Candida antarctica lipase B (CALB) is a commercially available versatile catalyst frequently used in organic synthesis for selective hydrolysis or acylation in both laboratory and large scale processes. The high enantioselectivity of this enzyme provided the opportunity to develop a process for the desymmetrization of 200 kg of diethyl 3-[3',4'dichlorophenyl]glutarate (DDG) a prochiral precursor for the synthesis of a series of neurokinin (NK) receptor antagonists. Although both soluble and immobilized forms of CALB were successfully utilized to deliver clinical drug supplies, low specific activity and limited stability at elevated temperature compromised the potential use of this enzyme for drug manufacturing. To circumvent these limitations and develop a more efficient process, CALB was modified through use of single and multi-gene directed evolution techniques to generate lipase B variants with improved activity and thermostability toward the hydrolysis of DDG. Seven chimeric lipase B proteins among 2500 clones screened were identified. In particular, variant C6 exhibited a 20-fold improvement in specific activity, an 11-fold improvement in half-life at 45°C and a 6.4°C higher melting point than the wild-type enzyme. Protein production of CALB variant C6 was successfully achieved with the methanol utilizing yeast Pichia pastoris. Enzyme expression was regulated by a modified methanol limited and temperature adjusted fed-batch fermentation strategy which produced up to 1.5 g /L protein in a 30-liter fermentor.