INTRODUCTION:

Enzymes are among the most important sequels obtained for human needs through plant, animal and microbial sources. Many of the enzymes have the lot of applications in industry, including food processing, brewery and baking^1-4. Chitin forms the exoskeleton of many invertebrates and is a major component of the cell wall of fungi. There are enormous chitin result in environmental problem all round the world. Different microorganism are capable of producing chitinase that degrades chitin directly to low-molecular-weight products. Almost all of the reported chitinase-producing strains will use chitin or colloidal chitin as a carbon source. Chitinases –a group of enzymes catalyse the hydrolysis of chitin to its monomer N-acetyl-D-Glucosamine. Chitinases may find important industrial applications to the treatment of chitin, especially derived from sea-food-processing units^5-7.

Received on 13.02.2015 Modified on 28.02.2015

Research J. Pharm. and Tech. 8(3): Mar., 2015; Page 280-284

DOI: 10.5958/0974-360X.2015.00047.5

Chitinases are used extensively in biological research for the generation of fungal protoplasts due to its ability to degrade fungal cell wall. The hydrolytic property of chitinases makes it an attractive alternative as an environmentally safe bio control agent. There are a variety of instances where chitinase producing organisms are used to inhibit the growth of phytopathogens. Chitinase is known to process diverse characteristics worthy of detailed enzymatic studies related to their biological role and structural elucidation^8-10.A wide range of microorganisms are known to produce chitinase which have a lot of industrial and environmental applications.Among the microbes,fungi play avital role in chitinase production because of the ease of cultivation on a wide range of media and high yield. Metarhizium anisopliae (M.) Sorokin is an important fungal biopesticidal agent against economic important pests. The fungal organism produces diverse metabolites during the pathogenesis of the insects.^[8].These entomopathogenic fungi produces extracellular chitinases only during host penetration and degrade chitin. It has the ability to grow in wide range of pH⁵.

Solid state fermentation (SSF) has grown importance recently in the production of microbial enzymes obtaining economic advantages over conventional submerged culture (SF). Several groups of microorganisms have been used in SSF, especially the filamentous fungi that have been exploited for their abilities to produce a wide range of extracellular enzymes and to grow on solid complex substrates. Several enzymes including amylases, cellulases, pectinases, proteases and glucoamylasas have been produced in solid state fermentation^18-,21.

In the present study, production and optimization of chitinase through submerged and solid state fermentation by Metarhizium anisopliae (M.) Sorokin was studied.

MATERIALS AND METHODS:

Fungal strain

Chitinase producing Metarhizium anisopliae was used in the present study was isolated from dead larval instars of Spodopteralitura by the modified method of Sahayaraj and Karthick Raja Namasivayam¹⁵.

The identification of isolated fungi was performed by macroscopic (colony morphology) and microscopic (conidia and conidiophores using a compound microscope)⁸.

Inocula preparation

Fungal inocula was prepared from Potato Dextrose Agar slant culture of M. anisopliae by scrapping the slant with Tween 20 using glass rod. Slurry thus obtained was filtered through crude filter paper, collected filtrate suspension was used as source of inocula

Chitinase production through submerged fermentation

Production and optimization

Production of chitinase was carried out by the modified method²⁰.Five millilitre of fungal spore suspension was inoculated into one litre of production media consist of colloidal chitin at different concentration, peptone 1 g/l, MgSO₄˙7H₂O 1 g/l, (NH₄)₂SO₄2 g/l,K₂HPO₄1g/l, NaCl 1 g/l,. Inoculated flasks were incubated under shaking condition at 300 RPM for 48 hours. After the incubation period, the media was filtered through cheese cloth to remove mycelia, filtered broth was centrifuged at 10,000 rpm and collected filtrate was used for enzyme assay.

Optimization

Optimization of nutrient factors of chitinase production media was studied by changing the compositions of chitin^11-14.

Chitinase production through Solid state fermentation

Estimation of Moisture content

The sample was weighed along with moisture and recorded as “wet weight of sample” Followed by the drying at 90^oC using hot air oven until the sample is dried. The sample was allowed to cool. The sample was weighed again and recorded as the “dry weight of sample”

The moisture content of the sample is calculated using the following equation:

Where: %W = Percentage of moisture in the sample, A = Weight of wet sample (grams), and B = Weight of dry sample (grams)

Production and optimization

The cultures under Solid State Fermentation were obtained by inoculating of 1 ml of spore suspension (10⁵ spores/mL) on 4 g of crushed fish waste as carbon source moistened with distilled water (Different concentration), yeast extract solution (1% v/v), salt solution. After incubation, the cultures were added with 50 ml of autoclaved distilled water^15-17.

The free cell filtrate, identified as extracellular crude extract, was dialyzed against distilled water at 4°C over-night and used for chitinase activity determination⁸.

