Expression Of Alkaline Polygalacturonate Lyase From Bacillus Subtilis In Prokaryotic System And Fermentation Optimization

Alkaline polygalacturonate lyase (E.C.4.2.2.2) is one of the pectinolytic enzymes, which cleaves theα-1,4-glycosidic bond of polygalacturonate and releases unsaturated soluble oligogalacturonates via a trans-elimination mechanism under alkaline condition. Alkaline polygalacturonate lyase(PGL) has been widely used in food production for improving yield, fruit juices clarification, and detergent manufacture, etc. In addition, alkaline pectate lyases have been used in the textile industry to release fibers from flax stems, as an alternative to conventional retting to resolve many problems such as environmental pollution and high energy cost. With the emerging of new applications for PGL, the demand for the commercial enzyme is increasing.As alkaline PGL gains increased industrial interest, its production has drawn extensive attention in recent years. The current researches mainly focus on the selection and construction of alkaline PGL producing strains and process optimization. Escherichia coli, Bacillus subtilis and Pichia pastoris have been used as hosts for the heterologous expression of alkaline PGL. In the previous work, a wild-type strain (Bacillus subtilis WSHB04-02) capable of secreting alkaline PGL was isolated and the enzyme activity reached 34 U mL^-1 in shake-flask. And then the PGL coding gene from Bacillus sp. WSHB 04-02 was integrated into P. pastoris, which achieved the highest extracellular yield of PGL ever reported. Nevertheless, P. pastoris has such disadvantages as long fermentation period and complicated process control technologies in comparison with prokaryotic expressing systems. Therefore, a recombinant prokaryotic expression system is required to achieve an efficient production of alkaline PGL.(1) An efficient recombinant E. coli expression system was constructed for the secretory production of alkaline PGL. The PGL coding gene was cloned from Bacillus sp. WSHB 04-02 and the strong inducible T7 promotor of the pET-20b (+) vector was used.(2) The influence of culture media, induction temperature, and IPTG concentration on the recombinant production of alkaline PGL was examined. The optimal media composed of glycerol 10 g L^-1, yeast extract 24 g L^-1, peptone 11 g L^-1, KH2PO4 17 mmol L^-1, K2HPO4 72 mmol L^-1, ampicillin 1 mmol L^-1. The results showed that :cell growth was inhibited at high IPTG concentration,total PGL activity reached the highest ,26 oC led to a better coordination between protein synthesis and translocation and adding 5¹0 mmol L^-1 Ca2+in cluture media could enhance the secretion of PGL.After optimation, the axtracelluar production of PGL was enhanced form 11.8 U mL^-1 to 127.4 U mL^-1 .(3) A two-stage glycerol feeding strategy was developed to reach the overproduction of alkaline PGL in a 3-L jar fermentor. During the pre-induction phase, the glycerol feeding rate was increased exponentially, and the cell growth was controlled at a specific growth rate (μset) of 0.20 h-1. When the DCW reached a certain value (e.g. 18 g L^-1), the post-induction phase began and the glycerol feeding rate was shifted to 12 mL h-1.(4) By combining a series of genetic engineering and process optimization approaches, the total alkaline PGL activity reached 371.9 U mL^-1, which is the highest alkaline PGL level produced in E. coli to date. (5) The gene encoding PGL from Bacillus sp. WSHB 04-02 was inserted into shuttle vecotor pMA09.11, pP43NMK and pHT43.And then recombinat plasmid were transformed into corresponding competent cells.The extracellular PGL activity of B. subtilis WB600/pMA/pgl(sp) was 28.7 U mL^-1. The PGL activity of B. subtilis WB600/pP43NMK/pgl(sp),B. subtilis WB600/pP43NMK/pgl ,B. subtilis 168/pHT43/pgl and B. subtilis 168/pHT43/pgl(BA) in supernatant were all below 3.0 U mL^-1.(6) The enzymatic properties of PGL from E. coli BL21 (DE3)(pET-20b(+)/pgl), B. subtilis WB600/pMA/pgl(sp) and Bacillus sp. WSHB 04-02 were investigated. Compared with the PGL form Bacillus sp. WSHB 04-02, the recombinant PGLs were preferable in optimal pH. The optimal pH of PGL from recombinat E. coli and B. subtilis were 8.6¹0.6 and 9.0¹0.0, respectively; the optimal temperature were 50⁶0 oC and 50⁶5 oC, respectively.Besides,both recombinant PGL exhibit good heat stability in 35⁵5 oC.These indicated that the recombinant PGL from E. coli and B. subtilis was stable for the industrial bioscouring application.