Optimization of media for improved production of recombinant T1 lipase using local substrates

Thermostable T1 lipase carries a lot of potential in industrial applications such as in diesel production and detergent formulation. However, the usage of laboratory media can cost a fortune when used at commercial scale (adding up to the final cost value of the enzyme). In order to create a cheaper enzyme product a new medium formulation from cheaper sources and readily available is crucial. This study was designed to formulate new medium and to develop an efficient large scale bioprocess strategie for thermostable T1 lipase from recombinant E. coli BL21. Different carbon and nitrogen sources from agro and industrial waste were screened. The compositions of the medium were optimized using response surface methodology (RSM). Isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose capability as inducer were also studied. The kinetics of T1 lipase production by recombinant E. coli were evaluated using Monod and Luedeking-Piret equations. The effects of dissolved oxygen tension (DOT) level on growth of recombinant E. coli and T1 lipase production were investigated in batch fermentation using 7.5 L stirred tank bioreactor. Fed-batch fermentation for T1 lipase production was initially developed in 7.5 L stirred tank bioreactor and then scaled up to 30 L. A newly formulated medium for production of T1 lipase was formulated using 5th grade molasses and fish processing waste as carbon and nitrogen sources. The medium consisted of molasses (2 g/L), fish waste (12%), NaCl (5 g/L), MgSO4 (0.5 g/L) and KH2PO4 (1 g/L). Through centre composite design (CCD), medium compositions using IPTG as an inducer showed higher T1 lipase production in predicted (172.89 U/mL) and actual run (164.37 U/mL) compared to lactose as an inducer in predicted (123.47 U/mL) and actual run (120.34 U/mL). Both R2 values calculated using RSM showed a good fit and the proposed models for T1 lipase production by recombinant E. coli were sufficient to describe the processes. T1 lipase production was found to be a growth associated process and 30% showed the optimal level of DOT for production of T1 lipase. The constant feed rate for fed-batch fermentation at 160 mL/h using 50% lactose as feeding medium was found to be optimal for production of T1 lipase (260.10 U/mL) and recombinant E. coli growth (51.30 g/L). The fermentation employing recombinant E. coli for T1 lipase production was successfully scaled-up to 30 L stirred tank bioreactor using a constant DOT approach, with DOT level controlled at 30% saturation. 50% of cost reduction was successfully achieved in production of T1 lipase when using new formulated medium and so far, this is the first report of using molasses and fish waste in the medium formulation. The information and findings obtained from this study are very useful in designing and in the preparation of standard operating procedure (SOP) for production of T1 lipase by recombinant E. coli at pilot plant and at industrial scale

Cloning and Expression of Staphylococcus Epidermidis AT2 Lipase in Yarrowia Lipolytica

Proteolytic degradation and the production of protein that accumulates as misfolded form always occur in bacterial expression system. In view of that, Yarrowia lipolytica is chosen as a host to express heterologous protein. The gene encoding sequence of Staphylococcus epidermidis AT2 lipase (1.2kb) was cloned into Y. lipolytica expression vector (pYLEX1) and placed under the regulation of the strong hybrid promoter (hp4d) carrying four tandem copies of an upstream activator sequence (UAS1B) from pXPR2 and a minimal pLEU2 fragment. Previously, primers were designed on the basis of S. epidermidis lipase precursor (geh1) gene (AF053006). PCR (Polymerase chain reaction) was used to amplify the gene and cloned into pJET 1.2/blunt-end vector (Fermentas) transformed into E. coli DH5α competent cell. After the gene was propagated in E. coli, the gene were purified and ligated into pYLEX1 vector (Yeastern Biotech). The recombinant plasmid was extracted and linearized before it was transformed into Y. lipolytica host strain Po1g. The recombinant Y. lipolytica was grown on YNB selection medium. Five positive transformants harboured the expected size of AT2 lipase gene were obtained and one of the transformants showed the highest expression. The expression of AT2 lipase enzyme was optimized at of 28 0C with the agitation speed of 200 rpm in optimized YNB medium. Process of breaking the cells or sonication profile was optimized at 7.5 min and the highest activity obtained was 14 U/mL. The crude proteins were electrophoresed on 12% (w/v) of SDS-PAGE and estimated protein band of 43 kDa was detected when stained with Coomassie brilliant blue. The expressed enzyme retained 100% of its activity after 30 min incubation (37 0C) in n-hexane, p-xylene and dimethyl sulfoxide

