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J. Trop. Agric. and Fd. Sc. 41(2)(2013): 319 – 327 A.K.R. Emmyrafedziawati Identification and characterisation of Bacillus subtilis as cellulase-producing bacteria (Pengenalpastian dan pencirian Bacillus subtilis sebagai bakteria penghasil selulase) A.K.R. Emmyrafedziawati* Keywords: carboxymethyl cellulose, filter paper assay, polymerase chain reaction, cloning Abstract Three cellulolytic bacteria were isolated from oil palm empty fruit bunch (EFB) compost under aerobic conditions. The genomic DNA extracted from isolates C4, C12 and EB6 were amplified using specific primers L15 and L73 to determine the presence of genes encoding cellulase CelL15 and CelL73 respectively. The presence of the expected lengths of nucleotide bands at 1,500 bp and 730 bp respectively, indicated the presence of the putative cellulase genes in these isolates. The genes encoding the cellulases CelL15 and CelL73 were cloned into the Bacillus subtilis expression vector Escherichia coli strain JM107 to identify the cellulase gene in the recombinant plasmids. The filter paper assay (FPase) on C4, C12 and EB6 were determined after 48 h of incubation period at 37 °C. C12 isolate showed the highest FPase activity at 1.733 ± 0.023 FPU/ml followed by C4 at 1.718 ± 0.006 FPU/ml. EB6 showed the lowest FPase activity at 1.695 ± 0.006 FPU/ml. However, the ANOVA results showed that all three isolates of B.subtilis had no significant differences (p >0.05) in the FPase activity. The sequence alignment of the 3 isolates C4, C12 and EB6 showed that C4 and EB6 were the same strain while C12 differed slightly and this was confirmed by the presence of 2 cellulase genes in C12 compared to only 1 in C4 and EB6. Introduction Bacillus subtilis is a ubiquitous bacterium commonly recovered from water, soil, air and decomposing plant residues. The bacterium produces an endospore that allows it to endure extreme conditions of heat and desiccation in the environment. The genus Bacillus consists of a large number of diverse, rod-shaped Gram positive (or positive only in early stages of growth) bacteria that are motile by peritrichous flagella. Like most members of the genus, B. subtilis is an aerobic bacterium, except in the presence of glucose and nitrate, some anaerobic growth can occur (Claus and Berkeley 1986). The bacterium is one of the most extensively studied model microorganisms, displays superior ability to produce various secretary enzymes. This ability has been widely applied to produce various useful enzymes in the industrial fields (Westers et al. 2004). Bacillus subtilis is considered a benign organism as it does not possess traits that cause diseases. It is not considered pathogenic or toxigenic to humans, animals or plants (U.S. EPA 2011). Extensive recent studies on proteins (such as cellulase, protease and amylase) secreted by Bacillus species have shown that the following *Strategic Resources Research Centre, MARDI Headquarters, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia Author’s full name: Emmyrafedziawati Aida Kamal Rafedzi E-mail: [email protected] ©Malaysian Agricultural Research and Development Institute 2013 319 Bacillus subtilis as cellulase-producing bacteria Bacillus species produce cellulases: B. cereus, B. licheniformis, B. subtilis and B. polymyxa (Sharma et al. 1990). Because these strains did not produce all three types of cellulase, they did not extensively hydrolyze crystalline cellulose (Han et al. 1995). Enzymes degrading (hemi) cellulose are found in microbes, plants and the digestive tracts of animals. Three classes of cellulases, including endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) are involved in the degradation of cellulose (Lynd et al. 2002). These enzymes, which cleave the β-1,4 bond of cellulose, belong to the large family of glycosyl hydrolases. Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant and renewable biopolymer on earth. The demand for microbial cellulases and related enzymes is growing more rapidly nowadays. Bacillus species in several respects has both endo- and exoglucanase activity. It degrades Cm-cellulose, cellotetraose and cellopentraose as an endoglucanase and cleaves aglycosidic bonds in p-nitrophenylbeta-D-cellobioside (pNPC) as an exoglucanase (Han et al. 1995). Reports on strains belonging to species such as B. sphaericus and B. subtilis express high cellulose degradation activities (Mawadza et al. 1996; Singh et al. 2004). In this study, the cellulolytic bacteria were isolated from oil palm empty fruit bunch (EFB) compost. Utilising the EFB as compost was a way of managing agricultural waste, besides it can also add value to EFB. We tried to isolate these bacteria from EFB because in theory, isolates from the original high cellulose substrate can potentially produce cellulolytic bacteria for industrial use. As reported by Sun et al. (1999), EFB are lignocellulosic composites which are comprised of cellulose, hemicellulose and lignin with 44% cellulose fraction. Cellulose degrading microorganisms can convert cellulose into soluble sugars either by acid 320 or enzymatic hydrolysis. Thus, microbial cellulose utilisation is responsible for one of the largest material flows in the biosphere (Lynd et al. 2002). However, pure cellulases from the cellulolytic bacteria are not commercially produced yet (Shabeb et al. 2010). The most studied group of cellulosedegrading microorganisms is the fungi, which are characterised by multicomponent, synergistic cellulolytic enzyme systems (Ulrich et. al 2008). The objectives of this study were to confirm the existence of the cellulase genes in the C4, C12 and EB6 isolates of B. subtilis and determine the isolates ability to produce cellulase enzyme using the filter paper assay or FPase (measures the total cellulase activity). B. subtilis has agricultural applications as a biopesticide and as a plant stimulant on various crops (Eeden and Korsten 2004). The Bacillus species showed potential to convert cellulose into reducing sugars which could be readily used in many applications such as animal feeds and feed stock for production of valuable organic compounds (Niranjane et al. 2007). Materials and methods Isolation of cellulolytic bacteria The B. subtilis used in this study were isolated from EFB compost using enrichment media in the pour plate technique (Van Soestbergen and Lee 1969). The main ingredients in the compost are empty fruit bunches (EFB) and palm oil mill effluent (POME). Ten-fold serial dilutions of compost sample were prepared in sterilised distilled water and 1.0 ml each of the sample dilutions was spread onto carboxymethyl cellulose (CMC) agar plates. The CMC agar medium consisted of CMC (10.0 g), (NH4)2HPO4 (2.0 g), K2HPO4 (0.5 g), MgSO4.7H2O (0.02 g), CaCl2.2H2O (0.02 g), FeSO4.7H2O (0.02 g) and distilled water up to 1,000 ml. The CMC was supplemented to react as a sole carbon source. The CMC medium plates were incubated at 37 °C for 24 h. A single colony was picked and streaked onto a new CMC A.K.R. Emmyrafedziawati medium plate. This procedure was repeated until a pure culture was obtained. Identification of bacterial isolates After obtaining the pure bacterial cultures, genomic DNA extraction was performed using a Macharey Nagel Kit (Germany) according to the manufacturer’s instructions. The genomic DNA of bacterial isolates was amplified using 16S rDNA universal primers; COM1: 5’- CAG CAG CCG CGG TAA TAC -3’ and COM2: 5’- CCG TCA ATT CCT TTG AGT TT - 3’ (Schwieger and Tebbe 1998). The PCR conditions were as follow: initial denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 61 °C for 45 s, extension at 72 °C for 2 min and a final extension at 72 °C for 10 min. The successful bands obtained on 1.0% (w/v) agarose gel (Figure 1) were outsourced to First Base Laboratories Sdn. Bhd., Selangor, for sequencing analysis. The nucleotide sequences obtained were then matched to sequences in the Genbank using the Basic local alignment search tool (BLAST) provided by the National Centre for Biotechnology Information (NCBI, USA) to identify the sequences. The nucleotide sequences were then aligned using the ClustalW2 program provided by the European Bioinformatics Institute (EBI). Amplification using specific primers to obtain the cellulase genes CelL15 