Enhanced stability of newly isolated trimeric l‐methionine‐N‐carbamoylase from Brevibacillus reuszeri HSN1 by covalent immobilization

Abstract

Newly isolated and partially purified trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 was immobilized by covalent coupling to a well-known support material, Eupergit® C. Approximately 80% enzyme activity yield was achieved with ≈61% binding of a soluble protein from a solution containing 5 mg/mL protein. The immobilized preparation was found to be quite unstable due to a poor multisubunit covalent interaction of trimeric enzyme. Additional cross-linking with polyaldehyde-dextran was done to sustain the biotechnological application of immobilized enzyme. The temperature and pH optima of immobilized enzyme were increased by 10°C and 0.5 unit, respectively. The enzyme was significantly stabilized and retained ≈93% enzyme activity when incubated at 60°C for 60 Min, whereas free enzyme lost ≈50% activity. It was recycled nine times with ≈100% conversion efficiency when batch experiments were carried out at 35°C, pH 7.5, for the 180 Min cycle, using 5% N-carbamoyl-l-methionine as the substrate. The half-life of the immobilized preparation was determined as 23 cycles and is significant. Approximately 50% of enzyme activity was retained even after 5 months of storage at 4°C, whereas free enzyme lost complete enzyme activity. Hence, we could enhance the stability of l-methionine-N-carbamoylase to make it a potential biocatalyst for biotechnological production of α-amino acids.

Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized l-N-carbamoylase and N-succinyl-amino acid racemase

“…Similar results were obtained when Deinococcus radiodurans NSAAR (DraNSAAR) was immobilized, resulting in a 90% decrease of its specific activity (Yen et al, 2010). Regarding L-carbamoylases, previous studies have shown that immobilization of the enzymes from Arthrobacer and Bacillus genera has resulted in a substantial decrease in their specific activity (Ragnitz et al 2001a and b;Yen et al, 2010;Nandanwar et al 2013), except in the case of a recombinant (Asp) 6tagged L-carbamoylase from Arthrobacter (AauBLcar), which maintained similar activities to the enzyme in solution (Ragnitz et al 2001 enzandmicrobtech). BsLcar could also be reused for several cycles as other Bacillus L-carbamoylases, maintaining similar significant half-lifes (Nandawar et al 2013;Yen et al 2010).…”

Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

“…Due to their thermal instability as well as oxidative and proteolytic sensitivity many efforts have been made for the immobilization of hydantoinases and carbamoylases with visible success (Pietzsch et al 1998; Ragnitz et al 2001b; Chiang et al 2008; Nandanwar et al 2013). Most investigations are dealing with encapsulation, covalent immobilization or non-covalent adsorption techniques.…”

Genome Sequence of Brevibacillus reuszeri NRRL NRS-1206 T , an l - N -Carbamoylase-Producing Bacillus -Like Bacterium

“…Up to now, very little research has been done on this spore-forming species. The only functional gene from B. reuszeri is l -methionine- N -carbamoylase, which will be a potential biocatalyst for the production of l -α-amino acids ( 3 , 4 ). Given its taxonomic history and no available genomic information of B. reuszeri , its type strain NRRL NRS-1206 T was selected as one of the research objects in our genome sequencing project for genomic taxonomy and phylogenomics of Bacillus -like bacteria.…”