Expression,Purification And Enzymatic Proporties Of Recombinant Hyaluronidase

Hyaluronidase (Hyaluronidase, HAase) is a general term for a class of enzymes which have the hyaluronic acid (HA) degradation activity. HAase can degrade the HA from a relative high molecular weight to a low molecular weight (LMHA) or oligo poly hyaluronic acid (O-HA) by acting at ?-1,3 or (3-1,4 glycosidic bond. LMHA and O-HA have been widely used in food, cosmetics and medicine. However, the preparation methods of LMHA and O-HA in commercial have many disadvantages and need to be modified.In this study, a pair of primers which designed according to the DNA sequence alignment results of the bacterial sourced HAase from GenBank. The hyaluronidase encoding gene (hyl) was amplified by PCR method using Streptococcus zooepidemicus MF002 genome as the template. The length of hyl was 2589 bp and the GenBank No. was KP661598. The amplified hyl fragment was inserted into a temperature-inducible expression vector named pBLMVL2. The recombinant plasmid was transformed into Escherichia coli BL21. Then, the recombinant HAase, which had a relative molecular mass of 9.7×104Da, was successfully expressed in recombinant E. coli named MF005. The results of temperature induction experiment and SDS-PAGE analysis showed that the optimum temperature for HAase expression was 41.5?. The HAase solution showed hydrolytic activity after renaturation from inclusion body. The specific activity of HAase solution reached 6.28×105 U/mg under an optimum condition at 37? and pH5.5. Besides, the existence of Ca2+?Mg2+?Co2+ had positive effect on the activity of recombinant HAase. And the recombinant HAase could degrade HA from a molecular mass of about 1.6×106Da to 104Da. Therefore, this study provided a gentle and efficient method to prepare LMHA or O-HA.To overcome the disadvantage on the complicated purification processes from the production of HAase through genetic engineering, the relatively low utilization efficiency of purified HAase and the complicated separation steps during production of LMHA and O-HA, a new method was developed in this study. A HAase surface display system was constructed using ice nucleation protein as anchoring motif. The aim of this system was to display HAase on the bacterial cell surface. This system could interact with HA substrate outside and produce LMHA and O-HA. The inaq-n which was responsible for transmembrane function was fused to hyl. The fusion gene was inserted into pBLMVL2 and transformed into Escherichia coli BL21. The recombinant strain named MF106 was used to hydrolyze the HA, directly. The optimum enzymatic activity was achieved at 37? and pH6.0. And the surface display system could degrade HA from a molecular mass of about 1.6×106Da to 7.4×104 Da. In conclusion, the surface display system can be used as a whole cell catalyst for the preparation of LMHA directly. This study provided a new and simple method for the production of LMHA.