Optimization of physical conditions for the production of thermostable T1 lipase in Pichia guilliermondii strain SO using response surface methodology

Abstract Background Pichia guilliermondii was found capable of expressing the recombinant thermostable lipase without methanol under the control of methanol dependent alcohol oxidase 1 promoter (AOXp 1). In this study, statistical approaches were employed for the screening and optimisation of physical conditions for T1 lipase production in P. guilliermondii. Result The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0. Conclusion The process of using RSM for optimisation yielded a 3-fold increase of T1 lipase over medium before optimisation. Therefore, this result has proven that T1 lipase can be produced at a higher yield in P. guilliermondii

Improving the efficiency of new automatic dishwashing detergent formulation by addition of thermostable lipase, protease and amylase

he use of T1 lipase in automatic dishwashing detergent (ADD) is well established, but efficiency in hard water is very low. A new enzymatic environmentally-friendly dishwashing was formulated to be efficient in both soft and hard water. Thermostable enzymes such as T1 lipase from Geobacillus strain T1, Rand protease from Bacillus subtilis strain Rand, and Maltogenic amylase from Geobacillussp. SK70 were produced and evaluated for an automatic dishwashing detergent formulation. The components of the new ADD were optimized for compatibility with these three enzymes. In compatibility tests of the enzymes with different components, several criteria were considered. The enzymes were mostly stable in non-ionic surfactants, especially polyhydric alcohols, Glucopon UP 600, and in a mixture of sodium carbonate and glycine (30:70) buffer at a pH of 9.25. Sodium polyacrylate and sodium citrate were used in the ADD formulation as a dispersing agent and a builder, respectively. Dishwashing performance of the formulated ADDs was evaluated in terms of percent of soil removed using the Leenert‘s Improved Detergency Tester. The results showed that the combination of different hydrolysis enzymes could improve the washing efficiency of formulated ADD compared to the commercial ADD “Finish” at 40 and 50 C

Production of thermostable T1 lipase using agro-industrial waste medium formulation

Large-scale production of T1 lipase using conventional culture media is costly. To reduce the cost of production, an alternative growth medium using local resources has been developed. In this study, the growth of recombinant Escherichia coli and expression of T1 lipase were tested using different agroindustrial wastes as carbon and nitrogen sources by conventional method. Subsequently, by using central composite rotatable design (CCRD), a set of 30 experiments was generated to evaluate the effect of different parameters, including the amount of molasses (as carbon source), fish waste (as nitrogen source), NaCl, and inducer concentration on production of T1 lipase. Response surface methodology (RSM) analysis indicated that all factors had significant effects on T1 lipase production. This statistical analysis was utilised to develop a quadratic model to correlate various important variables for the growth of the recombinant strain and regulation of gene expression to the response (T1 lipase activity). Optimum conditions for T1 lipase production were observed to be 1.0 g/L of molasses, 2.29 g/L of fish waste, 3.46 g/L of NaCl, and 0.03 mM of IPTG (Isopropyl β-d-1-thiogalactopyranoside). Based on these conditions, the actual lipase activity was found to be 164.37 U/mL, which fitted well with the maximum predicted value of 172.89 U/mL. Therefore, the results demonstrated that, the statistical analysis, performed using RSM, was efficient in optimising T1 lipase production. Moreover, the optimum conditions obtained can be applied to scale up the process and minimise the cost of enzyme production