“A Study on the Bacterial Production of Phospholipases C with Potential Industrial Application”

Abstract

One hundred soil samples were collected from seven different Egyptian governorates from which two hundred and thirty one isolates, with the characteristic morphology of Genus Bacillus, were recovered.The collected isolates were screened for phospholipase C (PLC) production by egg-yolk plate method, and 101 isolates were found to produce PLC of which 60 were considered very strong producers, 16 were relatively strong producers and the same number were mild producers. Only 9 isolates were considered weak producers. The sixty isolates that have shown very high PLC production were further assessed using a chromogenic assay method. The highest five producers, were identified by 16S rRNA gene sequencing as Bacillus thuringiensis, and their assembled sequences were deposited in Genbank database with assigned accession codes. PLC was purified to homogeneity using ammonium sulfate precipitation followed by Sephadex G-75 gel filtration chromatography and showed apparent molecular mass of 28.5 kDa as indicated by SDS-PAGE. B. thuringiensis B100 PLC was relatively thermostable with a maximum activity at 40°C and temperature above 50°C for loss of half the enzyme activity. Activity at the wide temperature range (20-80°C) was high (about 50% of maximum). This PLC could tolerate pH as high as 12 with only 30% loss of activity. Equivalent activities were recorded at low water tension. Specificity pattern of PLC from the same isolate showed equivalent activities toward phosphatidylcholine and phosphatidylinositol in addition to marked activity toward phosphatidylethanolamine. Optimization of PLC production by B. thuringiensis B100 was done using One-Factor-at-a-time (OFAT) method followed by response surface methodology (RSM) in submerged fermentation in shake flasks as well as in solid state fermentation. For optimization of production in submerged fermentation, the first step was optimization of process parameters concerning five variables; fermentation time, initial pH of fermentation medium, fermentation temperature, inoculum size and degree of aeration represented as the contained volume of medium in flasks. The optimal ranges obtained from the OFAT tests were employed in the RSM model. The model consisted of 51 experiments and had an R2 of 0.97.Fermentation under the optimized parameters of 21 h of fermentation at 28.5°C using an aeration level was 78% and an inoculum size of 9% added to a fermentation medium of initial pH 7.6 resulted in about 2-fold increase in PLC productivity (5.43 U/mg DCW) compared to production under unoptimized conditions (2.37 U/mg DCW). Second was the optimization of media composition. The OFAT study included the effect of some nitrogen sources, some carbon sources, potassium dihydrogen phosphate and its concentration, some surface active agents and some metal salts. Results have shown that the components that augmented PLC production were peptone, beef extract, malt extract and inorganic phosphate. Thus, these factors were studied at a concentration range and the concentrations that supported maximal production were the variables incorporated in the RSM model. A model consisting of 29 experiments and having an R2 of 0.96 was used to deduce the optimal medium composition. Using the optimized medium composed of peptone (1.8 g%), beef extract (0.50 g%), malt extract (1.00 g%; w/v) and KH2PO4 (0.1 mM) under optimized process parameters resulted in about 54-fold increase in PLC productivity (145.33 U/mg DCW) compared to the unoptimized medium (Luria-Bertani broth) under unoptimized process parameters. To the best of the available knowledge, this is the first report on the use of SSF for PLC production. A number of agro-substrates were screened for supporting PLC production by B. thuringiensis B100 and of them wheat bran was selected as it supported maximal production. The effects of different factors on production were studied prior to RSM optimization. These preliminary tests included the effects of level of moisturizing solution, inoculum size, incubation temperature, in addition to the effects of the type of moisturizing solution, its pH, and the addition of co-carbon and co-nitrogen sources to it. The factors that increased PLC production (level of moisturizing solution, inoculum size and incubation time at 5 levels each)were incorporated in a central composite RSM model consisting of 19 experimants. The model showed an R2 of 0.99 and resulted in optimized PLC productivity to a level of 429.35 U/mg Dry Cell Weight (DCW) corresponding to about 27-fold increase in productivity at a level of moisturizing solution of 200% (v/w), an inoculum size of 0.6 ml/ g dry matter and incubation for 96 h. The first step in the scale-up process is the move from production at the shake flask level to that in the laboratory fermentor. During this study, the fermentation process was conducted in a 14 L fermentor with its vessel containing 4 L of the optimized fermentation mediumwith the composition devised by the RSM model for optimization of medium components (Phosphate starved-Peptone-Beef Extract-Malt Extract Medium). Also, the optimum fermentation temperature (28.5°C) and inoculum size (9%) calculated by the RSM model for optimization of process parameters were those employed in the fermentor cycles.The effect of pH control was studied through two fermentation runs carried out under identical conditions (aeration rate 1 vvm and agitation rate 100 RPM). One run was conducted without on-line pH control, while in the other pH was kept constant at 7.6 which is the optimum pH for the fermentation medium as developed by the RSM model for optimization of process parameters on the shake flask level. Results have shown that pH control at 7.6 resulted in almost double the PLC production obtained under the same conditions but without pH control. When the aeration and agitation rates were increased to 2 vvm and 200 RPM, respectively, while controlling pH at 7.6, PLC production has increased to unprecedented high levels (1750 U/ml) which is about 10 folds higher than that obtained in the shake flask level. Since the benchmark for a successful scale-up process is managing to match at least the level of production obtained in shake flasks, in this scale-up study the shake flask production level was not only matched but also immensely exceeded.