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Catalase-peroxidases are bifunctional heam enzymes and the only so far known peroxidases with a high catalase activity at neutral pH. Different types of catalases are found in all aerobic organisms, from bacteria, archaeons and fungi to humans. Their role is to catalyse the decomposition of hydrogen peroxide to molecular oxygen and water.
Catalase-peroxidase AfKatG from Archaeoglobus fulgidus was the first enzyme of this kind, isolated from a hyperthermophillic archaeon in 2001. This enzyme shows peroxidase and catalase activity with the optimal conditions at pH 4.5 and 80°C for peroxidase activity and pH 6.0 and 70°C for catalase activity.
The bifunctionality allows the use of its excellent catalytic activity for the degradation of hydrogen peroxide, while its peroxidase activity opens new possibilities of application of this enzyme. Catalases are able to catalyse different types of reduction-oxidation reactions. Because most of catalases are robust enzymes with unusually fast and effective catalysis, they found application in many industrial processes as an effective tool for removal of toxic hydrogen peroxide.
Catalases are a group of enzymes with application in a wide spectrum of industrial fields, including textile industry, biosensors, corrosion, micromotors, polymers, biopolymers, food industry and medical and pharmaceutical technologies.
The object of the present invention is the production and purification of recombinant catalase-peroxidase AfKatG suitable for large scale production and for the use in hydrogen peroxide degradation. This aim has been reached by the novel production method of this enzyme. The invention allows relatively simple production and purification of recombinant catalase-peroxidase AfKatG by the expression of synthetic gene, modified to meet the needs of the production host.
The method of production of recombinant catalase-peroxidase polypeptide includes the following steps: • bioreactor cultivation of E. coli cells carrying the plasmid with the synthetic gene of AfKatG, • addition of hemin prior to expression induction allowing the production of target polypeptide in soluble form, • induction of target polypeptide expression with IPTG, • cultivation of the culture to desired (density) value, • centrifugation of the culture and storage of the biomass.
Isolation of recombinant catalase-peroxidase includes the following steps: • cell homogenization, • removal of fractions without recombinant polypeptide by centrifugation followed by heating, • purification using affinity chromatography.
After purification, the produced enzyme can be applied during cultivation of microorganisms, as an oxidizing agent for oxidation of aromatic compounds and also for waste water treatment (degradation of hydrogen peroxide).

The proprietors of the technology are looking for an industrial partner for licensing the technology. More details in the Partner Sought.

Advantages & innovations

• Inexpensive production with high yields of catalase-peroxidase AfKatG, • Simple and straightforward purification of catalase-peroxidase AfKatG leading to a highly pure product.

Stage of development

Under development/lab tested

Partner sought

Field and role of a partner: There are multiple potential applications, primarily during processes where degradation of hydrogen peroxide as a by-product is desired, in biocatalytic conversions of aromatic compounds and for the protection against oxidative stress. Large scale production of AfKatG would allow wide use of this enzyme with extensive heat tolerance even during processes where conventional enzymes cannot be used. The simple cultivation of the microbial producer and subsequent simple purification of the enzyme allow relatively low production costs and increase the added value of the final product, which can be then used for monitoring of microbial activity, for oxidation of aromatic compounds and for degradation of hydrogen peroxide in processes where its presence is not desired (such as waste water from bleaching solutions). Type of cooperation: License agreement - The proprietors of the technology are looking for an industrial partner for licensing the technology.