2. LITERATURE REVIEW
2.4 Cutinase sources and production
2.4.1 Cutinase production in wild type organisms
There are many cutinase producing fungi and bacteria isolated and screened till date (Table 2.2). However, only few of them are studied further in context of production, characterization and applications. As per production in wild strain is concerned, microbial cutinases are generally produced by submerged culture (Fett et al., 1992; Fett et al., 1999; Fett et al., 2000;
Ferreira et al., 2004; Macedo and Pio, 2005) albeit, solid state fermentation has also been reported (Macedo and Fraga, 2007). The production of wild type cutinase is influenced by several factors which include the type and concentration of carbon and nitrogen sources, culture pH and temperature, and dissolved oxygen concentration (Du et al., 2007). It was also reported that cutin can be used as a carbon source and/or an inducer for achieving high level
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of cutinase yield (Fett et al., 1992; Fett et al., 1999; Fett et al., 2000; Lin and Kolattukudy, 1978).
Fig. 2.2 Phylogenetic relationship of the cutinase producing organisms. The phylogenetic tree was constructed using UPGMA method on online construction tool from ExPASy online server (http://www.expasy.org/phylogeny_evolution).
Lin and Kolattukudy (1978) reported that cutin hydrolysate induces the secretion of an extracellular cutinase by F. solani f. sp. pisi. They observed that the rate of cutinase TH-1214_KHEGDE
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production depends on the amount of cutin hydrolysate added to the medium containing glucose and saturation was achieved at 80 μg mL-1 of cutin. Glucose was found to be a repressor for the production of cutinase. The production of cutinase was induced by cutin hydrolysate and exogenous labeled phenylalanine was found to be incorporated into cutinase.
The study also shown that the induction of cutinase by cutin hydrolysate was not inhibited by actinomycin D and stimulated (=100%) by cordycepin. Lin and Kolattukudy (1978) reported that extracellular cutinase production induced by cutin hydrolysate was a cycloheximide- sensitive process. Experiments with derivatives and analogues of ω-hydroxy C16 acid indicated that the free hydroxyl group at the ω-position was the most important factor for induction of cutinase activity. n-Aliphatic primary alcohols with 14 or more carbon atoms have displayed induction of cutinase, among which n-C16 was found to be the most effective inducer (Lin and Kolattukudy, 1978). These results are in the favor of the fact that cutin monomers act as a chemical signal, which induces extracellular cutinase production. Studies have been conducted on the production of cutinase with other substrates (viz., olive oil, soy oil, sunflower oil and palm oil) besides cutin (Pio and Macedo, 2007). A high level of enzymatic activity (from 11 to 22.68 µMol min-1 mL-1) was observed by Pio and Macedo (2007) after 48 h of fermentation from F. oxysporium using flaxseed oil as a carbon source.
This flaxseed oil is a low cost carbon source as compared to cutin. Rispoli and Shah (2007) observed that the K2HPO4 had a positive influence on the production of cutinase by C.
lindemuthianum and MgSO4 had a minimal effect on the production of cutinase. Due to change in the chemical activity of a particular chemical species like substrate concentration in the neighborhood of a microbial cell may cause any given physiological variable of the cell like specific growth rate to increase, to decrease, to fluctuate, or to show no apparent change.
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When the concentration of substrate is continuously varied from low to high values in an aqueous media of microbes, several patterns of dependence of physiological variables may be observed. At very low concentrations, the effect was negligible on gross measures of metabolic activity, such as specific growth rate, respiration rate, rate of protein synthesis, etc.
If the nutrient is essential for growth or the production of enzyme of interest, the initial value of the physiological parameter at very low concentrations may be zero or even negative if it represents a rate function. By increasing concentration of that nutrient, the physiological parameter (growth or enzyme production) will increase due to stimulation of the metabolism of the micro-organisms. Finally, a concentration of the nutrient is reached at which further increase in concentrations do not increase the physiological parameter. This occurs at this stage due to either there is limited supply of some other environmental factor or cells themselves have reached their own limit for the present culture conditions. Further increasing the concentration of the chosen nutrient or substrate will eventually cause decrease (the nutrient or substrate inhibition) in physiological parameter. This type of microbial behavior has vital role in the metabolic activities of microorganisms in industrial fermentation, biological waste treatment processes, infected plants or animals, and in other parts of the biosphere. In this case, reliable and tractable mathematical models relating the physiological state of microorganism to the state of its surroundings facilitates the analysis of such processes. So, there are several models reported to analyze the relation between specific growth rate and substrate concentrations. Among these, some models have focused either on the stimulatory range of concentrations or on inhibitory concentrations or analyze both stimulatory and inhibitory domains of substrate action.
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