title: Evolution of histamine oxidase activity for biotechnological applications.
abstract: Histamine is present to various degrees in many foods, and concentrations in fish samples are considered a good indicator of freshness and hygienic food quality. Seeking for innovative methods to quantify histamine in foods, we used a synthetic gene designed on the sequence of histamine oxidase from Arthrobacter crystallopoietes (HOD) as the starting point in this study to develop a biosensor. HOD was expressed in Escherichia coli cells with a yield of ∼7 mg protein/L of fermentation broth. Recombinant wild-type HOD oxidized histamine and tyramine whereas it was inactive toward putrescine and cadaverine (two amines present in fish samples). The putative residues involved in substrate binding were identified by an in silico docking procedure based on a model of the structure of HOD: site-saturation mutagenesis was performed on 8 positions. The most significant changes in kinetic properties were observed for the P143M HOD: this variant showed higher histamine affinity and lower substrate inhibition by tyramine than wild-type enzyme. Biosensor prototypes were produced using both the wild-type and the P143M variant HOD. These biosensors showed a good sensitivity and selectivity with respect to biogenic amines present in food specimens. Accordingly, the HOD-based biosensor was successfully used to assess histamine in fish samples, yielding values in good agreement with those obtained by HPLC analyses but in a few seconds and at a significantly lower cost per analysis.
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, via J.H. Dunant 3, 21100, Varese, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Rosini
authors.forename: Fabio
authors.initials: F
authors.lastname: Tonin
authors.forename: Natalia
authors.initials: N
authors.lastname: Vasylieva
authors.forename: Stephane
authors.initials: S
authors.lastname: Marinesco
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D000818:Animals; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D005399:Fishes; D005504:Food Analysis / Q000379:methods*; D015870:Gene Expression; D006632:Histamine / Q000032:analysis*; D008460:Meat; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D006632:Histamine; D000587:Oxidoreductases Acting on CH-NH Group Donors