Enhanced Expression Of Hyaluronidase Via Molecular Modification And Fermentation Optimization

Hyaluronidases(HAases) are a kind of glycosidase, which degrade macromolecule hyalurinan(HA) into lower-molecular weight HA oligosaccharides. HAases exist in mammals, insect, leech, pathogenic fungi and bacteria. It has been widely applied in the medical and food fields. At present, although the extracted bovine testicular hyaluronidase has been commercialized, the contained high content of miscellaneous proteins, low purity and strong immunogenicity increase the risk of treatment. These problems have been solved through the expression of leech HAase in recombinant P. pastoris GS115. Consequently, microbial production of the leech HAase without any potential risk of animal cross-infection will be the mainstream in future.In this work, to further improve the production of HAase in the recombinant P. pastoris, the secretion capacity of four different signal peptides was investigated towards the recombinant HAase. Then, six different amphipathic peptides(APs) were introduced at the N-terminal of the LHyal gene to evaluate their effects on the secretory production. After constructing a better recombinant HAase variant, the effect of induction temperature on HAase production was analyzed. The main conclusion of this study was as follows:(1) Improving the expression of HAase with the signal peptide ns BThe signal peptide α-factor of plasmid p PIC9K-LHyal was replaced by HKR1, YTP1, SCS3 and ns B signal peptides, respectively, obtaining the recombinant strain with different signal peptides. The recombinant strain with the ns B signal peptide produced a highest extracellular HAase production, which was reached 7.96×104 U·m L-1in flask cultivation. The titer of HAase wad increased by 26.0% compared with the recombinant strain with α-factor signal peptide. The yield of HAase was accumulated to 1.03×106 U·m L-1 with fed-batch fermentation in a 3 L fermenter, which was higher 12.9-fold than performed in flask cultivations.(2) Fusion AP2 into N-terminal of LHyal gene to improve the secretion of HAaseSix different amphipathic peptides were fused into the N-terminal of LHyal gene that containing the ns B signal peptide, constructing the recombinant strains with fusion amphipathic peptides. HAase activity was increased to 9.69×104 U·m L-1 in the recombinant strain with fusion AP2, which was increased by 21.7% compared with the recombinant strain with ns B signal peptide in flask cultivations. In addition, SDS-PAGE analysis of HAase production indicated that AP2 fused into N-terminal of LHyal gene improved the secretion of HAase.(3) Enzymatic properties of AP2-HAaseMoreover, the recombinant HAase were purified to further analyze the effects of AP2 on enzymatic properties. Unexpectedly, fusion with the AP2 produced higher optimum temperature(50°C) and lower optimum p H(5.0). Thermal stability was a little enhanced and p H stability was maintained between 3.0 and 8.0. Specific activity was 1.44×106 U·mg-1, the Km value of AP2-HAase was decreased, the kcat/Km value of AP2-HAase was increased. This indicated that fusion with AP2 enhanced the catalytic efficiency of HAase.(4) Applying variable temperature strategy to improve the production of HAaseThe cell concentration reached about 190.0 g·L-1 at different temperature, the maximum titer of HAase was accumulated to 1.48×106 U·m L-1at 22°C. Based on the investigation of cell viability and alcohol oxidase(AOX) activity, an appropriate induction strategy with variable temperature was applied to improve the production of HAase. In this strategy, temperature was controlled at 25°C during the beginning of induction phase 1-60 h, then decreased to 22°C after 60 h. The yield of HAase were accumulated to 1.68×106 U·m L-1 in a 3 L fermentor, which was higher 3.5-fold than performed at 30°C and the highest value reported to date.