and CelL73 Two pairs of DNA oligonucleotide primers designed by Li et al. (2009), L15F: 5’- AGG ATC CAT GAA ACG GTC AAT CT -3’ and L15R: 5’- CGA GCT CCT AAT TTG GTT CTG TT -3’and L73F: 5’- GGA TCC ATG CCT TAT CTG AAA C -3’ and L73R: 5’- GGC GAG CTC TTA TTT TTT TGT ATA - 3’ were used. These primer sets amplified the entire cellulase genes CelL15 and CelL73 with an expected size of about 1,500 bp and 730 bp respectively. Polymerase chain reaction (PCR) was performed in an Eppendorf gradient thermal cycler. The amplification was performed as follows: initial denaturation for 2 min at 94 °C, 30 cycles each of denaturation for 2 min at 94 °C, annealing for 1 min at 54 °C, and primer extension for 4 min at 72 °C and a final extension for 15 min at 72 °C. The PCR products were electrophoresed on a 1.0% (w/v) agarose gel, stained with 0.1% (v/v) SybrSafe ® (Invitrogen, US) and viewed using the Bio-Rad gel documentation system. Determination of cellulase activity A volume of 100 µl pure culture of the nutrient broth was inoculated into 5.0 ml of 0.05M sodium citrate buffer (pH 4.75). A Whatman number 1 filter paper strip (1 cm x 3 cm) was soaked in this buffer and two drops of glucose was added on it (Maki et al. 2011). The sample was incubated in a shaker incubator at 37 °C for 7 days at 100 rpm. For observation, the enzyme reaction was stopped immediately using 3, 5-dinitrosalicylic (DNS) acid. The mixture was then boiled for 5 min, cooled for 10 min and analysed using the GENESYS 10S UV-VIS spectrophotometer (Thermo Scientific, U.S.) at 540 nm against the blank. Glucose equivalents (reducing sugars) generated during the assay were estimated by using the 3,5 dinitrosalicylic acid (DNS) method (Miller 1959) with glucose as the standard. One international Filter Paper Unit (FPU) activity was defined as the enzyme required for releasing one μmol of glucose per min under the assay conditions and activities were reported as FPU/ml (Balamurugan et al. 2011). All samples were analysed in triplicates and the mean values were calculated. Cloning of genomic DNA PCR products The genomic DNA PCR products were purified and cloned using the CloneJETTM PCR cloning kit (Fermentas) with blue-white screening. The pJET1.2 (50 ng/µl) provided in the kit was used as the cloning vector. Transformation was carried out using the TransformAidTM Bacterial Transformation 321 Bacillus subtilis as cellulase-producing bacteria Kit (Fermentas). The cellulase genes were ligated into JM107 plasmid containing a promoter upstream of the insert site and digested with EcoRI to allow efficient binding of blunt ends. Statistical analysis All data obtained were analysed using Analysis Of Variance (ANOVA). The mean values ± standard deviations (SD) are presented in Tables 1 – 2. Results and discussion Thirteen isolates of the cellulase-producing bacteria were screened previously on carboxymethyl cellulose (CMC) plates stained with Congo red to observe the cellulolytic activity of the isolated strains under aerobic conditions. Three Bacillus isolates which produced the greatest clear zone diameters were selected as indicated in Table 1. Carboxymethyl cellulose (CMC) in medium served as the sole carbon source for the cellulolytic bacterial growth and the results showed strong evidence that cellulase was produced in order to degrade the cellulose. Carboxymethyl cellulose (CMC) as the endoglucanase substrate gave the highest yield of cellulase enzyme. Results showed that there were significant differences among the means of the clear zone diameters at the 0.05 level (Table 1). EB6 showed the greatest clear zone diameter (3.18 ± 0.1483 cm) with the putative cellulase gene CelL73. However, C12 which had the second greatest clear zone diameter, showed amplification of both specific products CelL15 (~1,500 bp) and CelL73 (~730 bp). Not all 13 isolates showed amplification of the ~500 bp product using the 16S rDNA universal primers COM1 and COM2 (Figure 1). Nucleotide sequences of isolates which gave the ~500 bp band were blast to the National Centre for Biotechnology Information (NCBI) gene bank database to identify the species. Three of the isolates with