Fermentation Optimization For Cutinase Production With A Recombinant Bacillus Subtilis

Cutinases are mutilfunctional enzymes and belong to the family of serine hydrolases containing the so-calledα/βhydrolase fold. As a lipolytic enzyme, cutinase has been presented as a versatile enzyme showing several interesting properties for applications in industrial products and processes. Hydrolytic and synthetic reactions catalyzed by cutinase have potential use in the dairy industry for the hydrolysis of milk fat, in house hold detergents, in the oleochemical industry, in the synthesis of structured triglycerides, polymers and surfactants, in the synthesis of ingredients for personal-care products, and the synthesis of pharmaceuticals and agrochemicals containing one or more chiral centers. The application of cutinase in the textile industry has become a new research direction.Compared with the traditional technique in cotton desizing, cutinase can improve cotton wettability and fabric hydrophilicity in addition to its ability to simplify process, reduce working time, savesteam-water-electricity and protect the environment.In this paper, recombinant Bacillus subtilis WSH 06-07, a strain could accumulate high concentration of cutinase, was selected for cutinase production. According to the principles of fermentation optimization including the optimization of culture conditions based on microbial reactions (outer factor), and the optimization based on metabolic flux analysis (quantitativeness of inner factor), a series of feasible approaches or strategies were carried out to achieve high product concentration, high yield and high productivity of cutinase in the optimization of B. WSH 06-07 cultivation processes, including:1. Seed aging has a extremely influence in fermentation, and the experiments results were following: the log growth stage was from 9 to 22 h, and the seed aging was 11h, which was beneficial to cutinase fermentation.2. For recombinant cell, plasmid and cellular insoluble fraction have an important effect on cutinase yield during fermentation. The experiment results showed that plasmid stability was over 90% during the whole fermentation course. Besides, there was no cellular insoluble fraction by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the objection proteins were included in supernant.3. The required nutrients for cutinase production by B. subtilis WSH 06-07 in shaking flasks were investigated. According to the single-factor experiments and Plackett–Burman design, sucrose, trytone and MgSO4 had a profound influence in cutinase fermentation, base on which, the optimal concentration ratio among them was defined by Response Surface Methodology, and the results were summed that sucrose and MgSO4 had a positive interaction, and their effects on cell growth and cutinase production were the most important elements. Finally, the essential culture medium for cutinase production was determined to be consisted of sucrose 32.5 g/L, trptonse 37 g/L, Na2HPO4 12.54 g/L, KH2PO4 2.31 g/L and MgSO4 4.4 g/L. The environmental conditions of cutinase fermentation were also investigated and an optimal combination was developed: an initial pH of 7.5, a medium content of 75 mL medium in 500 mL flask, and an inoculum size of 5%.4. The effect of dissolved oxygen (DO) concentration on the batch production of cutinase in a 3 L stirred fermentor by B. subtilis WSH 06-07 was studied. It was found that DCW and cutinase production, especilly for cutinase content, were not greatly influenced by the agitation rate under a fixed aeration rate. When the initial sucrose concentration was 32.5 g/L and the air flow rate was controlled at 1.5 vvm, the DO concentration, under the circumstance of the agitation rate not less than 600 r/min, was sufficient to satisfy the oxygen requirement for better cell growth and cutinae production during the batch fermentation.5. The modes of pH control for batch cutinase fermentation are extremely different. B. subtilis WSH 06-07 was cultivated by controlling pH, cell growth and cutinase production were improved comparing to without pH control. Based on specific cell growth rate and specific cutinase formation rate under different pH condition, a two stage pH control strategy was developed, in which pH was controlled at 7.5 for the first 4h and then shift to 6.5. By the utilization of this strategy, the yield of cutinase was significantly improved, the maximal cutinase activity and the productivity were 170 U/mL and 16.9 kU/(L·h), which were increased by 122.6% and 123.2%, respectively, compared to that the condition of constant pH 7.5.6. The effect of temperature, varied from 27 oC to 32 oC, on cell growth and cutinase production the batch fermentation of cultivation was investigated. It was found that cell growth was hastened along with the increase of temperature while lower temperature was more favorable for cutinase formation, however, plasmid lost greatly. Owing to the difference occurred in the optimal temperature for cell growth and cutinase production by thoroughly analyzing the kinetics of batch cutinase production under different temperatures, a two-stage temperature control strategy, in which the temperature was kept at 37 oC during the first 4 h of cultivation, followed by a shift from 37 oC to 30 oC, then maintained at 30 oC until the end of the cultivation, was developed in order to enhance the production of cutinase. Compared with the results under temperature 37 oC control, the two-stage temperature control strategy was confirmed to enhance the ability of cutinase synthesis. As a result, the maximum cutinase production and the productivily were 312.5 U/(mL), 13.02 KU/(L·h), increased by 83.4%,10.9% compareing to at 37 oC, respectively, Therefore, the strategy would have good feasibility in practice.7. Effects of diverse sucrose concentration on cell growth and cutinaes production were invested by batch fermentation, a sum was found that it was diffuclt to make high product concentration, high yield and high productivity of cutinase harmony by improving sucrose concentration. As to the cultivation of B. subtilis WSH 06-07 by batch or constant fed-batch process in a 3 L stirred fermentor was studied, the resrults shown that it was efficient to realize high cell and high cutinase concentration by two kinds of feeding strateges. However, comhensive comparision of them, constant feeding fermentation was ideal choice to enhance cutinase yield wether cell yield or high product concentration, high yield and high productivity of cutinase. The DCW and cutinase activity were 52.76 g/L, 545.87 U/mL aferte 31 h constant feeding batch cultivation, respectively.