Analytical methods

Enzyme assay

Monreal and Resse method¹⁸was used to determine chitinase enzyme quantification. The reaction mixture, which consisted of 1.5 ml enzyme solution, 2.5 ml of 1 % colloidal chitin in 0.075 M, pH 7 sodium phosphate buffer, was incubated at 50, 100 RPM for 10 min in a water bath. After the enzymatic reaction, 1 ml of DNS reagent (3, 5-dinitrosalicylic acid 10 g/l, phenol 2 mg/l, Na₂SO₃0.5 g/l, and NaOH 10 g/l) was added, followed by heating of the mixture at 100 for 5 min. Then the solution was cooled for 5 min, diluted with l ml of distilled water, and centrifuged at 3000 g for 15 min to remove the precipitate. The absorbance was measured in a spectrophotometers at 540 nm. One unit (U) of enzyme activity was defined as the amount of enzyme required to produce 0.5 μmol of N-acetyl-D-glucosamine for 1 h.

RESULTS AND DISCUSSION:

Identification:

Microbiological analysis (macroscopic and microscopic analysis) was performed to characterize the screened strain.

Macroscopic observation

Colonies grow rather gradually on PDA and pigmentation ranging from light to dark yellow diffusing into the medium. Conidia color may differ in colony size and condition. After 7 days of incubation, the culture produces a white mycelial margin with clumps of more or less verticillate branching conidiophores.

Microscopic observation

Conidial chains were round, columnar phialides in a dense parallel arrangement, and conidia were cylindrical to oval.

The above Macroscopic and Microscopic observation suggest the isolated organism was Metarhizium anisopliae.

Cynthia Barbosa Rustiguel et al 2012 ⁸used Metarhizium anisopliae was used as the culture organism for the production of chitinase. Many report suggest the usage of fungi and bacteria for the production.

Effect of chitin:

Different concentration of chitin was inoculated in the minimal media and the enzyme activity was calculated in different time intervals (12, 24, 36, 48, 60 and 72) (Table-1 & Figure -1). In the concentration of 5% (w/v) the enzyme activity was comparatively higher than the other concentrations. According to Mandana Zarei et al., 2010⁶expressed that the concentration of chitin was about 5% in the medium where Serratia marcescens was cultivated for the production of chitinase.

Table 1: Effect of Chitin on Enzyme Activity

S. No	Concentration of chitin (%)	Enzyme activity (unit per ml)
S. No	Concentration of chitin (%)	12	24	36	48	60	72
1	0.1	5.6	11.2	26.2	30.1	42.4	51.3
2	0.25	6	14.5	31.2	40.1	50.1	60.1
3	0.5	10	19	40.4	53.4	61.4	69.2
4	0.75	24	25	44.2	59.1	64.1	71.4
5	1	30.1	39.4	51.4	62.4	74.1	78.1
6	1.5	36.1	41.2	54.1	66.1	79.2	81.4
7	2	39.3	45.2	56.2	69.3	73.1	78.7
8	2.5	43.2	52.3	60.4	72.2	76.2	81.4
9	3	44.2	53.1	61.7	73	78.4	82.2
10	3.5	48.4	56.1	63.2	76.4	81.4	89.4
11	4	50.1	59.2	66.2	79.4	84.2	91.4
12	4.5	60.2	71.4	82.3	90.1	101	97.4
13	5	41.4	47.4	51.2	60.2	69.1	56.1
14	5.5	24.7	17.9	4	1.75	0.7	0.4
15	6	9.4	11.3	0.9	0.4	0.2	0.07

Figure 1.Theenzyme activity in (IU/ml) in different concentration of chitin with respect to time in hours

Table 2: Effect of Solid State Fermentation using Fish Cell Waste

S.NO	Moisture content%	Enzyme activity (Unit per ml)
S.NO	Moisture content%	12	24	36	48	60	72
1	0.0	9.1	24.5	32.4	41.1	49.0	53.1
2	1.0	9.5	21.0	30.5	40.4	47.4	49.2
3	2.5	10.6	11.2	32.2	38.1	47.4	54.3
4	5.0	9.2	19.3	28.4	38.3	43.4	46.4
5	7.5	8.7	13.4	24.4	32.4	39.1	41.1
6	10.0	3.2	11.2	14.1	16.1	9.2	3.4
7	12.5	1.0	4.2	6.2	8.3	4.1	1.7
8	15.0	0.05	1.01	2.02	2.53	0.09	0.07
9	17.5	0.05	0.09	0.09	1.90	0.08	0.07
10	20.0	0.05	0.07	0.09	1.02	0.07	0.06
11	22.5	0.03	0.07	0.09	1.00	0.03	0.03
12	25.0	0.03	0.05	0.06	0.05	0.03	0.03

Figure 2.The enzyme activity in (IU/ml) in different moisture content with respect to time in hours

The effect of solid state fermentation using fish cell waste:

The fish waste was added with suitable moistening agents to meet the water requirement and supplementary nutrients to the growing cultures. The Fig. 2 and Table-2 shows that the maximum enzyme production of ranges from 10.6 – 54.3 IU/ml with respect to the time was obtained using salt solution and enzyme secreation was subjective by the additional proportionate amount of mineral ions.

The cost-effective mechanism was needed for the production of enzyme and SSF is a suitable technology for economical production of chitinase using chitin as substrate.

CONCLUSION:

According to the present study, analyzed microorganisms exhibited the highest chitinolytic activity in the presence of 5% chitin. The moisture content for the solid state fermentation was determine the productivity of the enzyme.

