Development Of Hvaluronidase Bacterial Cell Surface Display Systems Using Ice Nucleation Protein Anchor Towards Biotechnological Appliaction

Hyaluronidase (Hyaluronidase, Hyal) is a general term for a class of enzymatic degradation of hyaluronic acid. Hyal can degradate (Hyaluronid acid, HA) for low molecular hyaluronic acid (LMHA) or oligo poly hyaluronic acid (shape on O-HA) by acting on β-1,3-or β-1,4 glycosidic bond. LMHA and O-HA have been widely used in food, cosmetics, medicine. In this study, a hyaluronidase bacterial cell surface display system was bulit through the bacterial cell surface display technology, and used for the preparation of LMHA and/or O-HA.According to the Streptococcus zooepidemicus Hyal coding gene sequence in GenBank, primers pairs for anmlifing hyal gene were designed. A 2.6kb hyaluronidase gene was obtained from Streptococcus zooepidemicus SM001 by polymerase chain reaction (PCR). Sequence analysis showed that the cloned hyal gene had similarities of 96.10%,97.2%and 98.15% compared with genes from Streptococcus equi subsp. zooepidemicus strain ATCC 35246、Streptococcus equi subsp. zooepidemicus strain NC88 and Streptococcus equi subsp. zooepidemicus cvcc23362 in GenBank, respectively. The amino acid sequence had similarities of 90.02%,91.13% and 90.13%, respectively. Through the analysis of the phylogenetic tree, Hyal protein is located in the Streptococcus equi subsp zooepidemicus.Plasmids harboring hyal and inaQ-N were digested with EcoRⅠ/BgⅡ enzymes, hyal and pTrcHisC-inaQ-N fragments were recycled and ligated. Recombinant plasmid pMF004 containing fusion gene inaQ-N/hyal was constructed. Meanwhile, an intracellular control plasmid pMF003 containing Hyal gene was constructed. Two plasmids were transformed into Escherichia coli host strain JM109. The screening and identification was used to finally obtain the recombinant strains MF003 JM109/pMF003 and MF004 JM109/pMF004.Expression condition of the recombinant strains MF004 was optimized by EPTG inducing concentration, temperature and reaction temperature, pH and Ca2+ concentration induced. The results showed that enzyme activity was optimal at 30℃,in 0.8mM IPTG induction concentration for 10h and reaction conditions under Ca2+ concentration of 0.8-1.0mM and pH6.0. The expression of recombinant protein was verified by SDS-PAGE and Western blot. The results showed that InaQ-N/Hyal fusion protein has a molecular weight of about the size of 116kDa, including a Hyal about 96kDa. The results showed that when the protease treated for 1h, enzyme activity decreased by 75%. The fusion protein InaQ-N/Hyal can sectred and anchored on the surface of Escherichia coli JM109 cell surface successfully.To detect the HA degradation activity, viscosity method, turbidimetric method and Morgan Elson method was compared. The results show that viscosity method has the best detection result. In surface display system, the enzymatic reaction conditions were explored. The results showed that in 37℃, pH6.0, 1.0mM Ca2+ concentration condition, recombinant strain MF004 has a stabilized HA degradation activity. The recombinant strain MF004, as a whole cell catalyst, could degradate high molecular weight HA from an average molecular weight of 1500kDa to 100kDa in lhour at a concentration of 0.2mg/ml HA. The average molecular weight of HA was finally reached 70kDa in 3h.