Thermostable Lipase Production By Geobacillus Thermodenitrificans In A 5-L Stirred-Tank Bioreactor

摘要

Lipases from thermophiles have gained interest in recent years as it has various applications in industries. It plays a significant role in industries as it has high stability and resistant to chemical denaturation. Its extensive application in industries requires its production in a large scale. In this study, thermostable lipase production by Geobacillus thermodenitrificans in a 5-L stirred tank bioreactor was evaluated. The cultivation was carried out for 96 hours at 55 oC in a cultivation medium containing (%; w/v or v/v): glucose 1.0; yeast extract 1.25; NaCl 0.45 and olive oil 0.1. Optimization of physical parameters in the bioreactor will improve the production of the thermosatble lipase. Different physical parameters affecting enzyme production and biomass concentration such as agitation rate (100, 200, 300, 400 and 500 rpm), aeration rate (1, 2, 3 and 4 lpm) and inoculum concentration [0.5, 1.0, 1.5, 2.0, and 2.5% (v/v; 5 x 106 cell/ml)] were evaluated. Thermostable lipase activity was enhanced by 53% in the bioreactor and the biomass concentration increased by 23% after optimizing the physical parameters in the stirred tank bioreactor. Maximum lipase activity of 180 U/ml was obtained at 72 hours of cultivation at 2 lpm, 400 rpm and 2% (v/v; 5 x 106 cell/ml) inoculum with a specific activity of 3.62 U/mg. Thermostable lipase production in the bioreactor was about six-fold higher than that attained in a shake flasks culture. From the results obtained in this study, it is possible of producing thermostable lipase in a large-scale which has an extensive application in industry. Introduction Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3), which acts only on an ester-water interface, is capable of catalyzing the hydrolysis of long-chain triglycerides with the formation of diacylglycerol and carboxylate, as well as the reverse reaction with the synthesis of esters formed from fatty acids and glycerols (1). Lipase is present in diverse organisms including animals, plants, fungi and bacteria; however, only microbial thermostable lipases are of commercial importance (2-3). Extracellular microbial lipases can be produced inexpensively in large quantity by fermentation (4). Thermophilic Bacillus species previously assigned to rRNA group 5 have recently been transferred to a new genus Geobacillus (5 – 7). The Geobacillus species form a phenotypically and phylogenetically coherent group of thermophilic bacilli with high levels of 16S rRNA sequence similarity (98.5-99.2%). This group comprises established species of thermophilic bacilli (Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans). Members of the genus Geobacillus may grow at temperatures ranging from 35 to 78°C and contain iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids (8). The major requirement for commercial lipases is thermal stability which would allow enzymatic reaction to be performed at higher temperatures and would be helpful to increase conversion rates, substrate solubility, to reduce the contamination of microorganism and the viscosity of the reaction medium (1). Geobacillus sp. has attracted industrial interest for their potential applications in biotechnological processes as sources of thermostable enzymes (9). The extensive application of thermostable lipases in industries requires its production in a large scale. In this study, production of a thermostable lipase by the thermophilic bacteria G. thermodenitrificans in a 5-L stirred tank bioreactor was investigated. Stirred-tank bioreactor is ideal for industrial applications as this unit is low in both capital and operating costs. The influence of agitation rate, aeration rate and inoculum concentration on biomass and thermostable lipase production were assessed. Materials and Methods Microorganism Geobacillus thermodenitrificans was cultured o