193 UTILIZATION OF WHEY AS A CHEAP SUBSTRATE FOR THE OPTIMIZATION OF LIPASE PRODUCTION BY Bacillus subtilis B10 ISOLATED FROM DAIRY INDUSTRY Kumaresan Kuppamuthu*, Sharath Soundiraraj, Kanmani Palanisamy Address(es): Department of Biotechnology, Kumaraguru College of Technology, Coimbatore – 641049, India. *Corresponding author: kumaresan.k.bt@kct.ac.in ABSTRACT Keywords: Box-Behnken design; lactose; lipase; one-factor-at-a-time; response surface methodology INTRODUCTION Whey, resulting from the processes of cheese making and casein manufacturing, is one of the by-products of dairy industry. It possesses only 6-7% of dry matter, which is far lower when compared to that of milk and hence commonly considered as a waste product. Its generation in large amounts represents a significant environmental problem as it exerts a high Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD). However, it contains much of the nutrients present in milk including lactose, lipids, functional proteins, peptides, minerals and vitamins. Lactose makes up a high proportion (47.5%) of the total whey solids and largely contributes to it being considered as one of the most polluting by-product streams. Whey offers immense potential as a source of value added compounds. Therefore, the industry should view the surplus availability of whey as a promising resource and not only as a problem of waste management. Various means of whey disposal and utilization have been described in many reviews. In this study, it has been utilized as a substrate for the production of an industrially important enzyme, lipase. Lipase is a triacylglycerol acylhydrolase (EC 3.1.1.3) that catalyses the hydrolysis of triglycerides into glycerol and free fatty acids. Unlike esterases (EC 3.1.1.1 carboxylic ester hydrolases), true lipases are able to hydrolyse esters of long-chain fatty acids. They exhibit enantioselective properties. Lipases are produced ubiquitously and possess considerable physiological significance and a wide range of industrial applications. Other than triglycerides, the enzyme possesses active site for other substrates such as aliphatic, alicyclic, bicyclic and aromatic esters and even esters based on organometallic sandwich compounds. The catalytic triad, Serine105-Histidine224-Aspartate187, is present at the enzyme’s active site (Uppenberg et al., 1994a,b). The natural substrates of lipases are practically insoluble in water and the reaction is catalysed only at the water-lipid interface. Besides being lipolytic, lipases also possess esterolytic activity and thus have a very diverse substrate range (Jaeger et al., 1999). The ability of lipase to undergo reverse reaction and synthesize triacylglyceride from glycerol and free fatty acids, discloses that it can catalyse a wide range of reactions such as hydrolysis, interesterification, esterification, alcoholysis, acidolysis and aminolysis (Joseph et al., 2008). It thus displays enzyme promiscuity, an ability to catalyse alternative reactions that differ from the natural physiological reaction. Microbial lipases are mostly extracellular and their production is greatly influenced by medium composition, besides physicochemical factors such as temperature, pH and dissolved oxygen. The major factor for the expression of lipase activity has always been reported as the carbon source, since lipases are inducible enzymes. The ability of lipases to perform very specific biochemical transformations has made them increasingly popular in food, detergent, cosmetic, organic synthesis and pharmaceutical industries (Hasan et al., 2006). The use of agro-industrial residues as substrates for lipase production undoubtedly favors the reduction of production costs and pollution associated with these substrates. Considering the above facts, production of lipase from whey is a pertinent area of research and this study encompasses optimization of the production process using response surface methodology. It enables effective medium optimization using a limited number of trials and has been widely employed for the optimization of a number of production processes. It has been successfully applied for lipase production as well (Gupta et al., 2007; Saxena and Saxena, 2004; Vohra and Satyanarayana, 2002). The objective of our study was to isolate bacterial strains from soil sample and screen them for lipase activity and lactose utilization. The best isolate was subjected to optimization of lipase production using One-Factor-at-a-Time (OFAT) and Response Surface Methodologies (RSM). MATERIAL AND METHODS Chemicals Bovine serum albumin (BSA) was purchased from SIGMA-ALDRICH, USA. Microbiological media components and all other chemicals were of reagent grade and procured from HI-MEDIA, India. Whey, a by-product of the dairy industry, acts as a prime source of environmental pollution due to its high organic load. Its utilization as a substrate for lipase production is an attractive option in facing the challenge associated with whey disposal. However, there are only very few studies that have reported the production of lipase using whey. In this study, 43 bacterial strains were isolated from dairy industry soil sample, of which, 26 were found to produce lipase. Since whey contains lactose as the sole carbon source, the ability of the isolates to utilize this sugar was tested and two of the isolates B3 and B10 were positive for growth in phenol red lactose broth. When they were cultured by submerged fermentation and their lipase activities quantified, strain B10 displayed 0.79U/ml of activity. In order to improve the production of lipase, one-factor-at-a-time method was used to study the impacts of oil inducers, nitrogen sources, mineral salts and whey concentration on the process. Statistical optimization was performed using the Box-Behnken design of Response Surface Methodology (RSM). From this design, 75% whey, 4% (v/v) sunflower oil, 0.625% (w/v) beef extract and 0.2% (w/v) CaCl 2 were inferred to be the optimal conditions that resulted in a maximum lipase activity of 0.954U/ml. Analysis of variance indicated statistical significance of the model. Lipase being an industrially sought-after enzyme owing to its unique properties, this cost-effective route to its production from a waste substrate holds paramount environmental and economic significance. ARTICLE INFO Received 25. 5. 2017 Revised 13. 6. 2017 Accepted 26. 8. 2017 Published 1. 10. 2017 Regular article doi: 10.15414/jmbfs.2017.7.2.193-198