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Biological Segment: 1(1) BS/1510, 2010
Optimization of chitinase production from Serratia marcescens - A classical approach.
The fermentative physicochemical parameters such as agitation speed, temperature and pH were optimized by classical approach method for the production of chitinase enzyme and growth of bacterial strain of Serratia marcescens. The parameters set up for reaching maximum response for production of chitinase and cell growth were obtained at 250 rpm agitation speed, 30 C temperature and the pH of 9. The chitinase production increased three fold using optimized culture compositions and culture conditions. The maximum production of chitinase obtained as 44.01 units/mL.

Kannan Natarajan* and V. Ramachandra murty
Department of Biotechnology, Manipal Institute of technology, Manipal-576104, Karnataka, India.

INTRODUCTION

Chitin, a h-1,4-linked homopolymer of N-acetylglucosamine is the second most abundant natural renewable polysaccharide and mostly is present in fungi, algae, insects and marine invertrebrates. Chitinase enzyme (EC 3.2.1.14) has capable of catalyzing the hydrolysis of chitin to its monomer N-acetyl-D-glucosamine from variety of sources, such as bacteria, fungi, yeast and plants (Patil et al., 2000). Chitinase enzyme is most promptly used in the biological research as a controlling agent for the generation of fungal protoplasts due to its degrading nature of fungal cell wall. Chitinase is a hydrolytic enzyme; this property makes them as an alternative environmental biological control agent (Ordentlich et al., 1988). And also chitinase find widespread applications in the field of agriculture, medicine, biochemical processing engineering, waste management, pesticide control and cell wall degrading enzyme.

Several microorganisms, including bacteria such as Bacillus lichiniformis, Bacillus pabuli, Bacillus thuringiensis, Serratia marcescens, Nocardia orientails ,Vibrio alginolyticus (Mabuchi et al., 2000; Someya et al., 2001; Wen et al., 2002 and Huang et al., 2005) and many species of fungi such as: Myrothecium verucaria , Stachybotrys elengans Streptomyces cinereorube, Streptomyces lydicuis, Trichoderma harzianum, Trichoderma viride, Verticillium lecanii (Mathivanan et al., 1998 and Viterbo et al., 2001) have a chitinase producing ability. Chitinase activity in plant and human serum has also been described recently (Mathivanan et al., 1998). Novel bacteria strains are described which are created by the introduction of DNA encoding for the production of chitinase.

In this present study a well known chitinolytic bacterial strain of Serratia marcescens has chosen to the production of chitinase, to ensure the maximum production of chitinase. The different medium culture was taken initially to find out the initial range of production of chitinase. And significance of the medium composition and other physicochemical parameters were optimized by the classical approach to screen the most prompt factors affecting the enzyme production by the chitinolytic bacterial strain.

MATERIALS AND METHODS

Microorganism maintenance and inoculum preparation

The chitinolytic bacterium was obtained from our laboratory, in Manipal Institute of technology, Manipal University, India. The organism was cultivated on LB medium consisting of (g/l) Agar 15; tryptone 10; sodium chloride 10 and yeast extract 5.0. The pH of the medium was adjusted to 7.2 using 1 M NaOH or 1 N HCl and sterilized by autoclaving at 121C for 15 min. The production medium was inoculated with 5% (v/v) of seed culture in the mid exponential phase at 30 h (Fig. 1). The flasks were incubated in an orbital shaker at 120 rpm and 30C for the fermentation period of 60 h. Aliquot of sample from the fermentation broth was withdrawn at 6 h interval without much change in the culture volume to maintain constant oxygen transfer. The cells were separated from the medium by centrifugation at 10,000 rpm for 15 minutes. The clarified supernatant was used for the analysis of chitinase activity.

The bacterial cell growth was determined by measuring the optical density at wavelength of 600 nm (Double beam UV Visible spectrophotometer).The biomass concentration was determined with a calibration curve made from the relationship between optical density at 600 nm and dry cell weight (Fig 1).

