Identification Of Lipase – Producing Psychrophilic Yeast, Leucosporidium Sp.

摘要

Cold-active enzymes have recently received great attention due to their potential applications in a broad range of industrial, agricultural and medical processes. One of the enzymes is lipase (triacyglycerol acylhydrolases E.C 3.1.1.3) which is unique in catalyzing the hydrolysis of triacylglycerols into free fatty acids and glycerol. In this particular research, an obligate psychrophilic microorganism was isolated from Casey Station, Antarctica. The growth of this microorganism has been tested at different temperatures, 4oC, 27oC and 37oC. At 4oC, the microorganism was able to grow whereas at 27oC and 37oC, there was no growth at all. The presence of lipase enzyme in this microorganism was detected by halo zone on palm oil (substrate) agar plates. Identification of this microorganism was done based on its morphological, biochemical and molecular characteristics. For the morphology analysis, two types of microscopy observation were carried out: phase contrast microscopy and Scanning Electron Microscopy (SEM). Both observations showed budding structures. This suggested that this particular microorganism is psychrophilic yeast. Biochemical tests were done based on its capability to ferment and assimilate sugar. In addition, assimilation of nitrate was also tested. In molecular approach, the genomic DNA (gDNA) of this microorganism was successfully extracted and the extracted gDNA was used for amplification via polymerase chain reaction (PCR) technique using Internal Transcribed Spacer (ITS) primers. The PCR product obtained was sequenced and submitted for Basic Local Alignment Search Tool (BLAST) at National Center for Biotechnological Information (NCBI). The analysis showed that this microorganism contained ITS sequences (highest identity with Leocosporidium sp.). Introduction Nowadays, the demand for industrial enzymes, particularly of microbial origin is greatly increased owing to their applications in a wide range of processes (Saxena et al., 1999). Microbial enzymes are often more useful than enzymes derived from plants or animals because of their inclusive significance such as great variety of catalytic activities available, high yields possible, ease of genetic manipulation, regular supply due to absence of seasonal fluctuations and rapid growth of microorganisms on inexpensive media (Hasan et al., 2006).Cold-adapted microorganisms are potential sources of cold-active enzymes that can exhibit higher catalytic activity at low temperature (Lo Giudice et al., 2006; Gerday et al., 2000). Psychrophilic enzymes function effectively at cold temperatures with high rates of catalysis in comparison to the enzymes from mesophiles or thermophiles (Joseph et al., 2008). These enzymes might offer novel opportunities for biotechnological exploitation based on their high catalytic activity at low temperatures, low thermostability and unusual properties (Russell, 1998).Lipases (triacylglycerol acylhydrolases, E.C 3.1.1.3) are one of the enzymes that have found a great number of biotechnological applications. These enzymes catalyze the hydrolysis of triacylglycerol to glycerol and free fatty acids at oil water interface (Kamini et al., 2000). The aim of this study was to screen the lipase activity and identify the psychrophilic microorganism isolated from Casey Station, Antarctica. Methodology MicroorganismMicroorganism used in this research was selected from the laboratory collection, previously isolated from Casey Station, Antarctica. The particular microorganism was initially named as Isolate S. Growth media and culture conditionIsolate S were grown in 100ml media in 250ml conical flask. The media contains 0.45% (w/v) NaCl, 1.25% (w/v) yeast extract, 1.00% (w/v) glucose and 0.10% (v/v) olive oil. The cultivation was carried out at 15°C with the agitation rate of 150rpm for 5 days. Temperature dependence of the isolateAt the initial stage of this research, the growth of isolate S has been tested at various temperatures. The temperatures