Commercially Important Microorganisms from Insects

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Author: N. Sangeetha Kamatshi*, C. K. Venil, P. Velmurugan and P. Lakshmanaperumalsamy

Commercially Important Microorganisms from Insects N. Sangeetha Kamatshi*, C. K. Venil, P. Velmurugan and P. Lakshmanaperumalsamy Department of Environmental Sciences, Bharathiar University, Coimbatore-641046, India.

* Corresponding Author *N. Sangeetha Kamatshi, Research Scholar, Department of Environmental Sciences, Bharathiar University, Coimbatore - 641 046. INDIA. Phone : 098426 99688 E.mail : sangeetha05@rediffmail.com

Commercially Important Microorganisms from Insects N. Sangeetha Kamatshi*, C. K. Venil, P. Velmurugan and P. Lakshmanaperumalsamy Department of Environmental Sciences, Bharathiar University, Coimbatore-641046, India.

Abstract Microorganisms produce industrially and commercially important compounds in the form of secondary metabolites. Most microorganisms surrounding us are still unknown and is likely to have significant and unpredictable economical consequences and effects on health. Insects are among the most successful animals of the world in terms of species richness as well as abundance. Their biomass exceeds that of mammals by far. Microbes within the insect gut are known to be important in break down, mineralization and cycling of a range of organic compounds. In search of new genera of microbes capable of producing some novel compounds, insect samples were collected and bacterial populations were screened. Micro flora inhabiting the gut of insects were found to be elaborating amylase, protease, phosphatase, lipase and L - asparaginase. Key words: Insects, amylase, protease, phosphatase, lipase, L - asparaginase.

1. Introduction Microbial enzymes have been used in various industries for many centuries. Recently, with the advent of biotechnology, there has been a growing interest in and demand for enzymes with novel properties. Currently, microbes from terrestrial sources are employed for industrial production of enzymes of which microbes from insect sources have been least studied. The interplay between insects and microorganisms inhabiting the gut was recognized as early as 1929. Gut microbes can adapt rapidly to changes in the diet of the insect by changing the population profiles and induction of requisite enzymes (Kaufman and Klug, 1991; Santo-Domingo et al., 1998). There is a paucity of information on the normal gut microflora of insects (Lysenko, 0. 1985). The vast number of insect species (estimated to be about one million [Lysenko, 0. 1985]), is serving as a good reservoir of microbes. Potent microbes associated with the gut of insects still remain an untapped resource in the microbial world and their potential gives ample scope for scientific study . Microbial enzymes are relatively more stable than corresponding enzymes derived from plants or animals. Furthermore, microbial enzymes provide a greater diversity of catalytic activities and be produced more economically using submerged fermentation. The majority of enzymes currently used in industry are of microbial origin. It has been estimated that only about 2% of the world's microorganisms have been tested as enzyme sources (Wiseman, 1985). It is for this reason that the isolation of new enzymatic activities from microorganisms is still of great interest. The role of enzymes in many process has been known for a long time. Their existence was associated with the history of ancient Greece where they were using enzymes from microorganisms in baking, brewing, alcohol production, cheese making etc., with better knowledge and purification of enzymes. The number of applications has increased drastically and with the availability of enzymes a number of new possibilities for industrial processes have emerged. Crude preparations from animal tissues were prepared which found technical applications in textile, leather, brewing and other industries. Once the favorable results of employing such enzyme preparations were established, a search began for better, less expensive and more readily available sources of such enzymes. It was found that certain microorganisms produce enzymes similar to other sources. This led to the development of processes for providing such microbial enzymes on commercial scale. Microbial enzymes such as protease, amylase, lipase, xylanse which are widely used in several industries (Wiseman, 1985) are mainly derived from terrestrial microorganisms. Increased awareness of the use of enzymes from microorganisms has made possible for the creation of new biologically based products. The search for better and more efficient microorganisms has been of concern. In the present study we have investigated the potential of gut microbiota. We have screened bacteria from the gut of 24 different insects which reveal that some bacteria are potent producers of amylase, protease, phosphatase, lipase and L - asparaginase.

