Pak. J. Bot., 49(2): 757-761, 2017. PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR LIPASE BY GEOTRICHUM CANDIDUM OF DAIRY ORIGIN ABUBAKAR MUHAMMAD 1 , SYED ALI IMRAN BOKHARI 2 , NAEEM ALI 1 , HAMID MUKHTAR 3* , ALI NAWAZ 3 AND MUHAMMAD IMRAN 1* *1 Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan 2 Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan 3 Institute of Industrial Biotechnology, GC University Lahore-54000, Pakistan * Corresponding author’s e-mail address: m_imran766@hotmail.com/mmimran@qau.edu.pk; hamidmukhtar@gcu.edu.pk Abstract In the present study, thermostable lipase from Geotrichum candidum UCMA 91(ATCC 204307) was purified and characterized. Lipase produced after optimization of the various cultural and physico-chemical conditions was purified to homogeneity by two step methods of purification: ammonium sulfate precipitation and column chromatography. The enzyme was purified by 60% ammonium sulfate precipitation and lipase activity of 5.77U mg -1 was attained. Then, Sephadex G-75 was used for gel filtration chromatography and 62.36fold purification was achieved. Molecular mass of lipase was estimated to be 59 KDa by using SDS-PAGE. It is also determined from the study that lipase showed stability at varying range of pH (5-12) and thermo stability (15-65 o C). The lipase was completely inhibited by EDTA (3.98%) confirming it as a metalloprotease, whereas the enzyme was found to be stable in various organic solvents. The results demonstrate that lipase hydrolyzes vegetable oils, which validates its technological relevance for use in the dairy, pharmaceuticals and bakery industry. Key words: Protein purification, Geotrichum candidum, Shake flask fermentation, Biocatalysis, Lipase. Introduction Enzymes are regarded as nature’s catalysts. Almost all enzymes produced nowadays (and perhaps almost all in the future) are produced by the fermentation of bio based materials. Lipase (EC 3.1.1.3), is a subclass of esterases and catalyzes the hydrolysis of triacylglycerol’s and convert them to diacylglycerols, monoacyglycerols, fatty acids and glycerol (Salihu et al., 2011; Soler et al., 2016). During hydrolysis, a lipase cleaves the acyl group from glycerides and transfers it to the –OH (Martinelle & Hult, 1995). There are region specific and non-specific lipases; region-specific as their name suggest they act on specific positions on lipid molecule while non-specific lipases can catalyze reaction at all positions (Sonnet & Gazzilo, 1991). Though, lipases can be acquired from animal, plant and microbial sources, microbial lipases have been considered most intensively as of their multipurpose properties, for example stability at elevated temperatures, activity at a wide-ranging pH values, selectivity in racemic mixtures as well as ease of mass production (Yang et al., 2005). Among microbial lipases, largely bacterial and fungal, represents the most broadly used class of enzymes that produces varied variety of extracellular lipase (Rigo et al., 2010; Jaeger et al., 1994). Lipases have numerous industrial applications in the food, degreasing formulation, dairy, medicinal, detergent industries and synthesis of fine chemicals (Gupta et al., 2004; Louwrier, 1998). In dairy products especially, cheese have Geotrichum candidum as dominant microorganism and known to impart significant impact on product organoleptic attributes. Studies on G. candidum lipase are scarce but are of particular interest for their widespread industrial demand and with respect to their use in the food industry (Kocabiyik & Ozel, 2007). Therefore, a need was felt to explore native fungal isolates, capable of producing lipase and at the same relatively stable at the operating conditions. The activity as well as stability of enzymes are significant factors to regulate the monetary viability in manufacturing procedures. High stability is important from the financial perspective due to lessened enzyme turnover (Gohel & Singh, 2012; Li et al., 2004). The high temperature resistance and greater specific activities along with physical and chemical characteristics makes enzymes potent candidate for future biotechnological applications (Temiz et al., 2008). The current study was aimed at the characterization and purification of lipase from G. candidum isolated from yogurt. It includes study of lipase properties, thermo stability, and solvent and pH resistance for its projected industrial applications. To date, there is no report to characterize and purify lipase from a G. candidum of dairy fermented product. Materials and Methods Yeast strains and maintenance: Twelve G. candidum was isolated from indigenous fermented milk product.. G. candidum cells were cultured at 25 o C in the presence of oxy-tetracycline glucose agar (OGA) medium (Merck). The medium pH was maintained to 5.0 (with 1N HCL/NaOH). Shake flask experiment (SFF): Lipase was produced in 1000 ml erlenmeyer flask containing 250 ml of oxy- tetracycline glucose broth supplemented with 1% tween 80 (Merck) and pH was regulated to 5.0. The flask after sterilization was aseptically inoculated with 2 days old slant of G. candidum. The inoculated flask was incubated at 30ºC in rotary shaker (VMR, Model No. 1572-2), at 150 rpm, for 48 h. One milliliter of yeast inoculum was transferred to optimized 250 ml of fermentation medium for lipase production (g/l: Peptone 30.0, Yeast extract