title: Thermus thermophilus as a cell factory for the production of a thermophilic Mn-dependent catalase which fails to be synthesized in an active form in Escherichia coli.
abstract: Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H(2)O(2)-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Centro de Biología Molecular Severo Ochoa CSIC-UAM, Campus de Cantoblanco, 28049 Madrid, Spain.
authors.forename: Aurelio
authors.initials: A
authors.lastname: Hidalgo
authors.forename: Lorena
authors.initials: L
authors.lastname: Betancor
authors.forename: Renata
authors.initials: R
authors.lastname: Moreno
authors.forename: Olga
authors.initials: O
authors.lastname: Zafra
authors.forename: Felipe
authors.initials: F
authors.lastname: Cava
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernández-Lafuente
authors.forename: José M
authors.initials: JM
authors.lastname: Guisán
authors.forename: José
authors.initials: J
authors.lastname: Berenguer
mesh_terms: D002374:Catalase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology*; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D008345:Manganese; D002374:Catalase