Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 19-32 ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 19-32 http://www.ijcmas.com Original Research Article Enzymatic Activities of some Streptomycetes Isolated from Soil at Taif Region Mohamed H. Sonya1,2, Altalhi A.D.1, El-Zahrani Gh. S. Badria1 and Sadik A.S.1,2* 1Department of Biology, Faculty of Science, Taif University, P.O. Box 888, Taif, KSA 2Soil, Water and Environmental Research Institute, Department of Agricultural Microbiology, Agricultural Research Center, P.O. Box, 12619, Giza, Egypt 3Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, P.O. Box 68, Hadayek Shobra 11241, Cairo, Egypt *Corresponding author A B S T R A C T This study was aimed at isolating, purifying and identifying some streptomycetes from soil of Taif region having enzymatic activities. Some streptomycete isolates were isolated from soil in Taif and their enzymatic activities and optimum K e y w o r d s conditions for producing the selected enzyme were studied. A number of 23 of streptomycetes-like isolates (SLI) were isolated from different locations (Al- Streptomycetes, Qaiem, Al-Qamaryiah, Al-Sail Al-Kabeer, Al-Sail Al-Sagheer, Al-Sawtah, Gabrah Taif, (1), Gabrah (2), Tarabah, Qaiah and Wadi-Bowa) at Taif region of KSA. All isolates Enzymatic were found to produce amylase. A number of 17 (73.9%) out of the 23 purified Activities, isolates showed excellent growth on starch casein agar medium. In case of Identification, cellulase, lipase and gelatinase, the growth was 15 (65.2%), 21(91.3%) and 17 Amylase (73.9%), respectively, and were active. A number of three isolates representing the Enzyme highest active isolates (10, 12 and 13), produce all the tested enzymes, and were Production selected for identification. The isolate 10 was identified as a strain of S. lateritius. The Streptomyces isolate 12, was very likely to be a strain of S. cacaoi subsp. asoensis. The tested isolate 13 was closely related to S. phaeopurpureus. Data showed that amylase enzyme production was affected by different culture conditions. It was observed that the optimum zone of hydrolysis in mm was obtained at pH 7.0 and temperature 30°C with the incubation period of 6.0 days. Introduction Streptomyces species were found worldwide industrial, environmental and food in soil and were important in soil ecology technology through microbial sources (Salahuddin et al., 2011). Much of the Mohamed Sonya (Boing, 1999; Gurudeeban characteristic earthy smell of soils arises by et al., 2011). Streptomycetes are considered Streptomyces species (Mohamed Sonya et as one of the most important bacteria, due to al., 2001). Enzymes are the most important their ability to develop the soil properties as products for human needs in the area of well as producing several extracellular 19 Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 19-32 substances (enzymes) as secondary products The present study was designed to isolate (Gandolfi et al., 2000; Techapun et al., and purify some Streptomycetes from soil of 2002; Brühlmann et al., 1994; Jang and Taif region, to determine the enzyme Chang, 2005; de Lima et al., 2005; Soares et activities of the purified isolates, and al., 2006; Saadoun et al., 2007; Altalhi and identify the selected isolates up to species Mohamed Sonya, 2010; Gulve and based on their cultural, morphological and Deshmukh, 2011; Mohamed et al., 2013; physiological characters. Some factors Mohamed Sonya et al., 2014). About three- affecting the activity of an enzyme was also fourths of the Streptomyces species may studied. produce antibiotics (Gupta et al., 1995). The Materials and Methods possibility of using streptomycetes for enzyme production had recently been Some soil samples were randomly collected investigated (Yang and Wang, 1999). A from five sites of different regions of Taif. serine protease from the keratin-degrading Plate technique as described by Mohamed Streptomyces pactum DSM 40530 was Sonya (1998) was applied using starch purified by casein agarose affinity nitrate agar medium (Waksman and chromatography. The enzyme was optimally Lechevalier, 1961) for isolation, purification active in the pH range from 7 to 10 and at o and maintenance of Streptomycete-like temperatures from 40 to 75 C (Böckle et al., colonies (SLC). Colonies were maintained 1995). on starch nitrate agar slants at 4-5oC until used. Sixty different strains of Streptomyces were tested for their enzymatic hydrolysis of Determination of Enzymatic Activities of cellulose and related materials. They found Streptomycetes Isolates that the most active three strains were S. mutabilis, S. chromofuscus and S. In these experiments, SLI were screened for cyanoviridis (El-Nasser and El-Shafei, their potential to produce fiber hydrolytic 1994). enzymes, i.e., amylases (Shaw and Ou-Lee, Pectinases are today one of the upcoming 1984; Santos and Martins, 2003), cellulases enzymes of the commercial sector (Kashyap (Carder, 1986; Sharma et al., 2001), lipases et al., 2001). It was proved that such species (Tendler and Burkholder, 1961) and of bacteria produces several enzymes such gelatinase (Stefka et al., 2004). In case of as: Amylase, cellulase, pectinase, chitinase, proteases, plates of skimmed milk casein agar medium were separately inoculated and protease, etc. Five Streptomycetes o obtained from Sinai sandy soil in Egypt, with each pure SLI, and incubated at 30 C±2 were identified based on the biological for five days. The appearance of clearing properties of Streptomycetes in the Key zones around colonies indicated the proposed by Pridham and Tresner (Pridham presence of proteolytic activity by these and Tresner, 1974). Results of the cultural, isolates due to hydrolysis of casein. morphological and physiological characteristics showed that Streptomyces Identification of selected Streptomycetes isolate Si-1 was a novel violet species. Four up to species isolates were identified as S. tuirus Si-4, S. lateritius Si-6, S. mauvecolor Si-9 and S. Identification of the highest active melanogenes Si-11. Streptomycete isolates in producing hydrolytic enzymes were subjected to 20 Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 19-32 identification up to species according to the Taif region proposed key of Pridham and Tresner (1974) in Bergey's Manual of Determinative Filamentous actinomycetes which formed Bacteriology (1974). Media as well as distinctive different colored colonies were methods used in these keys were described isolated from different soil samples by Shirling and Gottlieb (1966). (Mohamed Sonya, 1998; Duangmal et al., Identification based on cultural, 2005; El-Sherbiny, 2006; Ismail, 2006; Al- morphological and physiological characters, Askar et al., 2011; Atta et al., 2011; growth on Czapek-Dox agar medium; Mohamed Sonya et al., 2012; Shori Ghadeer Sensitivity to streptomycin and antibiosis et al., 2012). activities of the selected streptomycetes were carried out as exactly described by Data in Table 1 and illustrated by Figure 1 Mohamed Sonya (1998). show that a few numbers (23) of SLI were isolated from different locations (AlQaiem, Factors Affecting Amylase Activity of Al-Qamaryiah, Al-Sail Al-Kabeer, Al-Sail Streptomyces Isolate Al-Sagheer, Al-Sawtah, Gabrah (1), Gabrah (2), Tarabah, Qaiah and Wadi-Bowa) at Taif To determine the effect of different culture region of KSA. Numbers of 1, 1, 1, 1, 1, 3, conditions on -amylase enzyme production, 2, 4, 6 and 3 isolates were purified from three experiments were conducted using those locations, respectively. It worth to starch nitrate agar medium (Waksman and mention that a total number of 40 SLI were Lechevalier, 1961). Regarding the effect of isolated, but only, 23 out of them was incubation temperatures, plates of the clearly purified. normal agar medium were inoculated with growth discs of the selected Streptomycete Determination of enzymatic activities of isolates. The inoculated plates were the purified SLI incubated at each of 25, 30 and 35oC for 2 days. In case of incubation period, Data in Table (2) and illustrated by Figures inoculated plates were incubated at 30oC for (2), (3), (4), (5) and (6) showed that the 23 2, 4 and 6 days. To detect the effect of pH purified SLI were tested for their abilities to on enzyme production, the same medium hydrolyze starch, oil, casein, cellulose and was prepared with different pH degrees (5.0, gelatin as mentioned in the materials and 6.0, 7.0 and 8.0). The inoculated plates were methods. All isolates were found to produce incubated at 30oC for two days. Three plates amylase as 100% of the tested isolated were used as replicates for each treatment. showed a white-yellowish hallo around the All plates were flooded with iodine solution growth when the plates of starch nitrate agar and left for 30 min at room temperature medium inoculated with the SLI were (RT) and then washed with distilled H2O. A flooded with iodine. While, 17 (73.9%) out clear white-yellowish zone around the of them showed excellent growth on starch growth in blue medium indicated hydrolysis casein agar medium. In case of cellulase, of starch, and confirmed production of - lipase, gelatinase and 15 (65.2%), amylase enzyme (Shaw and Ou-Lee, 1984). 21(91.3%) and 17 (73.9%), respectively, SLI, were found to be able to produce the Results and Discussion mentioned enzymes. A number of three isolates representing the highest active Isolation, purification and maintenance of isolates (10, 12 and 13), produce all the Streptomycetes-like isolates from soil at 21 Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 19-32 tested enzymes, and were selected for proposed by Pridham and Tresner (1974), identification. Several investigators reported the experimental isolate 10 appeared to be the importance of streptomycetes as one of related to S. lateritius as demonstrated the most important bacteria, due to their although there were differences in the use of ability to develop the soil properties as well 3 sugars as sole carbon sources (L- as producing several extracellular substances arabinose, i-inositol and Sucrose).