Characterization of a Lipase from Sporidiobolus pararoseus 25-A Which Produces Cheese Flavor

Abstract

A lipase from a Sporidiobolus pararoseus strain was purified from culture filtrate by (NH 4 ) 2 SO 4 precipitation, and DEAE-Toyopearl 650M, Butyl-Toyopearl 650M, and Toyopearl HW-55 chromatography. The purified enzyme appeared as a single band with a molecular mass of 37 kDa by SDS-PAGE. The optimum temperature and pH were approximately 60℃ and 6.0, respectively. The specificity toward triglyceride was similar to that of pregastric esterase Lipase PGE T . Addition of the lipase during the mozzarella cheese-making process produced a strong cheese flavor. Taken together, the lipase produced by S. pararoseus is considered a potential replacement of pregastric esterase in the dairy industry.Keywords: Sporidiobolus pararoseus, extracellular lipase, flavor enhancement, cheese making *To whom correspondence should be addressed. E-mail: tmasej28@sugiyama-u.ac.jp IntroductionLipases (EC 3.1.1.3, triacylglycerol lipase) are enzymes that catalyze the hydrolysis of triacylglycerol and are widely found in animals, plants, and microorganisms. In the dairy industry, lipases are used for flavor development in specific cheese types, flavor enhancement, acceleration of the cheese ripening process, and for the production of cheese-like products (Arnold et al., 1975). In particular, pregastric esterase (Kwak et al., 1989) is an important lipase for the production of enzyme-modified cheese (EMC) which is used to formulate foods which require a cheese flavor. The short-chain fatty acids released by pregastric esterase not only function as flavor components themselves, but also serve as precursors for other flavor components. However, the fatty acid specificity of microbial lipases is different from that of pregastric esterase (Kanizawa et al., 1982). We were therefore interested in identifying a microbial lipase with qualities similar to pregastric esterase. In this study, we purified and characterized the extracellular lipase produced by S. pararoseus 25-A, and examined the possibility that it could serve as a replacement of pregastric esterase in the dairy industry. Materials and MethodsMaterials Lipase PGE T was purchased from Amano Enzyme Inc. (Nagoya, Japan). All other chemicals used were obtained from commercial sources. Microorganisms Strain 25-A used in this study was newly isolated from the digestive juice in the pitcher of Nepenthes truncata, a insectivorous plant, which was gathered from Higashiyama Botanical Garden in Nagoya, Japan. Strain 25-A was identified by its morphological characteristics and a gene sequence analysis based on the D1/D2 domain sequences of 26S ribosomal DNA as described previously (Hirose et al., 2009).Enzyme assay Lipase activity was measured at pH 7.0 and 30℃ using Lipase Kit S (DS Pharma Biomedical Co., Ltd., Japan) following the manufacturer's instructions.Cultivation A single colony of strain 25-A from an agar slant was inoculated into 100-mL shaking flasks containing 20 mL medium (pH 6.2) consisting of 21 g Difco YM broth and 10 mL olive oil in 1 L tap water. After incubation at 25℃ for 24 h,...

Characterization and Application of Lipase 39-A from Cryptococcus flavescens for Cheese Flavoring

“…We reported a lipase derived from S. pararoseus 25-A as a replacement for pregastric esterase (Mase et al, 2011). While the thermal stability of S. pararoseus lipase is higher than the current purified lipase, the short-chain fatty acid specificity toward milk fat is not as good.…”

“…The fatty acid composition released by lipase was defined as weight percentage. Cheese making Mozzarella cheese was made with a cheese-making kit (Auvelcraft Co., Ltd. Okazaki, Japan) using cow's milk as described previously (Mase et al, 2011). A total of 7000 units of lipase were added to 2 L of milk, and cheeses were prepared by maturation for 2 days in a refrigerator at 5℃.…”

“…Lipases catalyze the hydrolysis of triacylglycerol and are found widely in animals, plants, and microorganisms. Many yeasts, such as Sporidiobolus pararoseus (Mase et al, 2011), Geotrichum sp. (Monfort et al, 1999), and Candida rugosa (Linko and Wu, 1996), have been reported to produce lipase.…”