Expression Of Recombinant B-Agarase Ⅰ Daga In Prokaryotic Cell And Its Activity Identification

Agarase plays an important role in many fields because of its ability in decomposing agarose specifically. Agarase can disassemble agarose into agar oligomers which has special properties and functions and people pay more and more attention on it. The bacteria of decomposing agarose mainly exist in ocean, and the limitation of source restricts their application in large-scale. To bring agarase and its decomposing production into full and effective play, and to widen the source of agarase, in particular, by producing agarase through genetic engineering technology, it has certain value and significance to extend the application of agarase and to develop the usage of the production with active function decomposed by agarase.Prokaryotic expression system is the most well-manipulated expression system. The effectiveness of expressing recombined protein in E.coli by pET system has been demonstrated to be the highest in history. This system was used in my study, intending to obtain the target protein in large quantity. The full length encoding gene(M73783)ofβ-agaraseⅠdagA was published on NCBI in 1996, but no report about effective expression ofβ-agaraseⅠdagA has been covered in present. Comparingβ-agarases of different resources reported, though they have similar function, huge difference on amino acid sequence, molecular weight, substrate speciality and reaction speciality still exist. To explore more resources of agarase and develop the application ofβ-agaraseⅠDagA, process as the clone ofβ-agaraseⅠdagA gene, co-expression with molecular chaperone will be used to enhance the expression efficiency of target protein, which will make it possible greatly to construct the high efficiency expression system ofβ-agarase in E.coli under proper experimental conditions. The results of this study are as follows:1. Encoding gene ofβ-agaraseⅠdagA and coding sequence dagA(▽) which was sheared signal peptide sequence were cloned form Pseudoalteromonas atlantica 19262. the recombinant strains of ER2566-pET21a-dagA(▽)-DsbC and ER2566-pET21a-dagA(▽)–FkpA were isolated as high effective strains through vector construction, transforming and selecting identification, and made the expression ofβ-agaraseⅠDagA in prokaryotic cell become true.2. The culture and inducing conditions of the recombinant strain ER2566-pET21a -dagA(▽)-DsbC which expressed recombinant DagA mainly in the form of inclusion body were optimized, which, especially for industry, improved the yield of inclusion body, and initially explored the renaturation conditions of inclusion body. We have developed secreting expression of DagA through optimizing the inducing conditions of the recombinant strain ER2566-pET21a-dagA(▽) -FkpA, which lays an important foundation for in-depth study and industrial application ofβ-agaraseⅠDagA.3. The influence of signal peptide and molecular chaperone DsbC and FkpA on the expression level of recombinedβ-agaraseⅠDagA had also been studied. Using the same vectors and molecular chaperones in different host bacteria, and only successfully expressed of DagA in ER2566 in the form of inclusion body and secretion. The dagA(▽)gene which was sheared the signal peptide and added ATG promoter could highly improve the expression yield of target protein in ER2566. Co-expression with DsbC could not enhance the secretary expression of DagA, it is speculated that the DsbC was not active and unable to play its role of molecular chaperone. Molecular chaperone FkpA has markedly enhanced the expression of protein secretion in ER2566.4. The activity and characteristics of recombinant DagA were studied. When the pH value is between pH4.8~6.8, the activity of the enzyme maintained more than 60%, and the optimizing pH value is about 5.8; the enzyme is activated when the temperature is 37~60℃, the optimizing temperature is 55℃. Comparing with other agarases reported recently, the optimizing temperature of DagA improved more than 10℃. NaCl, MgCl2 and CaCl2 solution could improve the enzyme activity obviously, but Na2EDTA solution inhibits the enzyme activity of DagA.The research have established efficient prokaryotic expression system of the enzymeβ-agaraseⅠDagA and have basically studied the enzyme characteristics, which allows further biochemical studies and industrial production for DagA.