Production of chitinase from Verticillium lecanii F091 using submerged fermentation

Abstract

The effects of submerged cultivation parameters on the production of chitinase by Verticillium lecanii in a shaker-flask and bioreactors were investigated. The activity of chitinase was 9.95 mU/ml in an optimal culture medium with culture volume at 200 ml, agitation rate at 150 rpm, and 24 °C in shaker-flask cultivation. Based on this result, the scale-up cultivation with a 5-l stirred-tank bioreactor (STR) and a 30-l airlift bioreactor were conducted. The high chitinase activity 18.2 mU/ml was obtained under the optimal cultivation conditions of aeration rate at 0.6 vvm, pH 4, agitation rate at 150 rpm, and 24 °C, with a 5-l STR. The chitinase went up to 19.9 mU/ml by using 30-l airlift bioreactor with 24-mesh net-draft tube at aeration rate of 0.9 vvm, pH 4, and 24 °C. The study revealed that the pH and agitation rate were the most significant factors for the effects on chitinase production. Nevertheless, agitation rate and aeration rate could affect dissolved oxygen (DO) concentration that in turn affected V. lecanii growth and chitinase production.

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.