Production of chitinase from Verticillium lecanii F091 using submerged fermentation
Introduction
Chitinases (EC 3.2.1.14) catalyze the hydrolysis of chitin between the C1 and C4 of two consecutive N-acetylglucosamines. The enzyme is regarded important for the extensive carbon and nitrogen recycle in nature. Chitinase occurs widely in soil microorganisms and in some plants, fulfilling a possible defense role in the plant. Chitinase and/or chitosanase may find important industrial applications in the utilization of the enormous chitin and chitosan substrates, available from sea-food-processing units, for the generation of the size-specific chitosan oligomers required particularly in pharmaceutical industries. Chitinase having very diverse characteristics are known and may be of value in basic studies related to their biological role and the structural elucidation of natural chitin.
Several microorganisms, including bacteria such as Bacillus lichiniformis [1], Bacillus pabuli [2], Bacillus thuringiensis [3], Serratia marcescens [4], [5], Nocardia orientails [6], Vibrio alginolyticus [7], and many species of fungi such as: Myrothecium verucaria [8], Stachybotrys elengans [9], Streptomyces cinereoruber [10], Streptomyces lydicuis [11], Trichoderma harzianum [12], Trichoderma viride [13], Verticillium lecanii [14] have a chitinase-producing ability. Chitinase activity in plant [15], [16], [17] and human serum has also been described recently [18].
The entomopathogenic fungus, V. lecanii (Zimmerman) Viegas, has a wide range of insect hosts, including Homoptera [19], [20], [21], [22], [23], Coleoptera [24], Orthoptera [25], [26], and Lepidoptera [27]. The use of V. lecanii spores has received great attention in the biological control of insect and pests. Moreover, soybean cyst nematodes [28], cucumber powder mildew [29], and chrysanthemum rust fungus [30] can also be parasitized by this fungus. In addition, it could be used with the mixture of some insecticides or fungicides in integrated control programs to obtain a synergistic effect [31], [32].
It has been reported that agitation, pH, dissolved oxygen (DO) tension, and inoculum levels were the parameters that affect the productivity of a fermentation process. Among, agitation rate is the most critical parameter and plays a significant role in determining the productivity of the process [33]. The major roles of providing agitation were in improving the mixing, mass and heat transfer in the fermentor. Although increase in agitation may provide increased mixing and mass transfer, it may also have many negative effects in morphological state such as rupture of cells, vacuolation and autolysis, and causes the decrease in productivity [34]. Agitation rate becomes important, as it is one of the most critical parameters used for process scale-up [35]. Fenice et al. [14] reported the influence of temperature on chitinase activity from V. lecanii. However, the effects of agitation rate as well as aeration rate on the V. lecanii fermentation kinetics were not reported. The objective of this study is to investigate the desired combination of aeration, agitation, and pH that would yield the highest chitinase production by V. lecanii.
Section snippets
Microorganism
V. lecanii strain F091 was isolated from aphids by Dr. S.S. Kao of Taiwan Agricultural Chemicals and Toxic Substances Research Institute [36]. The culture was maintained on SMAY medium (Sabouraud maltose 4 g/l, agar 2 g/l, yeast extract 2 g/l, and neopeptone 1 g/l) or PDA slant at 4 °C. The fungus was transferred and propagated on a SMAY Petri dish at 24 °C for 8 days. The spores produced were suspended in 10% (v/v) glycerol of sterile distilled water by using a glass rod gently scraping the culture
Shaker-flask studies
In the electrophoretic system for crude product and partially purified enzyme showed three main bands and some other faint bands (Fig. 1). The major bands were between 35 and 50 kDa. The native chitinase was examined and exhibited a major band with molecular mass of 45 kDa as reported elsewhere [14]. This is in agreement with the present study (Fig. 1, lane 2: horizontal arrow). Another major band (∼50 kDa) in Fig. 1 may attribute to the chitosanase that has also shown the significant enzymatic
Acknowledgements
This work was supported by a research-funding grant (90AS-2.1.3-FD-Z2) from the Council of Agriculture, Executive Yuan, Taiwan, ROC.
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