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Applied Biochemistry and Biotechnology

, Volume 186, Issue 2, pp 459–475 | Cite as

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

  • Peng Huang
  • Jinlei Shi
  • Qingwen Sun
  • Xianping Dong
  • Ning Zhang
Article
  • 100 Downloads

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.

Keywords

Invertebrate-type lysozyme Strongylocentrotus purpuratus Pichia pastoris Antimicrobial activity Isopeptidase activity 

Abbreviations

sp-iLys

Strongylocentrotus purpuratus invertebrate-type lysozyme

AOX1

Alcoholoxidase 1

YPD

Yeast extract peptone dextrose

BMGY

Buffered glycerol-complex medium

BMMY

Buffered methanol-complex medium

MD

Minimal dextrose

PCR

Polymerase chain reaction

WCW

Wet cell weight

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

OD

Optical density

DO

Dissolved oxygen

PBS

Phosphate-buffered saline

RP-HPLC

Reverse-phase high performance liquid chromatography

HEWL

Hen egg white lysozyme

L-γ-Glu-pNA

L-γ-glutamine-p-nitroanilide

pNA

p-nitroanilide

MOPS

3-(N-morpholino) propanesulfonic acid

Notes

Acknowledgments

This work was supported by the Shanghai Municipal Commission of Health and Family Planning (Grant Number: 201740161) and the Natural Science Foundation of Shanghai (Grant Number: 15ZR1421800).

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no competing interests.

Supplementary material

12010_2018_2753_Fig8_ESM.gif (313 kb)
Fig. S1

Identification of pPIC9K-sp-iLys by digestion with restriction enzymes. Lane M: DNA marker; Lane 1: pPIC9K-sp-iLys digested with BamH I and EcoR I (GIF 313 kb)

12010_2018_2753_MOESM1_ESM.tif (2.2 mb)
High resolution image (TIF 2.22 mb)
12010_2018_2753_Fig9_ESM.gif (871 kb)
Fig. S2

Identification of positive P. pastoris transformants by PCR. Lane M: DNA marker; Lane 1: genomic DNA of P. pastoris GS115; Lane 2: empty pPIC9K vector; Lane 3: positive control; Lanes 4 and 6–10: positive transformants; Lane 5: negative clone (GIF 6.26 mb)

12010_2018_2753_MOESM2_ESM.tif (6.3 mb)
High resolution image (TIF 871 kb)

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Authors and Affiliations

  1. 1.School of Clinical MedicineShanghai University of Medicine and Health SciencesShanghaiChina
  2. 2.School of Life Science and TechnologyShanghaiTech UniversityShanghaiChina
  3. 3.School of Life SciencesFudan UniversityShanghaiChina
  4. 4.Department of Physiology and BiophysicsDalhousie UniversityHalifaxCanada

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