Qualitative and Quantitative Assessment of Cellulolytic Enzyme Production 2021 PDF

The Pharma Innovation Journal 2021; 10(2): 127-131 ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.03 Qualitative and quantitative assessment of cellulolytic TPI 2021; 10(2): 127-131 © 2021 TPI enzyme production in the microbial isolates from www.th...

The Pharma Innovation Journal 2021; 10(2): 127-131 ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.03 Qualitative and quantitative assessment of cellulolytic TPI 2021; 10(2): 127-131 © 2021 TPI enzyme production in the microbial isolates from www.thepharmajournal.com Received: 19-12-2020 environmental samples Accepted: 21-01-2021 BM Porkavi BM Porkavi, P Kalaiselvi, V Davamani, R Anandham and M Maheswari M. Sc Scholar, Department of Environmental Sciences, Tamil Nadu Agricultural University, DOI: https://doi.org/ 10.22271/tpi.2021.v10.i2b.5632 Coimbatore, Tamil Nadu, India Abstract P Kalaiselvi The biological treatment of lignocellulosic biomass through enzymatic hydrolysis serves as an Assistant Professor, Department ecofriendly and cost efficient method. As these biomass is the most abundant renewable resource which of Environmental Sciences, lacks proper utilization and disposal. This study was conducted to isolate and screen the presence of Tamil Nadu Agricultural cellulosic enzymes from the microbes of natural ecosystem under laboratory condition. The microbes University, Coimbatore, Tamil were isolated from elephant dung, forest soil, termites (gut) collected from Anaikatty forest range of Nadu, India Coimbatore district. Microbes were screened qualitatively and quantitatively for the presence of V Davamani endoglucanase, exoglucanase, β-glucosidase enzymes. Among the total microbes recovered, one bacteria Assistant Professor, Department (BT4), one action bacteria (AS3)and two fungal isolates(FE5,FE6)showed maximum cellulolytic of Environmental Sciences, efficiency during qualitative screening, those were further subjected for quantitative estimation of Tamil Nadu Agricultural endoglucanase, exoglucanase, β-glucosidase activity. University, Coimbatore, Tamil Nadu, India Keywords: Biomass, microbes, cellulase, hydrolysis R Anandham 1. Introduction Assistant Professor, Department of Agricultural Microbiology, With the increase in human population and food consumption the lignocellulosic biomass like Tamil Nadu Agricultural residues of rice, wheat, maize straw, etc. are generated enormously. Improper way of disposal University, Coimbatore, Tamil of these biomass leads to environmental pollution. Among the prevailing degradation process, Nadu, India biological treatment with the use of microbes to convert lignocellulose biomass is ecofriendly and cost efficient when compared to physical and chemical processing technology. M Maheswari Professor, Department of Lignocellulose is the fundamental structural component of all plants comprising cellulose (35- Environmental Sciences, Tamil 50 %), hemicelluloses (25-30%), lignin (25-30%) together they form a complex matrix (Betts Nadu Agricultural University, et al., 1991). In the order of degradation of the components of lignocellulosic material, Coimbatore, Tamil Nadu, India cellulose was the first structural component that needs to be degraded. Cellulose is the most abundantly available biopolymers on earth. It is the homopolysaccharide consisting of D-glucose linked by ß-1,4-glycosidic bonds forming microfibrils. Microfibrils are joined by intra and intermolecular hydrogen bonds imparting rigidity, structural and chemical stability. In the stalk, stem and woody parts of the plant, the cellulose content was high, their high molecular weight and crystalline structure makes it insoluble material and poor absorber of water, which in turn limits microbial degradation. The cellulose degrading hydrolytic enzymesare ubiquitous in nature found in different types of organisms including plants, bacteria, insects and fungi. Various subclass of cellulase enzymes includes(1) endoglucanases that randomly cleave intermonomer bonds (2) exoglucanases to remove mono and dimers from