Chitinase activity assay

Extracellular chitinolytic activity in the broth may calculate using chitin as the substrate. One milliliter of the supernatant broth was mixed with 1ml of 1% chitin in 0.05M phosphate buffer pH 7.0. The assay mixture was incubated at 40C and the end products of the reaction were analyzed using the DNS method. One unit of chitinase activity is defined as the amount of enzyme required to release 1 micro mol of GlcNAc in 1 min under the above mentioned conditions.

Effect of different media compositions

Three different media namely M1, M2 and M3 amended with 1% colloidal chitin were used to determine the growth of Serratia marcescens and chitinase production. 5% (v/v) of seed culture in the mid exponential phase at 30 h was inoculated with 100 ml of each medium and incubated at 100 rpm in a rotary shaker at room temperature. After two days of incubation, the cultures were harvested, centrifuged at 10000 rpm for 15 min and the supernatant was used for chitinase assay.

Optimization of physicochemical parameters

The optimized medium culture involves further optimization of fermentative physicochemical parameters, such pH, temperature and agitation speed.

RESULTS AND DISCUSSION

A amount of literature gives the detailed information about the microbial production of chitinase and inspection about the effect of important physicochemical and environmental factors, exclusively culture condition, culture composition, substrate concentration, pH, temperature, agitation speed, incubation period, inoculation volume, aeration rate, etc.,had significant effect on production of chitinase and biomass.

In biochemical investigation of enzyme production mainly depends on the culture compositions, it acts the important role for the growth as well as metabolites production by microorganisms. The kinetic profile of cell mass and chitinase production by S. marcescens grown in different media was illustrated in Fig. 2. The cell growth and chitinase production was high in the order M3>M2>M1 as shown in Fig. 2. The chitinase production by S. marcescens was found to be growth associated in all the fermentation runs conducted, as the chitinase production increased with the cell mass. Maximum cell mass was obtained in the media M3 which contains chitin as the major carbon source. Among the three medium compositions, M3 medium was found to be a best chitinase producing culture medium than other media M1 and M2 compositions. Medium M3 was chosen to further optimizing the physicochemical and environmental parameters. The composition of M3 medium contains the chitin as a sole carbon source combined with other nitrogen and mineral sources.The concentration of chitin is plays a major role, important factor to induce the chitinase production in microbial production. The different concentration of chitin enhances the production of chitinase, at 0.5% concentrations; chitin significantly enhanced the chitinase activity. Mathivanan et al., 1998 reported that the same concentration of chitin has the ability to produce chitinase in Fusarium chlamydosporum. The addition of chitin concentration above 0.5% also induced the maximum chitinase production in Bacillus sp. NCTV2 (Wen et al., 2002), Alternaria alternate (Sharaf, 2005) and Trichoderma harzianum (Sandhyal et al., 2005).

Effect of various carbon sources on chitinase production

For the growth of microorganism carbon source is the very essential with other nutrient sources, etc. The M3 medium was taken to further studies to evaluate the effect of carbon and nitrogen sources. Various carbon sources included chitin, arabinose, cellulose, glucose, galactose, maltose, fructose, sucrose, lactose and starch were examined at the concentration of 5 (g/L). The samples were taken at different time interval for the analysis of biomass and chitinase activity. The above mentioned carbon sources produces chitinase in very low, except chitin. Then the experiments were conducted in the presence and absence of chitin. The experimental result implies that presents of chitin activity increases the production of chitinase, but the absence of chitin doesnot improve the final reaction production. The exception of carbon sources like, cellulose and arabinose were shown the increased activity than the other carbon sources used.These experimental results were conformity with Vaidya, et al., 2001 reported that the production of chitinase with different carbon sources, no detectable activity in final production with some exceptional carbon sources. Gupta et al., 1995 reported that in Streptomyces viridificans, arabinose with chitin doubled the production of chitinase but arabinose alone failed to induce the chitinase. The carbon is very essential for the growth of the organism. The increased order of chitinase production by chitinolytic bacterial strain Serratia marcescens using different carbon sources was chitin>cellulose>arabinose>glucose>starch>sucrose>lactose>maltose (Fig 3). From the results of different carbon sources, found that chitin alone act as inducer and best carbon source to improve the chitinase production. Maximum production of chitinase obtained in M. verrucaria was observed with chitin as a carbon source and no detectable activity was seen in the culture grown with lactose, maltose, sucrose, chitosan, starch and cellulose (Vyas et al., 1991).

