Abstract
Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.
Highlights
Horseradish peroxidase (HRP) (EC 1.11.1.7) has high industrial relevance entailing an immense pool of published data, ranging from refolding of denatured plant enzyme to recombinant production in different hosts [1,2,3,4,5,6,7]
Plant HRP has a size of 44 kDa, but when the enzyme is produced in Escherichia coli it is unglycosylated resulting in a size of only 34.5 kDa [11]
In this study we present a novel production process for HRP from E. coli inclusion bodies (IBs) with upscaling potential to industrial dimensions
Summary
Horseradish peroxidase (HRP) (EC 1.11.1.7) has high industrial relevance entailing an immense pool of published data, ranging from refolding of denatured plant enzyme to recombinant production in different hosts [1,2,3,4,5,6,7]. Asad et al [41] published a comprehensive investigation on the refolding conditions of horseradish peroxidase, focusing on the buffer system, redox conditions and additives suitable for HRP stabilization They used one factor at a time and response surface methodology (RSM) to obtain a yield of 3.6 mg HRP from 15 mg solubilized protein. In 2016, we performed a comparative study on the production of HRP both in a soluble form and from IBs. We obtained a relatively high yield of 150 mg HRP/L culture medium for refolded HRP with a specific activity of 62.5 U/mg (ABTS), but only 15 mg/L could be recovered after concentration with spin filters [44]. We developed a novel protocol for salt precipitation and hydrophobic interaction chromatography (HIC) The combination of these optimized conditions resulted in a final process yield of 959 mg active HRP/L culture medium with a purity of ≥99% determined by SEC-HPLC. The developed process allows the scalable production of the unglycosylated, single isoenzyme HRP C1A at the highest yield reported far (Table 1)
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