In silico mining of sn-1,3 regioselective Rhizomucor miehei lipase homolog with strict pH sensitivity from Cordyceps militaris.

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Citation abstract

Regioselectivity is crucial for lipase-catalyzed lipid modification. Rhizomucor miehei lipase (RML) is a representative sn-1(3) regioselective lipase utilized in food industry, but it exhibits sn-1 stereo-preference. In this study, novel RML homologs (rmCMLs) from Cordyceps militaris were identified via in silico mining to discover enzymes with distinct catalytic features and expressed in yeast. Among them, rmCML2 displayed similar preference for both sn-1 and sn-3 positions, unlike RML, but maintained a similar catalytic efficiency to RML against trioleoylglycerol. Notably, rmCML2 was only active at acidic pH (pH 4.0-6.0) but lost its activity at neutral pH. Molecular dynamic simulation revealed that the extended C-terminus (P270-K304) may endow rmCML2 with unexpected pH sensitivity by changing the H-bond network to expose the active site in pH-dependent manner. The sn-1,3 regioselectivity and pH sensitivity are expected to confer dual advantages of rmCML2 for processing lipid-related foods that require pH-dependent enzymatic conversion (e.g., cheese ripening).