Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature

Abstract

The main goal of the present work is to investigate a novel process of purification and immobilization of a thermophilic catalase at high temperatures. The catalase, originated from Bacillus sp., was overexpressed in a recombinant Escherichia coli BL21(DE3)/pET28-CATHis and efficiently purified by heat treatment, achieving a threefold purification. The purified catalase was then immobilized onto an epoxy support at different temperatures (25, 40, and 55 °C). The immobilizate obtained at higher temperatures reached its maximum activity in a shorter time than that obtained at lower temperatures. Furthermore, immobilization at higher temperatures required a lower ionic strength than immobilization at lower temperatures. The characteristics of immobilized enzymes prepared at different temperatures were investigated. The high-temperature immobilizate (55 °C) showed the highest thermal stability, followed by the 40 °C immobilizate. And the high-temperature immobilizate (55 °C) had slightly higher operational stability than the 25 °C immobilizate. All of the immobilized catalase preparations showed higher stability than the free enzyme at alkaline pH 10.0, while the alkali resistance of the 25 °C immobilizate was slightly better than that of the 40 and 55 °C immobilizates.

Study of ALDH from Thermus thermophilus—Expression, Purification and Characterisation of the Non-Substrate Specific, Thermophilic Enzyme Displaying Both Dehydrogenase and Esterase Activity

“…Recombinant expression and purification of ALDH Tt proved an efficient method for the production of the full-length enzyme displaying high purity, while also obtaining high production yields. Heat precipitation protocols have been regularly incorporated as a selective purification step for thermostable proteins expressed in mesophilic hosts [ 53 , 58 , 59 , 60 , 61 ], and has proven effective with other T. thermophilus proteins expressed in E. coli, including other dehydrogenases and the ALDH family member 1-pyrroline-5-carboxylate dehydrogenase (P5CDH Tt ) [ 61 , 62 , 63 ]. The heat treatment step of purification of alcohol dehydrogenase, from T. thermophilus, at 75 °C for 15 min was deemed the most efficient step, enriching the enzyme 11-fold, and resulting in 47% yield of a homogeneous protein [ 61 ].…”

“…A previous report outlined how increased purity and yield of recombinant thermophilic catalase from Bacillus sp. expressed in E. coli was achieved via heat treatment purification at 65 °C for 2 h, resulting in a three-fold increase in specific activity of the cell supernatant [ 59 ]. Three temperatures were trialled for purification, 55, 60, and 65 °C, demonstrating increased catalase purity with increased temperature.…”

“…Recombinant expression and purification of ALDH Tt proved an efficient method for the production of the full-length enzyme displaying high purity, while also obtaining high production yields. Heat precipitation protocols have been regularly incorporated as a selective purification step for thermostable proteins expressed in mesophilic hosts [53,[58][59][60][61],…”

Covalent linkage of alkalothermophilic catalase onto functionalized cellulose

“…A comparative analysis was done with recently reported studies on the purication of catalase from other bacterial cultures. Xu and co workers 18 have also puried catalase from Bacillus sp. using conventional multistep processes.…”