Purification and characterization of chitinase produced by thermophilic fungi Thermomyces lanuginosus

Abstract

Background

In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed.

Result

The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of β-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa.

Conclusion

The characterization study concludes that Cu²⁺, Hg²⁺, and EDTA have an inhibitory effect on chitinase activity, whereas β-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste.

Keywords:

• Anion exchange chromatography
• chitinase
• enzyme kinetics
• purification
• SDS-PAGE

Acknowledgments

The authors gratefully acknowledged the National Institute of Technology Raipur, C.G., India for providing the necessary facility to carry out this work.

Additional information

Funding

Authors would like to thank the Council of Scientific and Industrial Research, New Delhi, India for providing the CSIR-JRF/SRF fellowship grant and the CSIR award [number is 09/1116(0002)/2016-EMR-I].