the end of glucose chain (3) β-glucosidase to hydrolase glucose dimers (Malherbe et al.,2002). All the three enzymes are together needed for the complete cellulose hydrolysis resulting in simple sugars formation. Cellulose degrading enzymes have wider applications in industries such as food processing, chemicals, paper and pulp production, detergent, textile, wine etc., Naturally, wide variety of microbes have the ability to degrade lignocellulosic biomass with Corresponding Author: the help of diverse enzymes and mechanisms. In which soft rot fungi and white rot fungi are P Kalaiselvi Assistant Professor, Department common degrader, apart from them bacteria belonging to genera Cellulomonas, Pseudomonas, of Environmental Sciences, Bacillus, Streptomyces etc., were widely reported as potential cellulase producers With mutual Tamil Nadu Agricultural interaction the microbes obtain carbon and energy sources through degradation of biomass, University, Coimbatore, Tamil they play a key role of carbon recycling in the environment. Nadu, India ~ 127 ~ The Pharma Innovation Journal http://www.thepharmajournal.com Materials and Methods containing test tubes and incubated for 30min at 50˚C. The Collection of environmental samples reaction was arrested by the addition of 3mldi-nitro salicylic The samples such as elephant dung (E), termites (gut) (T) and acid and placed in water bath at 100˚C for 5min. Blank was forest soil(S) were collected from Chinna Thadagam 11°4' N simultaneously prepared by adding all reagents except crude latitude and 76° 52' E longitude, Anaikatti forest range of enzyme. When the test tubes was still warm, 1ml of Rochelle Coimbatore district and the samples were stored at 4 °C for salt solution was added in all the tubes. The reducing sugar further study. released as the produce of the enzyme activity was measured at the optical density of 540nm. The enzyme activity was Enrichment Isolation of cellulolytic organisms expressed as one micromole of glucose released per minute Cellulolytic degraders in the samples such as elephant dung, and compared with glucose standard graph(Ghose 1987). forest soil and termites (gut) were enriched and isolated. In the termite sample, gut portion alone was dissected, surface Exoglucanase assay sterilized with ethanol, macerated with 0.9% NaCl and Cellobiohydrolase’s exoglucanase activity was measured centrifuged from which 5 ml supernatant was taken as adopting Mandels et al. (1976) , where in the reaction inoculum. In the other samples, 5 g was inoculated into the mixture contained 0.2 ml crude enzyme collected from the Basal Salt Medium (NaNO3 2.5 g; KH2PO4 2 g; MgSO4-0.2 g; supernatant, 1.5 ml of substrate obtained by dissolving 0.4% NaCl-0.2 g; CaCl2·6H2O-0.1 g in a liter) containing cellobiose in 0.1M citrate buffer of pH 4.8 and incubated at carboxymethyl cellulose 1% w/v (CMC) as the sole carbon 50˚C for 30min. The reducing sugar was measured at the source and incubated for 7 days in shaker cum incubator at optical density of 540nm using Spectrophotometer. The result 37˚C at 100 rpm, facilitating the enrichment of the cellulolytic was expressed as one micromole of glucose released per degraders. After the incubation period, employing serial minute dilution the enriched inoculum was plated in CMC agar medium. Based on the morphological characteristics, distinct β- Glucosidase assay bacteria (B), action bacteria (A) and fungal (F) colonies were The β- glucosidase activity was measured using p- picked and sub cultured using the CMC agar medium and nitrophenyl-β- D-glucopyranoside (pNPG) as the substrate further axenic culture was obtained by repeated sub culturing (Berghem et al.,1974). The cultures in the liquid CMC in the same medium and the isolates were stored in glycerol medium was centrifuged at 13,000 rpm for 4 min and the stocks at -80ο C for further studies. supernatant was incubated in 50 mM citrate buffer with 5 mMpNPG as substrate at pH 5.0 and a temperature of 50˚C Qualitative screening for cellulolytic property for 30 min. The reaction was arrested by adding 10 % sodium The confirmation test