2. Methods 2.1. Isolation and screening of microorganism The 24 different insect samples were collected and the bacterial populations were enumerated using pour plate technique. The bacterial cultures isolated were grouped to various genera based on their morphological and biochemical characteristics employing standard schemes on Bergey's Manual of Determinative Bacteriology and Systematic Bacteriology. The isolates were then screened for the production of amylase, protease, phosphatase, lipase and L-asparaginase. It was grown on nutrient agar slants for 24 h at 37oC, maintained at 0 - 2 oC and sub cultured every month. 2.2. Amylase activity Starch hydrolysis medium was prepared and were spot inoculated with the cultures to be tested and incubated at 30°C for 24hr. After incubation the plates were flooded with iodine solution and noted for the colour change. 2.3. Protease assay The isolated organisms were screened for protease production using casein and gelatin as substrates. 2.3.1. Caseinase Production Casein agar medium of Harrigan and McCane (1972) was used. Ten gram casein in 250 ml distilled water was sterilized separately and mixed with the basal medium before pouring in to the plates. After solidification, the isolates were made a single line of streak on the agar surface and the plates were incubated at 37oC for 24 - 48 hours. Positive reaction was indicated by the appearance of a clear halo zone around the colonies, which confirms the production of caseinase. 2.3.2. Gelatinase production Gelatin agar medium of Harrigan and McCane (1972). Gelatin agar plates were spot inoculated with the cultures to be tested and incubated at 30°C for 24hr. After incubation the plates were overlaid with 15% Mercuric chloride in concentrated hydrochloricacid. Clear transparent zones around the colony indicated gelatinase production.

2.4. Phosphatase assay Pikovskaya's Agar medium was prepared and were spot inoculated with the cultures to be tested and incubated at 30°C for 24hr. After solidification, the isolates were made a single line of streak on the agar surface and the plates were incubated at 37oC for 24 - 48 hours. Positive reaction was indicated by the appearance of a clear halo zone around the colonies. 2.5. Lipase assay Tween 80 agar medium was prepared and the isolates were made a single line of streak on the agar surface and the plates were incubated at 37oC for 48 - 72 hours. Positive reaction was indicated by the appearance of a clear halo zone around the colonies. 2.6. L-asparaginase assay L-asparaginase activity was determined by measuring the amount of ammonia formed by nesslerization. A 0.5 ml sample of cell suspension, 1.0 ml of 0.1 M sodium borate buffer (pH 8.5) and 0.5 ml of 0.04 M L-asparagine solution were mixed and incubated for 10 min at 37oC. The reaction was then stopped by the addition of 0.5 ml of 15% trichloroacetic acid. The precipitated protein was removed by centrifugation, and the liberated ammonia was determined by direct nesslerization. Suitable blanks of substrate and enzyme-containing sample were included in all assays. The yellow colour was read in a Beckman DB-G spectrophotometer at 500 nm. One international unit (IU) of L-asparaginase is that amount of enzyme which liberate 1 µmole of ammonia in 1 min at 37oC.

3. Results The bacterial populations were found to be high in cockroach (Eublaberus distanti) followed by earthworm (Eudrilus eugeniae) (Table 1). The predominant bacterial genera identified was Bacillus sp (44.92 %) followed by Pseudomonas sp (18.84 %), Staphylococcus sp (7.24 %), Lactobacillus (5.79 %), Proteus sp, Klebsiella sp (4.34 %), Serratia sp (2.89 %) and Micrococcus sp (1.24 %) (Figure 1). Among the bacteria isolated, Bacillus sp. (60%) was found to hydrolyze starch at significant levels followed by Pseudomonoas sp (15 %), Staphylococcus sp (11 %), Proteus sp (6 %), and Enterobacter (4 %) (Figure 2). Bacillus sp. (49 %) followed by Pseudomonas sp. (21 %), Staphylococcus sp (7 %), Lactobacillus sp (5%), Proteus sp and Enterobacteria sp (4 %), Micrococcus sp (2 %) and Serratia sp (1 %) were found to hydrolyze protease (Figure 3). Of all the bacterial strains isolated, Pseudomonas sp (67 %) and Lactobacillus sp (33 %) were found to produce phosphatase in enormous amounts (Figure 4). Bacillus sp, (40 %) Pseudomonas sp (40 %) and Serratia sp (20 %) were found to be the predominant genera of lipase producers (Figure 5). Distribution of L-asparaginase producing bacteria isolated from the gut of insects were found to be Pseudonmonas sp. (34 %) followed by Bacillus sp. and Serratia sp. (33 %) (Figure 6).

