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Expression, characterization, and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum.
Appl Microbiol Biotechnol 2019; 103(5):2229-2241AM

Abstract

L-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate L-amino acids and produce the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here we describe the heterologous expression of LAAO4 from the fungus Hebeloma cylindrosporum without signal sequence as fusion protein with a 6His tag in Escherichia coli and its purification. 6His-hcLAAO4 could be activated by exposure to acidic pH, the detergent sodium dodecyl sulfate, or freezing. The enzyme converted 14 proteinogenic L-amino acids with L-glutamine, L-leucine, L-methionine, L-phenylalanine, L-tyrosine, and L-lysine being the best substrates. Methyl esters of these L-amino acids were also accepted. Even ethyl esters were converted but with lower activity. Km values were below 1 mM and vmax values between 19 and 39 U mg-1 for the best substrates with the acid-activated enzyme. The information for an N-terminal aldehyde tag was added to the coding sequence. Co-expressed formylglycine-generating enzyme was used to convert a cysteine residue in the aldehyde tag to a Cα-formylglycine residue. The aldehyde tag did not change the properties of the enzyme. Purified Ald-6His-hcLAAO4 was covalently bound to a hexylamine resin via the Cα-formylglycine residue. The immobilized enzyme could be reused repeatedly to generate phenylpyruvate from L-phenylalanine with a total turnover number of 17,600 and was stable for over 40 days at 25 °C.

Authors+Show Affiliations

Biochemie III, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.Biochemie I, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.Organische und Bioorganische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.Organische und Bioorganische Chemie, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.Biochemie I, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany.Biochemie III, Fakultät für Chemie, Universität Bielefeld, Universitätsstrasse 25, 33615, Bielefeld, Germany. gabriele.mollard@uni-bielefeld.de.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30631897

Citation

Bloess, Svenja, et al. "Expression, Characterization, and Site-specific Covalent Immobilization of an L-amino Acid Oxidase From the Fungus Hebeloma Cylindrosporum." Applied Microbiology and Biotechnology, vol. 103, no. 5, 2019, pp. 2229-2241.
Bloess S, Beuel T, Krüger T, et al. Expression, characterization, and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum. Appl Microbiol Biotechnol. 2019;103(5):2229-2241.
Bloess, S., Beuel, T., Krüger, T., Sewald, N., Dierks, T., & Fischer von Mollard, G. (2019). Expression, characterization, and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum. Applied Microbiology and Biotechnology, 103(5), pp. 2229-2241. doi:10.1007/s00253-018-09609-7.
Bloess S, et al. Expression, Characterization, and Site-specific Covalent Immobilization of an L-amino Acid Oxidase From the Fungus Hebeloma Cylindrosporum. Appl Microbiol Biotechnol. 2019;103(5):2229-2241. PubMed PMID: 30631897.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression, characterization, and site-specific covalent immobilization of an L-amino acid oxidase from the fungus Hebeloma cylindrosporum. AU - Bloess,Svenja, AU - Beuel,Tobias, AU - Krüger,Tobias, AU - Sewald,Norbert, AU - Dierks,Thomas, AU - Fischer von Mollard,Gabriele, Y1 - 2019/01/11/ PY - 2018/08/27/received PY - 2018/12/28/accepted PY - 2018/12/21/revised PY - 2019/1/12/pubmed PY - 2019/1/12/medline PY - 2019/1/12/entrez KW - Aldehyde tag KW - E. coli KW - Enzyme immobilization KW - Formylglycine KW - Formylglycine-generating enzyme KW - Heterologous expression KW - L-amino acid oxidase SP - 2229 EP - 2241 JF - Applied microbiology and biotechnology JO - Appl. Microbiol. Biotechnol. VL - 103 IS - 5 N2 - L-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate L-amino acids and produce the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here we describe the heterologous expression of LAAO4 from the fungus Hebeloma cylindrosporum without signal sequence as fusion protein with a 6His tag in Escherichia coli and its purification. 6His-hcLAAO4 could be activated by exposure to acidic pH, the detergent sodium dodecyl sulfate, or freezing. The enzyme converted 14 proteinogenic L-amino acids with L-glutamine, L-leucine, L-methionine, L-phenylalanine, L-tyrosine, and L-lysine being the best substrates. Methyl esters of these L-amino acids were also accepted. Even ethyl esters were converted but with lower activity. Km values were below 1 mM and vmax values between 19 and 39 U mg-1 for the best substrates with the acid-activated enzyme. The information for an N-terminal aldehyde tag was added to the coding sequence. Co-expressed formylglycine-generating enzyme was used to convert a cysteine residue in the aldehyde tag to a Cα-formylglycine residue. The aldehyde tag did not change the properties of the enzyme. Purified Ald-6His-hcLAAO4 was covalently bound to a hexylamine resin via the Cα-formylglycine residue. The immobilized enzyme could be reused repeatedly to generate phenylpyruvate from L-phenylalanine with a total turnover number of 17,600 and was stable for over 40 days at 25 °C. SN - 1432-0614 UR - https://www.unboundmedicine.com/medline/citation/30631897/Expression,_characterization,_and_site-specific_covalent_immobilization_of_an_L-amino_acid_oxidase_from_the_fungus_Hebeloma_cylindrosporum L2 - https://dx.doi.org/10.1007/s00253-018-09609-7 DB - PRIME DP - Unbound Medicine ER -