Fermentation Optimization For Cutinase-CBM Production With Recombinant E.coli

As a multifunctional lipolytic enzyme, cutinase could degenerate the cutin which is located on the plant surface, thus it is being used as a key additive in the textile industry. Cellulose-binding module (CBM) could adsorb highly on cellulose. Thus, compared to the cutinase, the enzyme fused with CBM can get higher catalysis efficiency by enhancing the affinity between the enzyme and fiber substrate. Our lab has successfully fused the gene of CBM to the cutinase and obtained two types recombinant strains: the TypeⅠrecombinant str- ain, Tfu₀883-CBM-HlyAs/pET20b(+)/E. coli BL21(DE3), and the TypeⅡrecombinant strain, Tfu₀883-CBM/pET20b(+)/E. coli BL21(DE3).In this paper, in order to increase the yield and productivity of cutinase-CBM in the medium by scale-up fermentation, we investigated different media and culture conditions on the extracellular production of cutinase-CBM in shaking flasks and 3-L fermentator. The main results are listed as follows:1. Studying the effects of the seed age on the fermentation process of the TypeⅡ, the growth curve was introduced to describe the growth condition of the seed; the seed was cultivated for 8-10 hours before inoculating into the TB medium. An extracellular enzyme activity of 27 U/ml was reached at the fermentation time of 48 H.2. Studying the effects of the nutritional condition on the growth and cutinase-CBM production of the TypeⅡrecombinant strain on the flask level,. an optimized fermentation strategy was developed through single factor analysis and orthogonal design, which can be illustrated as follows: glycerol 5 g/L, peptone 16 g/L, MgSO₄?7H₂O 2.5 mmol/L, K2HPO₄ 13.7 g/L, KH₂PO₄ 1.53 g/L, 1 g/L lactose and 0.75 g/L glycine (final concentration) were added at the pro-metaphase of logarithmic phase. On the basis of above-mentioned medium, investigating the influence of other factors, such as heat-shock, osmotic agent, and tempera -ture, on the extracellular expression of cutinase-CBM of the TypeⅡ, it was found that with the addition of 75 mmol/L L- proline, heat-shock for 1h at 37°C or 0.5h at 47°C, and then shifted to 25°C, the final extracellular expression of cutinase-CBM reached 90 U/ml.3. The feeding strategies of carbon source in correlation with the effect of DO-stat and pH–stat on the growth and cutinase production from Type II strain was investigated. As the results from the use of differentμset of exponential feeding on the growth and extracellular experession of the cutinase-CBM showed, employing an exponential feeding strategy,μset of 0.25 at the end of the batch phase.Studying the induction conditions of the TypeⅡ, including induction points, induction temperatures, pH in induction phase and the feeding strategies of inducer, the results shows that, starting the induction at OD 30, temperature at 37°C, pH at 7.1 and intermittent feeding of inducer, the final extracellular production of cutinase-CBM reached 180 U/ml, which was a 50% increase when compared to consistent feeding. 4. For the low efficiency and extracellular production of the typeⅡsecretion, the growth and production of TypeⅠwas measured, a final extracellular production of cutinase–CBM reached 492 U/ml when two-stage exponential feeding were employed. .