Preparation of Stable Cross-Linked Enzyme Aggregates (CLEAs) of a Ureibacillus thermosphaericus Esterase for Application in Malathion Removal from Wastewater

Abstract

In this study, the active and stable cross-linked enzyme aggregates (CLEAs) of the thermostable esterase estUT1 of the bacterium Ureibacillus thermosphaericus were prepared for application in malathion removal from municipal wastewater. Co-expression of esterase with an E. coli chaperone team (KJE, ClpB, and ELS) increased the activity of the soluble enzyme fraction up to 200.7 ± 15.5 U mg−1. Response surface methodology (RSM) was used to optimize the preparation of the CLEA-estUT1 biocatalyst to maximize its activity and minimize enzyme loss. CLEA-estUT1 with the highest activity of 29.4 ± 0.5 U mg−1 (90.6 ± 2.7% of the recovered activity) was prepared with 65.1% (w/v) ammonium sulfate, 120.6 mM glutaraldehyde, and 0.2 mM bovine serum albumin at 5.1 h of cross-linking. The biocatalyst has maximal activity at 80 °С and pH 8.0. Analysis of the properties of CLEA-estUT1 and free enzyme at 50–80 °C and pH 5.0–10.0 showed higher stability of the biocatalyst. CLEA-estUT1 showed marked tolerance against a number of chemicals and high operational stability and activity in the reaction of malathion hydrolysis in wastewater (up to 99.5 ± 1.4%). After 25 cycles of malathion hydrolysis at 37 °С, it retained 55.2 ± 1.1% of the initial activity. The high stability and reusability of CLEA-estUT1 make it applicable for the degradation of insecticides.

Thermoalkalophilic recombinant esterase entrapment in chitosan/calcium/alginate‐blended beads and its characterization

“…43 Also, CLEA-estUT1 activity was reduced as much as 70% by Triton X-100 whereas SDS did not change its activity. 54 Additionally, 1% SDS inhibited almost all of its activity of the immobilized esterase from Paenibacillus sp. (CLEAs-PsEstA).…”

Polymer supports for the removal and degradation of hazardous organic pollutants: an overview

“…In this carrierless method of enzymatic immobilization, the enzymes are crosslinked covalently by the use of bifunctional reagents such as glutaraldehyde, diisocyanates and diaminoesters. 28,95 Additionally, macromolecular crosslinkers, such as chitin, chitosan, dextran polyaldehyde and L-lysin, 28 have been used. These reactants react mainly with the NH 2 groups of lysine on the surfaces of the enzymes.…”

Immobilized Biocatalysts

“…An application example of biocatalyst immobilization for wastewater treatment was provided by Samoylova et al [9]. They prepared stable cross-linked enzyme aggregates (CLEAs) of the thermostable esterase, estUT1, of the bacterium Ureibacillus thermosphaericus, and used these CLEAs for removal of the organophosphate insecticide, malathion.…”