greatest clear zone diameters were selected for amplification using specific primers to obtain the cellulase genes CelL15 and CelL73. Isolates C4 and EB6 were identified as B. subtilis strain BAB-2742 (gene bank accession no. KF053069.1) and C12 was identified as B. subtilis strain SBRh5 (gene bank accession no. KF053071.1). The multiple sequence alignment of genomic Table 2. Mean FPase activity of cellulaseproducing bacterial isolates Isolate C4 C12 EB6 FPase activity (FPU/ml) 1.7182 ± 0.006a 1.7331 ± 0.023a 1.6951 ± 0.006a The FPase activity values shown are the mean ± standard deviation (SD) of triplicates. Means with the same letter are not significantly different at p <0.05 Table 1. Screening of Bacillus isolates on CMC plate and PCR amplification using specific primer sets Isolate Identification Diameter of clear zone (cm) PCR product obtained using specific primers GenBank acession no. C4 Bacillus subtilis (strain BAB-2742) 2.2 ± 0.1871c +ve L73 (~ 730 bp) KF053069.1 2.6 ± 0.1225b KF053071.1 Bacillus subtilis (strain BAB-2742) 3.18 ± 0.1483a +ve L15 and L73 (~ 730 bp and 1,500 bp) C12 EB6 Bacillus subtilis (strain SBRh5) +ve L73 (~ 730 bp) KF053069.1 Note: The values shown for diameter of clear zone are the mean values ± standard deviation (SD) of triplicates. Means with different letters are significantly different at p <0.05 322 A.K.R. Emmyrafedziawati Figure 1. PCR products of COM1 and COM2 16S rDNA of all cellulase-producing bacteria isolates. Lanes 1 and 11 represent 1kb DNA ladder (Invitrogen, USA). Lanes 2 – 9 (upper gel) and lanes 12 – 16 (lower gel) show genomic DNA bands of all isolates. Lane 10 is the negative control. The genomic DNA amplified is about 500 bp DNA from all 3 isolates C4, C12 and EB6 are shown in Figure 2. Figure 3 shows the gel images of PCR products amplified by both L15 and L73 primer sets. Primer L15 encoded a cellulase gene of about 1,500 bp while primer L73 encoded beta-glucan of about 730 bp (Li et al. 2009). C12 gave a positive result for both PCR products, CelL15 and CelL73 with an expected size of about 1,500 bp and 730 bp respectively. The C4 and EB6 isolates amplified only the CelL73 PCR product with an expected size of about 730 bp. As described by Li et al. (2009), the C12 sequence contained both cellulase and beta-glucan hydrolysing enzyme primer sequences while C4 and EB6 sequences contained only beta-glucan hydrolysing enzyme primer sequence. The sequence analysis of the recombinants determined that C12 was 99% identical to B. subtillis cellulase gene in the Genbank with accession no: NC014976 under the reference genome sequences database (refseq). Under the protein cluster database, this sequence showed the presence of the endo-1,4-beta-glucanase enzyme (accession numbers: YP004203745, YP003866220 and NP389695). C12 was also 99% identical to B. subtilis ssp. subtilis (accession no. NC017195) which had the beta-glucanase gene (accession no. YP005558949). C4 and EB6 showed 99% identity to B. subtilis cellulase gene with accession no. NC014976. A study by Li et al. (2009) showed that the predicted amino acid (protein) sequence of the CelL15 gene was cellulase from sequence position 43 to 302 while sequence position from 355 to 437 was cellulose binding domain (CBM3). Meanwhile for CelL73, gene sequence position 32 to 242 was a multi-domain of glycosyl-hydrolases 16 superfamily which is a more complicated gene. In this study, the total cellulolytic activity was assayed by filter paper. Other assays to determine the specific cellulase activity (e.g.beta-glucosidase) were not done since the endoglucanase or endo-1, 4-betaglucanase was the major cellulase found in all 3 isolates C4, C12 and EB6. Results in Table 2 shows that C12 isolate had the highest mean FPase activity (1.733 ± 0.023 FPU/ml) followed by C4 isolate (1.718 ± 0.006 FPU/ml). EB6 showed the lowest mean FPase activity (1.695 ± 0.006 FPU/ ml). However, ANOVA (Table 2) indicated that there was no significant differences in FPase activity amongst the 3 isolates (p >0.05). Results from this analysis further strengthened the report of Desvaux (2005) who stated that a single cellulase gene is