ACKNOWLEDGMENT:

The authors would like to thank the Management and Faculty of Bio and Chemical Engineering, Sathyabama University, Chennai for their support.

REFERENCES:

1. Abdel-Fattah Y. Optimization of thermo stable lipase production from a thermophilic Geobacillus sp. using Box–Behnken experimental design. Biotechnology Letter. 2002;24: 1217–1222.

2. Abel H, Marie D, Nathalie R, Danielle D, Louis S, Louis C. Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated From palm fruit. Enzyme Microbial Technology. 2000;26:421–430.

3. Canganella F, Andrade C, Antranikian G. Characterization of amylolytic and pullulytic enzymes from thermophilic archaea and from a new Ferividobacterium species. Applied Microbiology Biotechnology. 1994;42:239–245.

4. Bertoldo C, Duffner F, Jorgensen P, Antrakianin G. Pullulanase type I from Ferividobacteriumpennavorans VEN5: cloning, sequencing, expression of the gene and biochemical characterization of the recombinant enzyme. Applied Microbiology Biotechnology.2012; 65:2084–2091.

5. Keiko Shirai Matsumoto, Advances in Agricultural and Food Biotechnology. Editors: Ramón Gerardo Guevara-González and Irineo Torres-Pacheco; 2006.

6. Mandana Zarei, Saeed Aminzadeh, Hossein Zolgharnein, Alireza Safahieh, Ahmad Ghoroghi, Abbasali Motallebi, Morteza Daliri, Abbas Sahebghadam Lotfi,, Serratia marcescens B4A chitinase product optimization using Taguchi approach, Iranian journal of Biotechnology. 2010;8:424-484.

7. Bergquist P, Love D, Croft J, Streiff M, Daniel R, Morgan H. Cloning of genes involved in cellulose hydrolysis. In: Herbert., Sharp, R. (Eds.), Molecular Biology and Biotechnology of Extremophiles. Chapman and Hall, NY; 1992.

8. Cynthia Barbosa Rustiguel, João Atílio Jorge, Luis Henrique Souza Guimarães, Optimization of the Chitinase Production by Different Metarhiziumanisopliae Strains under Solid-State Fermentation with Silkworm Chrysalis as Substrate Using CCRD, Advances in Microbiology, 2012;2:268-276.

9. Carlsen M, Nielsen J, Jorgensen SB, Zangirolami TC. Growth and enzyme production during continuous cultures of a high amylase-producing variant of Aspergillus oryzae. Braz. J. Chem. Eng. 2002;19: 55–67.

10. Patel B, Gohel V, Raol B. Statistical optimization of medium components for chitinase production by Paenibacillussabina strain JD2. Annuals of Microbiology. 2007;57:589–597.

11. Takayanagi T, Ajisaka K, Takiguchi Y, Shimahara K, Isolation and characterization of thermostable chitinases from Bacillus lichiniformis X_7u. Biochem. Biophys. Acta. 1991; 1078:404–410.

12. Chen C, Adolphson R, Dean F, Eriksson K, Adamas M, Westpheling J. Release of lignin from kraft pulp by a hyperthermophilicxylanase from Thermotogamaritema. Enzyme Microb. Technol.1997;20:39–45.

13. De Mot R, Andries K, Verachtert H. Comparative study of starch degradation and amylase production by Ascomycetes yeast species. Syst. Appl. Microbiol. 1984;5:106–118.

14. De Rosa M, Morana A, Riccio A, Gambacorta A, Trincone A, Incani O. Lipids of the archaea: a new tool for bioelectronics. Biosens. Bioelectr. 1994;9:669–675.

15. Sahayaraj K, Karthick RNS, Mass production of entomopathogenic fungi using agricultural products and by products. African Journal of Biotechnology. 2008;7: 1907-10.

16. Tsujibo H, Endo H, Miyamoto K, Inamori Y. Expression in Escherichia coli of a gene encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520. Biosci. Biotechnol. Biochem. 1995;59: 145–146.

17. Aloise PA, Lumme M, Aynes CA. N-Acetyl-d-Glucosamine Production from Chitin-Waste Using Chitinases from Serratia marcescens, In: Muzzarelli RAA, editor. Chitin enzymology, vol. 2.Italy: Ncona, 1996;581–594.

18. Monreal J, Reese ET. The Chitinase of Serratia marcescens, Canadian Journal of Microbiology,1969;15:689–696.

19. Ulhoa CJ, Peberdy JF. Regulation of chitinase synthesis in Serratia marcescens. Journal of General Microbiology, 1991;14:2163–9.

20. Suresh PV, Chandrasekaran M. Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation. World Journal of Microbiology and Biotechnology. 1998;14:655–660

21. Rattanakit N, Plikomol A, Yano S, Wakayama M, Tachiki T. Utilization of shrimp shellfish waste as a substrate for solid-state cultivation of Aspergillus sp.S1-13: Evaluation of a culture based on chitinase formation which is necessary for chitin-assimilation. Journal of Biosciences and Bioengineering. 2002; 93:550–556.