Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme

Abstract

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of > 99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.

Structural insights into thrombolytic activity of destabilase from medicinal leech

“…Destabilase remained the only known i-type lysozyme with isopeptidase activity until a number of similar lysozymes were found in other invertebrates (Table 1 ). Among them are enzymes isolated from the annelid worm ( Eisenia andrei ) 15 , the sea cucumber ( Apostichopus japonicus ) 16 , the purple sea urchin ( Strongylocentrotus purpuratus ) 17 , the sea snail ( Haliotis discus discus ) 18 and the saltwater clam ( Tapes japonica ) 19 .…”