.ml −1 [32]. Comparatively, in the present investigation B. aryabhattai TFG5 produced 1.34 U.ml −1 of tyrosinases and when induced for laccase it had produced 1.8 U.ml −1 without any optimization studies.…”

A New Strategy for Effective Succinic Acid Production by Enterobacter sp. LU1 Using a Medium Based on Crude Glycerol and Whey Permeate

“…Although the capability of bacteria of the genus Enterobacter to produce large quantities of succinic acid has recently been reported in the literature [10], the quantities that were produced are relatively low compared to those obtained using most well-described bacterial producers of this acid [27]. One of the factors that allows higher concentrations of succinic acid to be produced in the post-culture liquid is the bioreactor culture conditions [28][29][30]. In the present study, succinic acid production to a level of 35 g/L was achieved in the first bioreactor culture run, in which glycerol analytical grade and lactose were added to the medium at doses of 50 g/L and 25 g/L, respectively.…”