title: Novel thermostable amine transferases from hot spring metagenomes.
abstract: Hot spring metagenomes, prepared from samples collected at temperatures ranging from 55 to 95 °C, were submitted to an in silico screening aimed at the identification of novel amine transaminases (ATAs), valuable biocatalysts for the preparation of optically pure amines. Three novel (S)-selective ATAs, namely Is3-TA, It6-TA, and B3-TA, were discovered in the metagenome of samples collected from hot springs in Iceland and in Italy, cloned from the corresponding metagenomic DNAs and overexpressed in recombinant form in E. coli. Functional characterization of the novel ATAs demonstrated that they all possess a thermophilic character and are capable of performing amine transfer reactions using a broad range of donor and acceptor substrates, thus suggesting a good potential for practical synthetic applications. In particular, the enzyme B3-TA revealed to be exceptionally thermostable, retaining 85% of activity after 5 days of incubation at 80 °C and more than 40% after 2 weeks under the same condition. These results, which were in agreement with the estimation of an apparent melting temperature around 88 °C, make B3-TA, to the best of our knowledge, the most thermostable natural ATA described to date. This biocatalyst showed also a good tolerance toward different water-miscible and water-immiscible organic solvents. A detailed inspection of the homology-based structural model of B3-TA showed that the overall active site architecture of mesophilic (S)-selective ATAs was mainly conserved in this hyperthermophilic homolog. Additionally, a subfamily of B3-TA-like transaminases, mostly uncharacterized and all from thermophilic microorganisms, was identified and analyzed in terms of phylogenetic relationships and sequence conservation.
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, C.N.R, Via Mario Bianco 9, 20131, Milan, Italy. erica.ferrandi@icrm.cnr.it.|University of Copenhagen, Ole Maaløesvej 5, 2200, Copenhagen N, Denmark. erica.ferrandi@icrm.cnr.it.
authors.forename: Erica Elisa
authors.initials: EE
authors.lastname: Ferrandi
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, C.N.R, Via Mario Bianco 9, 20131, Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Previdi
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, C.N.R, Via Mario Bianco 9, 20131, Milan, Italy.|Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133, Milan, Italy.
authors.forename: Ivan
authors.initials: I
authors.lastname: Bassanini
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, C.N.R, Via Mario Bianco 9, 20131, Milan, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Riva
authors.affiliation: University of Copenhagen, Ole Maaløesvej 5, 2200, Copenhagen N, Denmark.
authors.forename: Xu
authors.initials: X
authors.lastname: Peng
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, C.N.R, Via Mario Bianco 9, 20131, Milan, Italy. daniela.monti@icrm.cnr.it.
authors.forename: Daniela
authors.identifier: http://orcid.org/0000-0002-3399-7973
authors.initials: D
authors.lastname: Monti
mesh_terms: D000588:Amines / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D045482:Hot Springs / Q000382:microbiology*; D007055:Iceland; D007218:Industrial Microbiology; D007558:Italy; D054892:Metagenome; D056186:Metagenomics; D010802:Phylogeny; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D000588:Amines; D000637:Transaminases
keywords: Amine transferases; Chiral amines; Industrial biocatalysis; Metagenomics; Thermostability