for the presence of cellulase enzymes carbonate which was followed by the release of p- nitrophenol from the isolates was done by preliminary screening methods. from pNPG, that was in turn detected at 405nm optical The isolated colonies were grown individually in CMC liquid density using UV-Spectrophotometer. One unit of enzyme medium at 28±2˚C for 2, 3, 5 days for bacteria, fungi and activity was defined as the amount of enzyme required to action bacteria respectively. About10µL inoculum was spot release 1 micromole of p-nitrophenol per minute. drie dat the center of CMC agar medium and incubated for 2, 3, 5 days at 28±2˚Cfor the growth of bacteria, fungi, action Results and Discussion bacteria respectively. Then the plates were flooded with 1% Isolation of cellulolytic microbes Congo red dye and left for 15min and washed with 1M NaCl Enrichment favoured the growth of natural microorganisms. for des taining the Congo red dye (Gupta et al.,2011). The Plating the samples of basal enrichment medium into CMC efficiency of hydrolysis was calculated using the following agar plates, a total of 24 microbial strains were recovered. The formula, colonies were selected based on distinct morphological characters. Among them 6 strains of fungi (FE1, FE2, FE3, Clear zone diameter − Colony diameter FE4, FE5, FE6) and 4 strains of bacteria (BE1, BE2, BE3, Cellulolytic efficiency = × 100 Colony diameter BE4) were isolated from elephant dung. 3 strains of fungi (FT1, FT2, FT3) and 3 strains of bacteria (BT1, BT2, BT3) Quantitative estimation of cellulolytic enzymes isolated from termites (gut) and 2 strains of fungi (FS1,FS2), Mass multiplication, crude enzyme extraction and enzyme 2 strains of bacteria (BS1, BS2) and 4 strains of action assays bacteria (AS1,AS2, AS3, AS4) were isolated from forest soil. The isolates were inoculated into CMC liquid medium for In recent years, action bacteria was proved to be potential cellulase assay and incubated for 2, 3 and 5 days for bacteria cellulose degraders which was supported by the study of and fungi. The inoculum was centrifuged at 10,000 rpm for 10 Větrovský et al. (2014) by isolating seventy six min at 4˚C. The pellets were discarded and the supernatant actinobacterial strains from soil in which 31% of portion assumed to contain the extracellular cellulolytic crude actinobacteria produced both cellobiohydrolase and 1,4-β- enzymes that were used for estimation of enzyme production. glucosidase enzyme. A study by Tsegaye et al. (2019) revealed that microbiome from termite (gut) found to possess Endoglucanase assay potential cellulose degrading population. Similarly, eight new Endoglucanase activity was estimated employing Mandels et fungi isolated from Asiatic elephant dungsamples proved to al. (1976) method with the substrate modified to CMC be the potential cellulolytic degraders (Farouq et al., 2012). instead of filter paper. The cellulolytic activity was measured using UV Spectrophotometer, where in the reaction mixture Screening of isolates for cellulolytic activity contained 1.8 ml of extracted crude enzyme, 1% w/v CMC The Congo red plate assay was done for the 24 isolates, of dissolved in 0.1M citrate buffer maintained at pH 4.8, from which 10 isolates showed positive results by formation of which 1.8ml solution was added to crude enzyme sample halo zone around the colony using Congo red dye indicator. ~ 128 ~ The Pharma Innovation Journal http://www.thepharmajournal.com Based on the colony diameter and halo zone formation, the Enzymatic profile of isolates cellulolytic efficiency was calculated and provided in the Primary screening provides information on the overall Table.1.Among the isolates, BT4 bacterial culture isolated cellulolytic activity of the organism, whereas secondary from termite has the maximum cellulolytic efficiency of 65% screening was a quantitative estimation of the presence of and a total of 4 isolates (BT4,FE1, FE6, AS3) showed high cellulase enzymes in isolates. cellulolytic efficiency was taken for further enzyme The previously screened four isolates were subjected for estimation. This result