Effectivity of tyrosinase purification by membrane techniques versus fractionation by salting out

Abstract

The main goal of this study was to select micro-and ultrafiltration membranes that can be used for the purification of mushroom tyrosinase, replacing salting-out dual-step processes followed by centrifugations. In experiments, a raw extract from white mushrooms was used with high level of ballast proteins and brownish impurities. Four microfiltration membranes for the removal of undesired high molecular weight compounds were screened and that made of nitrocellulose was selected due to high recovery of enzymatic activity. Then diafiltration and concentration on the membrane made of polyethersulphone (300 kDa) was selected to recover 8% of proteins and 58% of tyrosinase activity with five-to seven purification fold, 10% of proteases, and 8% of brown impurities. It was shown that tyrosinase can be pre-purified by selected membranes yielding the enzyme quality at least comparable to that after double salting-out method but in one device. In both cases, subsequent purification by ion-exchange chromatography slightly increased purification degree of the enzyme and brown impurity removal. The surplus of membrane pre-purification is substantially higher thermal stability of the enzyme, enlarged after the chromatographic step, due to very low content of proteolytic enzymes.

1Progress, applications, challenges and prospects of protein purification technology

“…Dialysis is a method to separate colloidal substances such as proteins from other small molecular substances including inorganic salt ions and reducing agents based on the principle that protein and other macromolecular particles are large, have Frontiers in Bioengineering and Biotechnology frontiersin.org colloidal properties, and cannot freely pass through the semipermeable membrane (Echave et al, 2021). The activity of purified protease can be improved by attaching specific substances targeting the target protease to the semi permeable membrane (Labus et al, 2020). In order to improve the recovery rate of the target proteins during dialysis, factors such as buffer exchange time, the design of dialysis system, and the chemical and morphological characteristics of dialysis membrane should be considered.…”