Abstract
Cross-linked enzyme aggregates (CLEAs) is an immobilization technique that can be used to customize enzymes under an optimized condition. Structural analysis on any enzyme treated with a CLEA remains elusive and has been less explored. In the present work, a method for preparing an organic solvent tolerant protease using a CLEA is disclosed and optimized for better biochemical properties, followed by an analysis of the structure of this CLEA-treated protease. The said organic solvent tolerant protease is a metalloprotease known as elastase strain K in which activity of the metalloprotease is measured by a biochemical interaction with azocasein. Results showed that when a glutaraldehyde of 0.02% (v/v) was used under a 2 h treatment, the amount of recovered activity in CLEA-elastase was highest. The recovered activity of CLEA-elastase and CLEA-elastase-SB (which was a CLEA co-aggregated with starch and bovine serum albumin (BSA)) were at an approximate 60% and 80%, respectively. The CLEA immobilization of elastase strain K allowed the stability of the enzyme to be enhanced at high temperature and at a broader pH. Both CLEA-elastase and CLEA-elastase-SB end-products were able to maintain up to 67% enzyme activity at 60 °C and exhibiting an enhanced stability within pH 5–9 with up to 90% recovering activity. By implementing a CLEA on the organic solvent tolerant protease, the characteristics of the organic solvent tolerant were preserved and enhanced with the presence of 25% (v/v) acetonitrile, ethanol, and benzene at 165%, 173%, and 153% relative activity. Structural analysis through SEM and dynamic light scattering (DLS) showed that CLEA-elastase had a random aggregate morphology with an average diameter of 1497 nm.
Highlights
Enzymes are considered one of the most notable biocatalysts and are extensively used in the industry considering its wide applications and advantages
The highest recovered activity of Cross-linked enzyme aggregates (CLEAs)–elastase was recorded at 60% based on Figure 1
While immobilization using the CLEA method has often been reported as causing a significant loss of enzyme activity, CLEA–elastase has been shown to retain up to 70% the enzyme activity
Summary
Enzymes are considered one of the most notable biocatalysts and are extensively used in the industry considering its wide applications and advantages. The use of enzymes in industrial bioprocesses is desirable as enzymes offer a greener alternative to chemical catalysts when taking into consideration biodegradability, cost-effectiveness and the production of non-toxic byproducts [1]. The enzymes in this group, which include protease, lipase, and amylase, have been commonly used as biocatalysts in the industry for many years. Other extreme conditions that may affect the stability of protease include extreme temperature, pH, and organic solvents which may be present in the reaction media [5,6]. Organic solvents can be classified according to their log Po/w value, where any organic solvent showing log Po/w < 2 is a polar organic solvent and any organic solvent showing log Po/w > 4 is a non-polar organic solvent [7,8,9]
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