Heterologous expression, purification, and characterization of a recombinant Cordyceps militaris lipase from Candida rugosa-like family in Pichia pastoris

Lipases (EC 3.1.1.3) are serine hydrolases that catalyze the hydrolysis and esterification of various water-insoluble substrates [1]. Microbial lipases have unique catalytic propeties such as substrate specificity, enantioselectivity, regioselectivity, and stability under harsh conditions [2], [3], making them suitable for catalyzing various reactions in food processing, cosmetics, detergents, and pulp [4]. Among them, catalytic properties of lipase isoforms from Candida rugosa have been extensively reported [5], and their crystal structure provided mechanistic insight into their unique properties at the molecular level [6], [7], [8].

In general, C. rugosa lipase-like lipases (homologous family 6 in the Lipase Engineering Database v. 4.1.0) share conserved signature motifs and structural features [9]; e.g., lipase isoforms from Geotrichum candidum [10], sterol esterase from Melanocarpus albomyces [11], and sterol esterase from Ophiostoma piceae [12]. Attempts have been made to isolate esterases/lipases putatively classified as C. rugosa lipase-like lipases, and their catalytic properties have been demonstrated from fungal origins such as Nectria haematococca, Trichoderma reesei, and Aspergillus niger [13]. They were recently redefined as versatile lipase for showing broad hydrolytic activity towards a wide range of substrates [14]. They feature lipase, aryl esterase, or sterol esterase activity [13], [15], but substrate acceptability and hydrolytic efficiency varies due to differences in substructural elements such as intramolecular tunnel or lid [14]. Lipase isoforms from C. rugosa show diverse chain length selectivity and substrate preferences for triacylglycerols, p-nitrophenyl alkanoates, and cholesteryl esters despite their high sequence identity (77–88%) and similar molecular masses (~ 60 kDa) [16]. This is a result of sequence variation in the lid region and differences in the shape, slope, and depth of the acyl-binding tunnel [5], [17].

Global sequence coverage analysis of three extracellular lipase isoforms from the entomopathogenic fungus, Cordyceps militaris, revealed that they share a sequence (NCBI accession number: 18162943), characteristic of the C. rugosa lipase-like group [18]. Attempts were made to heterologously express the lipase from C. militaris (CML) in Escherichia coli system [19], but failed due to the absence of post-translational modification; extracellular proteins of filamentous fungi are modified and expressed as active forms by folding, proteolytic cleavage, and glycosylation [20]. Expression in baculovirus-insect cell system yielded active CML, albeit with lower catalytic efficiency (kcat/Km) of 4.89 mM-1·min-1 [19] than the native enzyme (931.22 mM-1·min-1) [18]. Therefore, research on enzymatic characterization and industrial applications has been hampered by the low yield of active enzyme.

In this study, the methylotropic yeast, Pichia pastoris was used to express CML in active form. A sequential purification was perfomred and the catalytic properties of recombinant CML (rCML) was compared to the native protein. Analysis of substrate specificity, interfacial activation, and binding pocket using a structural model revealed that rCML has catalytic features unlike other lipases from C. rugosa lipase-like group despite its sequence similarity and structural commonality.

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