correlated well with the study of quantitative enzyme activity studies. After mass multiplying Mahalingam et al. (2014) , in that study, solubilization the cultures in the CMC liquid medium for 8 days, at regular index was measured ascellulolytic efficiency among fungal intervals, the supernatant collected by centrifugation strains, in particular Aspergillus fumigates had the highest contained the crude enzyme portion that was subjected for solubilization index of 57.96 ± 0.02. quantitative estimation studies at regular intervals.Among The halo zone formation indicates the positive result for the them, isolateFE6 showed the maximum endoglucanase cellulolytic activity, in contrast the congo red dye still activity of 6.032 IU/ml on 5th day of incubation (Fig.2).The remaining in the plate reveals the presence of non hydrolysed exoglucanase activity recorded highest (4.142 IU/ml) for the β-1,4-D-glucosidic bonds (Lamb et al., 2005). In a isolate FE5 on 6th day of incubation (Fig.3) and the β- previous study, preliminary screening was opted as a vital glucosidase activity recorded maximum (5.024IU/ml) for the method for selecting potential 14 cellulolytic bacteria from 30 isolate BT4 on 4thday of incubation(Fig.4).Overall, all the isolates (Zhang et al., 2017). selected isolates showed an increase in cellulase enzyme activity over a period of 4 to 6 days followed by a gradual Table 1: Cellulolytic efficiency of microbial isolates decline. Microbial Isolates Cellulolytic efficiency (%) The active period (up to 7 days) for cellulase production Bacteria corroborates with a previous study in which CMC and filter BE2 15 paper were used as substrate, in this study also the enzyme BT3 26.9 secretion was noted upto 8 days of incubation period (Adsul BT4 65 et al., 2004). A study by Deschamps et al., (1985) Fungi observed that using filter paper as cellulose substrate, FE1 55 Trichodermaharzianum produced maximum cellulolytic FE5 10 activity of 11 IU /g after 3 days of incubation. Similarly, FE6 60 highest production of cellulolytic enzymes was noticed in FT2 12 Trichodermaressei over an incubation period of 5 days with Actino bacteria Endoglucanase, Exoglucanase and β- glucosidaseactivity AS2 15 4.692±0.04, 2.759±0.04, 6.01±0.06respectively(Bilal et al., AS3 50 2015). (B-Bacteria, F-Fungi, T-Termite, E-Elephant dung, A-Actino bacteria, S-Soil) Fig 1: Screening of cellulose degrading bacteria, fungi, actino bacteria ~ 129 ~ The Pharma Innovation Journal http://www.thepharmajournal.com Fig 2: Endoglucanase enzyme activity of isolates over different time period Fig 3: Exoglucanase enzyme activity of isolates over different time period Fig 4: β- Glucosidaseenzyme activity of isolates over different time period Conclusion measurements proved that cellulolytic activity was mediated This study highlights that organisms isolated from various through endoglucanase, exoglucanase and beta glucosidase forest resources including elephant dung, degraded wood, activity. Screening of microbial population from natural termites (gut) found to harbour cellulolytic microbial sources remains as the vital step for further research in population. Four isolates comprising of 1bacteriumisolated production of bio based products through biomass conversion. from termite gut, 1 actino bacterium isolate recovered from forest soil, 2 fungi isolated from elephant dung origin were Acknowledgements proved to be cellulolytic in nature through qualitative and I am thankful to Head of the department, Department of quantitative measurements. In particular, quantitative Environmental Science, TNAU, Coimbatore for providing lab ~ 130 ~ The Pharma Innovation Journal http://www.thepharmajournal.com facility and support. References 1. Betts WB, Dart RK, Ball AS, Pedlar SL. Biosynthesis and structure of lignocellulose. In Biodegradation Springer, London 1991, 139-155. 2. Mandels M, Andreotti R, Roche C. Measurement of saccharifying cellulase. In Biotechnol. Bioeng. Symp. (United States) Army Natick Development Center, MA 1976, 6. 3. Gupta P, Samant K, Sahu A. 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