4. Discussion By screening bacterial populations isolated from the gut of different insects, a strain of Bacillus sp was found to be the best producers of most enzymes. Almost all animals possess their own gut microflora consisting of a number of bacterial and other microbial species, in their alimentary tracts. Most of the gut bacteria are parasitic or commensal associates of the host organisms, but some of them have beneficial effects on the hosts (Dillon and Dillon, 2004; Ley et al.,2006; Xu and Gordon, 2003). In our study, it is conceivable that even in gut symbiotic associations, potent bacteria may be acquired. Insect acquires a specific bacterial symbiont of a beneficial nature from the environment. The potential of the microbes on insect gut flora is currently unknown. Starch industry is one of the largest users of enzymes for the hydrolysis and modification of this useful raw material. The starch hydrolytic enzymes comprise 30% of the world's enzyme consumption (Van der Maarel et al., 2002). A number of attempts were made to isolate and characterize amylolytic enzymes from diversified sources (Daniel, 1979; Norman, 1979; Fogarty and Kelly, 1995) to meet the requirements of the starch industry. In our study, we have reported that among various genera isolated from the gut of insects, Bacillus sp. was found to be the maximum producers of amylase. Proteases are becoming major industrial enzymes and constitute more than 65 % of the world market (Rao et al., 1998). These enzymes are extensively used in the food, pharmaceutical, leather and textile industries (Cowan, 1996; Fan et al., 2001; Mozersky et al., 2002). The applications will keep increasing in the future as with the need. Extremely thermostable proteases are produced by the hyperthermophilic archaeum Desulfurococcus strain (Hanazawa et al., 1996) and thermostable proteases are reported from a gram negative thermophilic bacterium (Pawinee and Wipapat, 1996). In our investigation, we found Bacillus sp was found to be the predominant genera of protease producers followed by Pseudomonas sp. Phosphatase is the first reported enzyme from marine environments of India. Its occurrence and distribution was studied (Ayyakkannu and Chandramohan 1971). However, in our study, we have reported phosphatase activity from the gut flora of insects. Pseudomonas sp followed by Lactobacillus sp. was found to be the best phosphatase producers. Lipases of microbial origin are the most versatile enzymes and are known to bring about a range of bioconversion of reactions (Vulfson, 1994), which includes hydrolysis, interesterification, esterification, alcoholysis, acidolysis and aminolysis (Jaeger et al., 1994; Pandey et al., 1999; Nagao et al., 2001; Kim et al., 2002). Lipases are of widespread occurrence throughout earth's flora and fauna. Most abundantly, they are found in bacteria, fungi and yeasts (Wu et al.,1996). Several Bacillus sp. were reported to be the main source of lypolytic enzymes (Kim et al., 1994; Schmidt et al., 1994; Luisa et al., 1997). In our study, we have attempted to screen bacteria from the gut of different insect and found Bacillus sp., Pseudomonas sp., and Serratia sp. to be the best producers of lipase. Bacterial L - asparaginases are enzymes of high therapeutic value due to their use in leukemia treatment. L - asparaginase received increased attention in recent years for its anticarcinogenic potential. Cancer cells differentiate themselves from normal cells in diminished expression of L - asparagine (Swain et al., 1993; Manna et al., 1995). At present, the principal source of L - asparaginases for clinical trials is the bacterium Escherichia coli (Adamson and Fabro, 1968). Although production and purification techniques have been developed, they generally provide a quantity of enzyme sufficient for only limited trials. Possibly, alternative sources of L-asparaginases could overcome the problem of antigenic reactions found in some patients (Ottgen et al., 1967). Hence we have investigated the alternate source of L-asparaginase from the gut of different insects which reveal that Pseudomonas sp. was found to be the best L-asparaginase producers followed by Bacillus sp. and Serratia sp. Acknowledgement The authors are thankful to Bharathiar University for providing facilities to undertake the work. References Adamson, R.H., Fabro, S., 1968. Antitumor activity and other biologic properties of L- asparaginase. Cancer Chemother. Rep. 52, 617 - 626. Ayyakkannu, K., Chandramohan, D., 1971. Occurrence and distribution of phosphate solubilising bacteria and phosphates in marine sediments of Portonovo. Mar Biol 11: 201 - 205. Cowan, D., 1996 Industrial enzyme technology. Trends Biotechnol. 14 (6), 177 - 178. Daniel, L., 1979. Fermentation and Enzyme Technology. John Willey and Sons, NY. Dillon, R. J., Dillon, V.M., 2004. The gut bacteria of insects: nonpathogenic interactions. Annu. Rev. Entomol. 