difficult to degrade cellulose even though it may have high activity under specific conditions (e.g. clear zone, CMC activity). Cellulase is an inducible enzyme and it is affected by the nature of the substrates used for their production (Haung and Monk 2004). When filter paper was used as a substrate, further incubation period was found to decrease the enzyme activity. The filter paper assay (FPA) has been widely used to measure total cellulase activity because it is readily available and inexpensive. Coward-Kelly et al. (2003) reported that assay performed with filter 323 Bacillus subtilis as cellulase-producing bacteria Figure 2. The multiple sequence alignment of the 16S rDNA (amplified from isolates C4, C12 and EB6 using universal primers COM1/COM2) obtained using ClustalW2 program. C4 and EB6 have the same sequence (480 bp) while C12 sequence differs slightly (443 bp) paper provided reliable and reproducible results. The degradation of the filter paper would imply multiple cellulase activities including exoglucanase activities because these enzymes work in crystalline regions (Dashtban et al. 2010). Several studies have shown that B. subtilis can be used as a host to secrete heterologous cellulases, and naturally occurring strains have been identified that secrete cellulases (Joliff et al. 1989). Cellulase enzymes have also been targeted 324 to the membrane, enabling protoplasts of B. subtilis to degrade carboxymethylcellulose (Kim et al. 2005). Conclusion The presence of the cellulase genes was successfully demonstrated in three locally isolated cellulolytic bacterial strains (C4, C12 and EB6) from palm oil empty fruit bunch (EFB) through amplification using specific primers and were identified as Bacillus subtilis. Two of these isolates, A.K.R. Emmyrafedziawati a) 730 bp b) 1,500 bp Figure 3. a) 1% agarose gel image of PCR product 730 bp amplified using L73 pair of primers in isolates C4 (lane 2), C12 (lane 6) and EB6 (lane 8). Isolates C6 (lane 3), C7 (lane 4), C10 (lane 5) and EB4 (lane 7) were not amplified by L73. Lane 1 is 1 kb DNA ladder (Invitrogen,US) and lane 9 is the negative control. b) 1% agarose gel image of PCR product 1,500 bp amplified using L15 pair of primers in isolate C12 (lane 4). Isolates C4 (lane 2) and EB6 (lane 3) were not amplified by L15 pair of primers. Lane 1 is 1 kb DNA ladder (Invitrogen, US) C4 and EB6, were identified to be the same strain based on the 16S rDNA gene sequences. Amplification using two primer sets encoding CelL15 and CelL73 genes showed that only C12 contained both the cellulase genes while C4 and EB6 contained only one cellulase gene. Assays on their individual enzyme activities (total cellulase activity) were not significant. These findings suggest that combination of the 3 isolates may be capable of producing higher enzyme activities. Further studies have to be carried out to confirm the capability of the mixed culture by quantification of their enzyme activities. Acknowledgment The author would like to thank Ms Noor Shita Desa for her assistance in this project. This study was funded by RMK-10 development fund under White Agriculture project, No.148-2012. References Balamurugan, A., Jayanthi, R., Nepolean, P., Vidhya Pallavi, R. and Premkumar, R. (2011). Studies on cellulose degrading bacteria in tea garden soils. African Journal of Plant Science 5(1): 22 – 27 Claus, D. and Berkeley, R.C.W. (1986). Genus Bacillus Cohn 1872. In: Bergey’s Manual of Systematic Bacteriology, (Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G., eds.), p. 1105 – 1139. Baltimore MD: Williams and Wilkins Co. Coward-Kelly, G., Aiello-Mazzari, C., Kim, S., Granda, C. and Holtzapple, M. (2003). Suggested improvements to the standard filter paper assay used to measure cellulase activity. Biotech. and Bioeng. 82: 745 – 749 Dashtban, M., Maki, M., Leung, K.T., Mao, C. and Qin, W. (2010). Cellulase activities in biomass conversion: measurement methods and comparison. Critical Rev. Biotechnology 30: 302 – 309 Desvaux, M. (2005). The cellulosome of Clostridium cellulolyticum. Enzyme Microb. Technol. 