Effect of various nitrogen sources on chitinase production

Nitrogen sources are very important for the microbial growth and to maximize the final reaction product next to carbon sources. For nitrogen sources the medium was supplemented with peptone, yeast extract, potassium dihydrogen phosphate, ammonium sulphate and ammonium chloride in equivalent concentrations to the production medium.

The order of chitinase production using various nitrogen sources follows peptone> yeast extract> ammonium sulphate> ammonium chloride> potassium dihydrogen phosphate (Fig 4). Among the various nitrogen sources involved in reaction, the experimental results were shown pepote and yeast extract has the significant increasing order than other sources added to the medium. The optimal value of nitrogen source of the media for chitinase production was 1 g/L. Addition of yeast extract to the medium has been reported to enhance enzyme production in Serratia marcescens (Monreal and Reese, 1969) and Aspergillus carneus (Sherief et al., 1991). The ammonium sulphate and ammonium chloride also gave the response next to the above mentioned nitrogen sources. Vyas and Deshpande, 1991 reported that in Myrothecium verrucaria, 0.14% ammonium sulphate and 0.010.05% urea increased the chitinase production up to fourfold, while sodium nitrate favored chitinase production in Stachybotrys elegans (Tweddell et al., 1994).

The analyzed various carbon and nitrogen sources gives the neat information about to optimize the culture medium compositions, for further studies of optimizing the culture environment to maximize the chitinase production. The optimized medium composition obtained with the chitinase activity of 24.2 units/mL.

Optimization of physicochemical parameters by classical approach

Effect of pH on chitinase production

Normally the action of initial pH of fermentation medium influences the final yield of chitinase. Any change in pH affects the protein structure and a decline in enzyme in activation or its instability. The effect of different levels of pH was studied to evaluate the final production of chitinase from the nature of product profile. While in the fermentation aeration rate, agitation rate and temperature were kept in the constant levels, the pH levels varied from 3 to 10.The culture broth was grown to exponential growth phases and inoculated on a separate culture medium containing optimized culture compositions. Fig 5 shows the fermentative production of chitinase activity with different pH levels. The cell growth and chitinase activity were measured in different pH levels, the maximum chitinase activity of 43.61 units/mL occurred at pH 8. Monreal and Reese, 1969 and Bergeys Manual of systemic bacteriology gives the idea in general S. marcescens had a tendency to be more acidic than pH 7.5.The pH for optimal production of chitinase using Serratia marcescens was close to pH 8 reported by Moneal and Reese, 1969. This experimental results leads to increased stability at high pH with following order 8>7>9>6>5>10>4>3 (Fig 5). The process with pH might force it to change its metabolic pathway and cause a decrease in the chitinase activity during the cultivation.

Effect of temperature on chitinase production

The rate of the reaction increases as the temperature is raised. The enzyme reaction activity gets increase to two fold higher with increase of temperature. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. As shown in Fig 6, the chitinase enzyme activity increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most enzymes rapidly become denatured at temperatures above 40C, widely the enzyme determinations are carried out somewhat below that temperature.

The optimized medium was selected to study the effect of temperature on chitinase production by changing the temperature and kept all other fermentation conditions were constant. The optimum growth temperature for chitinase production by S. marcescens was found to be in the range of 30C (Fig. 6). The activity achieved at 20C was lower than the optimum temperature. The enzyme activity decreased as the temperature increased above 30C and even at 35C a 70% decrease in chitinolytic activity was observed.

Effect of agitation speed on chitinase activity

The optimized culture compositions were taken for the further reaction carried out to evaluate the optimal shaking speed on chitinase production by subjective the different agitation speed, with the optimized culture pH and temperature. The optimum agitation speed for the maximum chitinase activity was found to be 250 rpm (Fig. 7) upon varying the agitation speed (0- 350 rpm). Agitator speed of 250 rpm was found to be most suitable for cell growth as well as for chitinase production. Chitinase yield decreased rapidly at higher agitator speed, while decrease in cell yield at higher agitator speed was not rapid. Probably, mass transfer limitation was predominant in the fermentation process at lower agitator speed. Higher agitator speed appears to reduce chitinase production. The increase in agitation rate beyond 250 rpm resulted in a drastic fall in specific enzyme activity. The agitation speed below 150 rpm resulted in inadequate mixing of the broth towards the later stages of growth. The reaction against agitation speed indicates that oxygen adjustment is required for the maximum production of chitinase enzyme (Mathivanan et al., 1998).

