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Enhanced stability of newly isolated trimeric l ‐methionine‐ N ‐carbamoylase from Brevibacillus reuszeri HSN1 by covalent immobilization
Author(s) -
Nandanwar Hemraj S.,
Vohra Rakesh M.,
Hoondal Gurinder S.
Publication year - 2013
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1082
Subject(s) - immobilized enzyme , enzyme , covalent bond , methionine , enzyme assay , substrate (aquarium) , chemistry , biocatalysis , chromatography , biochemistry , amino acid , organic chemistry , catalysis , biology , reaction mechanism , ecology
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.