37: 373 – 385 325 Bacillus subtilis as cellulase-producing bacteria Eeden, M.V. and Korsten, L. (2004). Effect of additives and copper fungicide on Bacillus subtilis to control avocado (Persea americana Mill.) fruit diseases. South African Avocado Growers’ Association Yearbook 27: 11 – 16 Han, S.J., Yoo, Y.J. and Kang, H.S. (1995). Characterization of a bifunctional cellulase and its structural gene: The cel gene of Bacillus sp. D04 has exo- and endoglucanase activity. J. Biol. Chem. 270: 26012 – 26019 Haung, X.P. and Monk, C. (2004). Purification and characterization of a cellulase from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov.sp. World journal of Microbiology and Biotechnology 20: 85 – 92 Joliff, G., Edelman, A., Klier, A. and Rapoport, G. (1989). Inducible secretion of a cellulase from Clostridium thermocellum in Bacillus subtilis. Appl. Environ. Microbiol. 55: 2739 – 2744 Kim, J.H., Park, I.S. and Kim B.G. (2005). Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis. Biochem. Biophys. Res. Commun. 334: 1248 – 1253 Li, W., Huan, X., Zhou, Y., Ma, Q. and Chen, Y. (2009). Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden takin (Budorcas taxicolor Bedfordi). Biochem. and Biophysical Research Comm. 383: 397 – 400 Lynd, L.R., Weimer, P.J., Willem, H.Z. and Pretorius, I.S. (2002). Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev. 66: 506 – 577 Maki, M.L., Broere, B., Leung, K.T. and Qin, W. (2011). Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers. Int J Biochem Mol Biol. 2(2): 146 – 154 Mawadza, C., Boogerd, F.C., Zvauya, R. and Van Verseveld, H.W. (1996). Influence of environmental factors on endo-β-1,4glucanase production by Bacillus HR 68, isolated from a Zimbabwean hot spring. Antonie Van Leeuwenhoek 69(4): 363 – 369 Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426 – 428 326 Niranjane, A.J., Madhou, P. and Stevenson, T.W. (2007). The effect of carbohydrate carbon sources on the production of cellulase by Phlebia gigantean. Enzyme and Microbial Technology 40: 1464 – 1468 Schwieger, F. and Tebbe, C.C. (1998). A new approach to utilize PCR-single-strand conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl. Environ. 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Diversity and activity of cellulosedecomposing bacteria; isolated from a sandy and a loamy soil after a long-term manure application. Microb Ecol. 55: 512 – 522 U.S. Environmental Protection Agency. (2011). Bacillus subtilis; Final Risk Assessment. Retrieved on 12 June 2012 from http://www. epa.gov/oppt/biotech/pubs/fra/fra009.htm Van Soestbergen, A.A. and Lee, C.H. (1969). Pour plates or streak plates? Appl. Microbiol. 18(6): 1092 – 1093 Westers, L., Westers, H. and Quax, W. J. (2004). Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochem. Biophys. Acta. 1694: 299 – 310 A.K.R. Emmyrafedziawati Abstrak Tiga bakteria selulosa telah dipencilkan daripada kompos tandan kelapa sawit (EFB) dalam keadaan aerob. DNA genomik yang diekstrak daripada pencilan C4, C12 dan EB6 telah diamplifikasi menggunakan primer spesifik L15 dan L73 untuk menentukan kehadiran gen pengekodan selulase CelL15 dan CelL73. Kehadiran band nukleotida pada 1,500 dan 730 bp menunjukkan kehadiran gen selulase berpontensi dalam setiap pencilan. Gen pengekodan selulase CelL15 dan CelL73 telah diklon dalam Bacillus subtilis dengan vektor Escherichia coli strain JM107 untuk mengenal pasti gen selulase yang terdapat dalam plasmid rekombinan. Cerakin kertas penapis (FPase) dalam C4, C12 dan EB6 ditentukan selepas 48 jam tempoh pengeraman pada 37 °C. Pencilan C12 menunjukkan aktiviti FPase tertinggi iaitu 1.733 ± 0.023 FPU/ml diikuti oleh pencilan C4 pada 1,718 ± 0.006 FPU/ml. EB6 menunjukkan aktiviti FPase yang lebih rendah pada 1.695 ± 0.006 FPU/ml. Bagaimanapun, keputusan ANOVA menunjukkan bahawa ketiga-tiga strain B. subtilis tidak mempunyai perbezaan yang signifikan (p >0.05) dalam aktiviti FPase. Penjajaran jujukan bagi ketiga-tiga pencilan (C4, C12 dan EB6) menunjukkan C4 dan EB6 adalah strain yang sama manakala C12 berbeza sedikit dan ini disahkan dengan kehadiran 2 gen selulase dalam C12 berbanding dengan hanya satu yang hadir dalam C4 dan EB6. Accepted for publication on 10 September 2013 327