CONCLUSION

From these studies it has been found that the composition of medium plays a crucial role in the production of fermented products and optimized levels of carbon and nitrogen sources from the large number of experimental runs were determined. The chitinase fermentation process utilizing chitin as the sole carbon source was investigated in a submerged fermentation process.

The best physicochemical parameter conditions for maximum production of chitinase

pH 9,

Temperature 30C,

Agitation speed 250 rpm with the combination of optimized medium

compositions contains (g/l): chitin, 5.0; peptone, 1.0; (NH4)2SO4, 1.0; MgSO47H2O, 0.3; KH2PO4, 1.36. Fungal species are most potent producer of this enzyme but the production time is larger and the initial cost also very high, but in case of bacterial strain the time consuming for production if comparatively very low than the fungal species also the range of initial cost also differs. The cultivation time required for bacterial culture is low and easily can grow and produce huge amount of enzyme.Further works on optimization of process parameters using statistical designs, scale up process using fermenter, purification of enzyme by suitable methods like ATPs, RMs, characterization and regulation of the enzyme synthesis is in progress.

Acknowledgement

The authors are thankful to Department of Biotechnology, Manipal Institute of Technology, Manipal University, Manipal, India for continuous encouragement and problem solving assistance.

REFERENCES

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Huang, C.J., Wang, T. K., Chung, S. C., and C. Y. Chen. 2005. Identification of an antifungal chitinase from a potential biocontrol agent, Bacillus cereus. Journal of Biochemistry and Molecular Biological Science 38: 82-88.

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Sharaf, E. F. 2005. A potent chitinolytic activity of Alternaria alternate isolated from Egyptian black sand. Polish Journal of Microbiology. 54: 145-51.

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  Graphs / Images / Plates / Tables
<b>Fig. 1:</b> Growth profile of <i>Serratia marcescens</i> in the seed LB medium (g l<sup>-1</sup>): agar 15.0; yeast extract, 5.0; tryptone, 10.0 and NaCl<sub>2</sub> 10.0. Initial pH 7.4 at 25C, temperature.
Fig. 1: Growth profile of Serratia marcescens in the seed LB medium (g l-1): agar 15.0; yeast extract, 5.0; tryptone, 10.0 and NaCl2 10.0. Initial pH 7.4 at 25C, temperature.
 
<b>Fig. 2:</b> Effect of different medium composition on chitinase production
Fig. 2: Effect of different medium composition on chitinase production
 
<b>Fig. 3:</b> Effect of various carbon sources on chitinase production
Fig. 3: Effect of various carbon sources on chitinase production
 
<b>Fig. 4:</b> Effect of nitrogen source concentration on chitinase production
Fig. 4: Effect of nitrogen source concentration on chitinase production
 
<b>Fig. 5:</b> Effect of pH on chitinolytic enzyme production by <i>S.marcescens</i>. Each point represents the mean of three independent experiments.
Fig. 5: Effect of pH on chitinolytic enzyme production by S.marcescens. Each point represents the mean of three independent experiments.
 
<b>Fig. 6:</b> Effect of temperature on chitinolytic enzyme production by <i>S.marcescens</i>. Each point represents the mean of three independent experiments.
Fig. 6: Effect of temperature on chitinolytic enzyme production by S.marcescens. Each point represents the mean of three independent experiments.
 
<b>Fig. 7:</b> Effect of agitation speed on chitinolytic enzyme production by <i>S.marcescens</i>. Each point represents the mean of three independent experiments.
Fig. 7: Effect of agitation speed on chitinolytic enzyme production by S.marcescens. Each point represents the mean of three independent experiments.
 
<b>Table 1:</b> Different medium compositions involved for production of chitinase.
Table 1: Different medium compositions involved for production of chitinase.