Screening Of Thermophilic Neutral Lipase-Producing Pseudomonas Sp. ZBC1and Improving Its Enzymatic Activity

Lipases (triacylglycerol acylhydrolases, EC3.1.1.3) are carboxylesterases which catalyze the hydrolysis of triacylglycerols to glycerols, monoglyceride, diglyceride and free fatty acids, while the reverse reaction is also driven by lipases in non-aqueous conditions. Due to their stability, selectivity and broad substrate specificity, microbial lipases are broadly used in industry. Besides their widespread use in cosmetic and organic synthesis industries, lipases have become increasingly prevalent in food industry, specifically for interesterification of oils and fats to produce baker’s margarine, cake shortening and vanaspati fat. Moreover, interesterification is one of the best methods for trans-ftee fats production, while trans fatty acids were reported to be risk factors involved in coronary heart disease, diabetes mellitus and cancer.Lipases are widely found in nature, but only microbial lipases are commercially significant. It has been reported that commercial lipases are mainly produced from bacteria, fungi and yeast. Pseudomonas sp. lipases, belong to bacterial lipases, have unique properties to meet the demands of business applications, particularly in food industry. However, the lipases production of Pseudomonas sp. have relatively low yield and high cost, so the enhancement of the production becomes an important step for commercial application. Statistical experimental design is the methodology of conducting and planning experiments to obtain the maximum lipase production in the fewest number of runs. In this study, the screening of lipase, medium optimization and lipase properties were investigated for bacteria associated with the oil-contaminated soil.Among forty microorganisms isolated from oil-contaminated soil, sixteen of them were found to be with lipase activity. Three strains (ZBC1, ZBC2and ZBC3) were selected as good lipase producers using Rhodamine B-olive oil plate agar and liquid state fermentation in shake flask cultures. They were identified as from Pseudomonas, Burkholderia and Klebsiella genera by morphological, biochemical characterization and16S rRNA gene sequence. The Pseudomonas sp. ZBC1maximum lipase activity reached8.5U/mL at48h. The Burkholderia sp. ZBC2and Klebsiella sp. ZBC3maximum lipase activity reached8.25U/mL at120h and72h, respectively. Thus, Pseudomonas sp. ZBC1was selected for medium optimization and lipase property.Pseudomonas sp. ZBC1was found to produce thermophilic, neutral lipase which exhibits the maximum activity the optimum temperature at80℃and highly stable at80℃, with over90%residual activity for25min and65%for75min. Pseudomonas sp. ZBC1lipase exhibits the maximum activity the optimum pH7.0and stability at pH6.5-9.0, almost70%of the residual activity retained after pre-incubation for24h at4℃.Through the single factor experiment optimize Pseudomonas sp. ZBC1fermentation conditions. The results showed that maltose was the best carbon source for maximal lipase production, followed by sucrose, starch, glycol and yeast powder, whereas the catalytic reaction was repressed by glucose as the carbon source. A combination of organic (Soybean flour) and inorganic nitrogen sources (NH4NO3) was used for lipase production. The maximum lipase activity was at initial pH6.50mL of basal medium in250mL Erlenmeyer flasks led to maximum lipase production. A high inoculum density of15%v/v (A550=0.8) and culture time of seed at16h led to maximum lipase production.0.7mg/mL MgSO4and0.5mg/mL Na2HPO4is more advantageous to the lipase production. The classical optimization method (single factor optimization) does not depict the complete effects of the parameters in the process and ignores the combined interactions between physicochemical parameters. In order to overcome this major problem, optimization studies are done using Response Surface Methodology (RSM) which has gained lot of impetus for medium optimization and for understanding the interactions among various physico-chemical parameters using a minimum number of experiments. Plackett-Burman design (PB design) was applied to select the most significant factors of MgSO4, inoculum size and medium volume for RSM analysis, resulting an optimal composition of MgSO4(0.85g/L), inoculum size (6.59%) and medium volume (51.57mL), respectively. Using the optimal conditions, lipase production by Pseudomonas sp. ZBC1was enhanced by approximately2.3fold (from8.5to19.5U/mL).