Intergeneric fusant development using chitinase preparation of Rhizopus stolonifer NCIM 880.

Kailas D Sonawane,Narayan R Dandagal,Piyush T Gurav,Naiem H Nadaf,Akibjaved G Naikwadi,Deepak B Jadhav,Samar V Anapat,Shailesh R Waghmare

doi:10.1186/s13568-016-0287-8

Abstract

Fungal chitinase have tremendous applications in biotech industries, with this approach we focused on extracellular chitinase from Rhizopus stolonifer NCIM 880 for the formation of fungal protoplasts. The maximum chitinase production reached after 24 h at 2.5% colloidal chitin concentration in presence of starch as an inducer. Chitinase was extracted efficiently at 65% cold acetone concentration and then purified by using DEAE-Cellulose column chromatography. Purified chitinase having molecular weight 22 kDa with single polypeptide chain was optimally active at pH 5.0 and temperature 30 °C. The purified chitinase revealed kinetic properties like Km 1.66 mg/ml and Vmax 769 mM/min. Crude chitinase extract efficiently formed protoplasts from A. niger, A. oryzae, T. viride and F. moniliforme. The formed protoplasts of A. niger and T. viride showed 70 and 66% regeneration frequency respectively. Further, intergeneric fusants were developed successfully and identified at molecular level using RNA profiling. Thus, this study could be useful for strain improvement of various fungi for biotechnological applications.

Highlights

Chitinase (E.C. 3.2.1.14) is a glycosyl hydrolase which catalyzes degradation of chitin polymer (Henrissat and Bairoch 1993)
It was found that R. stolonifer produces maximum chitinase production at 24 h incubation periods, which indicates that chitinase was produced during logarithmic phase and chitin used as sole source of carbon to obtain energy
The extracellular chitinase was extracted from fungi R. stolonifer National Collection for Industrial Microorganisms (NCIM) 880 in presence of chitin as a substrate similar to earlier studies using R. oligosporus (Yanai et al 1992) and R. oryzae (Chen et al 2013)

Summary

Introduction

Chitinase (E.C. 3.2.1.14) is a glycosyl hydrolase which catalyzes degradation of chitin polymer (Henrissat and Bairoch 1993). Plant chitinases function in self defense against pathogens having chitinous cell wall, whereas yeast and fungal chitinases are required for development and growth of the respective organisms (Gohel et al 2006). Chitinase from Autographa californica nucleopolyhedrovirus was successfully expressed in Protoplast fusion is an important tool to perform strain improvement as well as to manipulate and develop hybrid strains in filamentous fungi (Lalithakumari 2000). Fungal protoplasts have been used as an effective experimental biochemical tool to study cell wall synthesis, enzyme synthesis and their secretion, as well as in strain improvement for biotechnological applications. Dahiya et al (2005) reported the effectiveness of Enterobacter sp. NRG4 chitinase in the generation of protoplasts from Trichoderma reesei, Pleurotus florida, Agaricus bisporus, and A. niger (Kitamato et al 1988; Dahiya et al 2006).

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Results

Conclusion