Identification, expression and characterization of genes encoding nitrilases from Trichoderma reesei
thesisposted on 28.03.2022, 19:54 by Rajeev Koundinya
Nitrilases are attractive as industrial catalysts due to their ability to break down toxic nitriles into carboxylic acids and ammonia. Although nitrilases have been identified from several sources, identification of relevant nitrilases with attributes of enantio- and regioselectivity remains a challenge and research continues in this area. The current research explores the filamentous fungus Trichoderma reesei as a potential source of nitrilases. Identification of genes encoding nitrilases in the T. reesei genome was performed in silico using bioinformatics and phylogenetic techniques. The in silico analysis enabled identification of four T. reesei genes that could potentially encode enzymes from branch 1 of the nitrilase superfamily; the gene nit1 was judged as most likely to encode a nitrilase. To induce the expression of the nitrilase gene(s), T. reesei was grown in media supplemented with nitriles such as adiponitrile and benzonitrile. Due to the predicted intracellular nature of the enzymes, the mycelial protein was examined for the presence of nitrilases using a combination of approaches including nitrilase activity assays, high performance liquid chromatography (HPLC), 1 and 2-dimensional gel electrophoresis and mass spectrometry. Verification of the expression of the nit1 gene by T. reesei grown in adiponitrile-supplemented minimal medium was achieved and the protein was found to be functionally active as a nitrilase, enabling the complete hydrolysis of adiponitrile to adipic acid. Thereafter, to allow the recombinant expression of the nit1 gene in E. coli, generation of the intron-less nit1 complementary-DNA (cDNA) was attempted using reverse transcription polymerase chain reaction (RT-PCR) and overlap extension polymerase chain reaction (OE-PCR). The nit1 cDNA was successfully obtained by RT-PCR using the total RNA extracted from T. reesei mycelia. The isolated nit1 cDNA was cloned into a pET expression vector and transformed into two E. coli strains, OrigamiTMB(DE3) and TunerTMDE3. Expression of the nit1 cDNA was successfully achieved in OrigamiTMB(DE3) cells induced with 0.4 mM IPTG at 22 °C. Although the recombinant nitrilase appeared to aggregate in inclusion bodies in the cell, the protein could be solubilized and refolded to an enzymatically active form, as shown by nitrilase activity assays. The recombinant T. reesei nitrilase was purified using the Ni-NTA His-tagged protein purification system. Optimal activity of the nitrilase was achieved at 40 °C, pH 7.5, against the substrate adiponitrile. The enzyme could be of interest for future improvement and development for industrial processes involving the biocatalysis of adiponitrile such as in the manufacture of nylons and resins.