Fermentation Optimization And Purification Of ?-carrageenase Produced By Recombinant Strain BL21-HTa-cgkZ

Carrageenans are linear sulfated polysaccharides extracted from the extracellular matrix of red marine algae such as Eucheuma, Chondrus and Hypean. And the mainly three types of carrageenans are ?-, ?-, and ?-carrageenan. Researches on the structure and activities of the kappa-carrageenan oligosaccharides in recent years, found that the kappa-carrageenan oligosaccharides and some of its derivatives have many important physiological activities. A large amount of references and materials reported the biological activities of the kappa-carrageenan oligosaccharides, including antiviral, anti-tumor, antioxidant, inhibition of angiogenesis, immune regulation and so on. The kappa-carrageenan oligosaccharides show a greater development and better application prospect in the field of biological medicine because of its advantages of smaller molecular weight, good solubility and absorption compared with carrageenan The kappa-carrageenase, as a polysaccharide hydrolase, degrades the kappa-carrageenan to kappa-carrageenan oligosaccharides by ?-1,4 glycosidic bond. And for the mild means, the advantages of simple operation and highly specificity of the enzymatic degradation, kappa-carrageenase is an ideal means to get the kappa-carrageenan oligosaccharides compared with other methods.A recombinant strain BL21-HTa-cgkZ had been successfully constructed by the application of gene recombination technology in the laboratory, which can express kappa-carrageenase by exocytosis. On the premise of it, this paper studied the optimal fermentation conditions of the recombinant strain BL21-HTa-cgkZ, separation and purification of the kappa-carrageenase, and the process of enzyme preparation. The main research and results were as follwes:1.The optimal fermentation conditions of recombinant strain BL21-HTa-cgkZ? induced by IPTG were determined by using single factor design and response surface analysis in the flask. As a result, the optimal medium compositions were composed of lactose 7 g/L, tryptone 10 g/L, yeast extract powder 5 g/L, NaCl 10 g/L and CaCl2 0.666 g/L. The optimal induction condition was adding IPTG 0.89 mmol/L after inoculation for 1.55 h and maintaining the fermentation at 23 ? for 24 h. After the condition optimization, extracellular enzyme activity of kappa-carrageenase produced by recombinant strain BL21-HTa-cgkZ can reach 10.76 U/mL, while the enzyme activity of kappa-carrageenase produced by wild type bacterium Zobellia sp. ZM-2 was 0.33 U/mL. That means the enzyme producing ability of recombinant strain BL21-HTa-cgkZ was 32.6 times of the original strain. The distribution of the kappa-carrageenase expressed by recombinant strain BL21-HTa-cgkZ was also affected by the additives such as lactose, Triton X-100 and Tween-80. The result showed that the way of increasing the enzyme activity by lactose was different with the way by Triton X-100 and Tween-80 in the medium. Lactose in the medium was conducive to the total amount of kappa-carrageenase by promoting the growth of recombinant strain BL21-HTa-cgkZ, while Triton X-100 and Tween-80 promoted the release of kappa-carrageenase from intracellular to extracellular by changing the permeability of cell membrane.2. The kappa-carrageenase produced by recombinant strain BL21-HTa-cgkZ was successively purified by ion exchange chromatography of CM Sepharose Fast Flow, ultrafiltration concentration and gel filtration chromatography of Sephadex G-75. Using polyacrylamide gel electrophoresis to get a very clear band, its molecular weight was approximately 62 kDa, which was very close to the deduced molecular weight according to the DNA sequences of the kappa-carrageenase. Then this electrophoresis strip and the protein sequences deduced by the DNA sequences were delivered to Beijing Protein Institute to mass spectrometry identification The result showed that strip was kappa-carrageenase by LC-MS/MS mass spectrometry identification. After purification, the specific activity of kappa-carrageenase was 94.23 U/mg,12.33 times that of berore. On the basis of knowing the definite molecular weight of kappa-carrageenase, the inclusion body protein of recombinant strain BL21-HTa-cgkZ was extracted and electrophoresed to determining the molecular weight. The result showed the kappa-carrageenase was not expressed in the form of inclusion body protein.3. The enzyme preparation of kappa-carrageenase was preliminary prepared. The enzyme activity of kappa-carrageenase produced by recombinant strain BL21-HTa-cgkZ in a scale-up experiment using a 10 L fermentor can reach 10.82 U/mL. The extraction of kappa-carrageenase was done by three methods, ammonium sulfate precipitation, ethanol precipitation and acetone precipitation, respeceively. The result revealed the enzyme activity lost a lot when using ammonium sulfate precipitation or acetone precipitation to extract kappa-carrageenase, and ethanol precipitation was a moderate way. The optimal extraction conditions of ethanol precipitation were determined by using single factor design and response surface analysis. The result showed the optimum extraction conditions were:42.6% ethanol,4.83 h for ethanol precipitation and pH 5.84 of the crude enzyme before adding ethanol. In these conditions, the total enzyme avtivity recovery of kappa-carrageense was 72.4% and the enzyme activity of enzyme preparation was 38.23 U/mL.As an important marine enzyme, kappa-carrageenase contributes a lot to the research and practical application, but it is difficult to large-scale development and utilization because of the lower enzyme activity. This paper was devoted to increasing the enzyme activity of kappa-carrageenase, to lay the foundation for industrial application of carrageenase.