49: 71 - 92. Fan, Z., Zhu, Q., Dai, J., 2001. Enzymatic treatment of wool. J. Dong Hua University (English edition) 18, 112 - 115. Fogarty, W., Kelly, C., 1995. Amylases, amyloglucosidases and related glucanases. In: Enzyme and microbiol technology. In: Enzyme and microbial systems involved in starch processing, vol. 17, pp. 770 - 778. Hanazawa, S., Hoaki, T., Jannasch, H., Maruyama, T., 1996. An extremely thermostable serine protease from hyperthermophilic archaeum Desulfurococcus strain sy, isolated from deep sea hydrothermal vent. J. Mar. Biotechnol. 4, 121 - 126. Jaeger, K., Ransac, S., DIjktra, C., Colson, C., Heuvel, M., Misset, O., 1994. Bacterial lipases. FEMS Microbiol. Rev. 15, 29 - 63. Kaufman, M.G., Klug, M.J., 1991. The contribution of hindgut bacteria to dietary carbohydrate utilization of crickets (Orthoptera, Gryllidae). Comparative Biochemistry and Physiology 98 A, 117 - 123. Kim, H., Sung, M., Kim, M., Oh, T., 1994. Occurrence of thermostable lipase in thermophilic Bacillus sp. strain 398. Biosci. Biotech. Biochem. 58. 961 - 962. Kim, I., Kim, H., Le, K., Chung, S., Ko, S., 2002. Lipase catalyzed acidolysis of perilla oil with caprylic acid to produce structured lipids. JAOCS 79, 363 - 367. Ley, R.E., Peterson, D.A.,Gordon, J.I., 2006. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124: 837 - 848. Luisa, M.R., Schmidt, C., Wahl, S., Sprauer, A., Schmid, R.,1997. Thermoalkalophilic lipase of Bacillus thermocatenulatus. Large scale production, purification and properties: aggregation behavior and its effect on activity. J. Biotechnol. 56, 89 - 102. Lysenko, 0., 1985. Non-sporeforming bacteria pathogenic to insects: incidence and mechanisms. Annu. Rev. Microbiol. 39:673-695. Manna, S., Sinha, A., Sadhukhan, R., Chakrabarty, S.L.,1995. Purification, characterization and antitumor activity of L-asparaginase isolated from Pseudomonas stutzeri MB-405. Curr Microbiol 30, 291 - 298. Mozersky, S., Marmer, W., Dale, A.O., 2002. Vigorous proteolysis. Rlining in the presence of an alkaline protease and bating (Post Liming) with an extremophile protease. JALCA 97, 150 - 155. Nagao, T., Shimada, Y., Sugihara, A., Murata, A., Komemushi, S., Tominaga, Y., 2001. Use of thermostable Fusarium heterosporum lipase for production of structured lipid containing oleic and palmitic acids in organic solvent free system. JAOCS 78, 167 - 172. Norman, B., 1979. The application of polysaccharide degrading enzymes in starch industry. In: Berkley, R. et al.(Eds.), Microbial polysaccharides. Academic Press, New York, pp. 339 - 376. Oettgen H.F., Old L.J., Boyae E.A., Campbell H.A., Philips F.S., Clarkson B.D., Tallal L., Leper R.D., Schwartz M.K., Kim J.H., 1967. Inhibition of leukemias in man by L-asparaginase. Cancer Res. 27, 2619 - 2631. Pandey, A., Benjamin, S., Soccol, C., Nigam, P., Krieger, N., Soccol, V., 1999. The realm of microbial lipases in biotechnology: a review, Biotechnol. Appl. Biochem. 29, 119 - 131. Pawinee, K., Wipapat, K., 1996. Thermostable metalloproteases excreted by gram negative thermophillic bacterium.. In: Poster presentation: 10th International Biotechnology symposium. Sydney, Australia. Rao, M., Tankasale, A., Ghatge, M., Desphande, V., 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597 - 634. Santo-Domingo, J.W., Kaufman, M.G., Klug, M.J., Holben, W.E., Haris, D., Tiedje, J.M., 1998. Influence on diet on the structure and function of the bacterial hindgut community of crickets. Molecular Ecology 7, 761 - 767. Schmidt, C., Sztajer, H., Stocklein, W., MEnge, U., Schimid, R., 1994. Screening, purification and properties of thermophilic lipase from Bacillus thermocatelnulatus. Biochem. Biophys. Acta 1214, 43 - 53. Swain, A.I., Jaskolski, M., Housset, D., Mohana Rao, J.K., Wlodawer, A., 1993. Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc Natl Acad Sci USA 90, 1474 - 1478. Van der Maarel, M., Van der Veen, B., Uitdehaag, H., Leemhuis, H., Dijkhuizen, L.,2002. 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S.No. Organisms Bacteria Common Name Scientific Name 105 cfu 1 Ant-black Paratrechina longicornis 15 2 Ant-Red Monomorium pharaonis 36 3 Centipede Scutigera coleoptrata 22 4 Cockroach Periplaneta americana 26 5 Dragonfly Libellula luctosa 8 6 Earthworm Eudrilus eugeniae 61 7 Earthworm Eisenia fetida 30 8 Grass hopper - Green Tettigonia viridissima 40 9 Grass hopper-Brown Patanga japonica 25 10 Honey bee Apis mellifera 20 11 Housefly Musca domestica 14 12 Millipede Narceus spp 41 13 Silk worm Bombyx mori 9 14 Silkworm Cocoon Bombyx mori - cocoon 34 15 Snail Helix aspersa 25 16 Soil cockroach Eublaberus distanti 84 17 Spider Achaearanea tepidariorum 18 18 Stick insect Carausius morosus 51 19 Sulphur Butterfly Kricogonia lyside 12 20 Termite Reticulitermes virginicus 29 21 Wasp Aleiodes indiscretus 30 22 Wasp moth Cosmosoma myrodora 3 23 Weevil Otiorhynchus sulcatus 38 24 Wolf spider Trite planiceps 27

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