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Ali, I., Sultan, S., Tahir Mahmood, R., Tariq, M., Shamim, Z., Mushtaq, A., Asiri, M. (2023). "Production and characterization of α-amylase from indigenously isolated Streptomyces sp.," BioResources 18(1), 6-18.


Streptomyces species have been exploited widely as microbial cell factories, especially for antibiotic production. However, their potential for alpha-amylase production has not been extensively studied. This study reports the isolation, molecular identification, and optimization of the physiological conditions for alpha-amylase production from Streptomyces sp., isolated from soil in Kotli Azad Kashmir. The maximum growth of Streptomyces MI-1 was observed at a neutral pH and a temperature of 35 °C. An amylase activity of 1.15 IU/mL was observed when 4% starch was added to the nutrient medium. During the submerged state fermentation, the maximum amylase activity of 2.136 IU/mL/min was observed after 144 h of incubation. The characterization of bacterial amylase revealed an optimum temperature of 40 °C, and its optimum pH was 7.0. Furthermore, during the study, examining the effect of different metal ions found that Mg2+ and Ca2+ ions had a positive effect, while Cu2+ and Fe2+ ions had an inhibitory effect compared to the control. This preliminary study provides basal line information for the discovery of novel microbes from the unexplored natural resources for amylase production, which will be used for many purposes.

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Production and Characterization of α-Amylase from Indigenously Isolated Streptomyces sp.

Imran Ali,a,* Shastah Sultan,a Raja Tahir Mahmood,a,* Muhammad Tariq,a Zeeshan Shamim,a Aamar Mushtaq,a and Mohammed Asiri b

Streptomyces species have been exploited widely as microbial cell factories, especially for antibiotic production. However, their potential for alpha-amylase production has not been extensively studied. This study reports the isolation, molecular identification, and optimization of the physiological conditions for alpha-amylase production from Streptomyces sp., isolated from soil in Kotli Azad Kashmir. The maximum growth of Streptomyces MI-1 was observed at a neutral pH and a temperature of 35 °C. An amylase activity of 1.15 IU/mL was observed when 4% starch was added to the nutrient medium. During the submerged state fermentation, the maximum amylase activity of 2.136 IU/mL/min was observed after 144 h of incubation. The characterization of bacterial amylase revealed an optimum temperature of 40 °C, and its optimum pH was 7.0. Furthermore, during the study, examining the effect of different metal ions found that Mg2+ and Ca2+ ions had a positive effect, while Cu2+ and Fe2+ ions had an inhibitory effect compared to the control. This preliminary study provides basal line information for the discovery of novel microbes from the unexplored natural resources for amylase production, which will be used for many purposes.

DOI: 10.15376/biores.18.1.6-18

Keywords: Streptomyces; Amylase; Fermentation; Nutrient medium

Contact information: a: Department of Biotechnology, Mirpur University of Science & Technology (MUST), Mirpur 10250 (AJK) Pakistan; b: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, P.O. Box 61413, Abha 9088, Saudi Arabia;

* Corresponding authors:;


Amylases are high-value biocatalysts that are used in the starch processing industry for the hydrolysis of starch. Amylases are found in all life domains, but microbial amylases are primarily used in industrial processes because of their easy culturing and economical enzyme production at a massive scale. Other advantages of microbial enzymes include the easy directed manipulation in terms of introducing/enhancing desired characteristics (Sidhu et al. 1997; Li et al. 2007). Amylases have found their applications in starch saccharification, clinical and analytical chemistry, and the food, beverage, and textile industries (Paneley et al. 2007). Because of their prodigious demand and application, interests are increasing in isolating new microbes with amylase production abilities.

Amylase may be generated by a variety of bacteria; however for commercial purposes, amylase is mostly sourced from the genus Bacillus (Pandey et al. 2000). The well-known thermostable amylase producers, Bacillus subtilis, Bacillus licheniformis, Bacillus stearothermophilus, and Bacillus amyloliquefaciens have been extensively used in the manufacture of the enzyme for a variety of commercial purposes (Prakash and Jaiwal 2009).

There has been a continued interest among scientists to discover and gain insight into new bacterial strains (actinobacteria) from diverse environments, such as from deserts and soils in temperate region, for producing this high demanding enzyme (Kurapova et al. 2012; Ding et al. 2013). Multi prospective demands and applications in different sectors pave the way for searching for more useful processes for native amylase production (Hoque et al. 2003). There have been a few reports on amylase production by Streptomyces species from various environments such as brick-kiln soil (Kar and Ray 2008), mushroom-compost (Singh et al. 2011), marine-soil and marine sponge of Ircinia sp. (Krishnan and Sampath Kumar 2015), desert (Nithya et al. 2017), and fertile soil (Al-Dhabi et al. 2020).

Streptomyces is the largest and very important genus from the actinobacteria family and has considerable economic importance. A majority of them are chemotrophic and ubiquitously found in soil and water. Approximately half of the discovered metabolites are produced by this group, e.g., antibiotics, enzymes, enzyme inhibitors, immunosuppressant agents, vitamins, hormones, and many other secondary metabolites (Berdy 2005). However, there are few reports describing the starch hydrolyzing activities from the Streptomyces species (Hoque et al. 2006). Since there are no reports on isolation and characterization of Streptomyces species from the soil of Kotli, Azad Kashmir, Pakistan, the present study has been designed to isolate and screen actinomycetes and determine the amylase activity from Streptomyces found in soil.


Isolation and Selection of Streptomyces

Streptomyces MI-1 was isolated from soil samples collected from different localities of the Kotli Azad Kashmir district. Casein KNO3 agar media was used for the isolation of the strain. The isolated strain was monitored for amylase production on a starch agar plate containing starch as the carbon source. The strain was identified on the basis of 16S rDNA sequence analysis as well as its morphological and biochemical properties, e.g., colony morphology, gram staining, acid fast staining, catalase production, and starch hydrolysis. The polymerase chain (PCR) product was sequenced using an automatic sequencer.

Physical Properties

The temperature tolerance and pH requirements of the selected strain were determined by growing the strain at different temperatures and a wide range of pHs.

Enzyme Production

For the production of amylase, a starch (1%) containing medium was used. The medium (100 mL) was inoculated with 1 mL of fresh overnight culture of Streptomyces MI-2, incubated for 4 d at a temperature of 37 °C in a shaking incubator at 150 rpm. Bacterial cells were removed via centrifuge and the supernatant was used for the amylase activity assay.

Amylase Activity Assay

The amylase activity of the supernatant was assayed by the detection of reducing sugars. The reaction mixture contained 800 μL of 1% soluble starch and 200 μL of enzyme solution. The enzyme reaction was stopped with a dinitrosalicylic acid (DNS) solution. The absorbance was measured at 540 nm in a spectrophotometer, and the activity was calculated from the standard curve. One unit of amylase activity was defined as the amount of enzyme that released 1 μmol of reduced sugar per minute under assay conditions.

Optimization Conditions for Amylase Activity

In order to elucidate the optimum conditions for the alpha amylase activity, assays were performed at different temperature and pH ranges and different concentrations of substrates under standard conditions.

Effect of Metal Ions on the Amylase Activity

Salts of various metal ions, e.g., Mg2+, Ca2+, Hg2+, Cu2+, Fe2+, and Zn2+ (10 mM each) were added to the reaction mixture and then incubated at a temperature of 40 °C for 25 min before performing the activity assay under standard conditions. The enzyme activity was determined as compared to the control without metal ions.


Isolation and Identification of the Starch Hydrolyzing Isolate

In total, 12 isolates were selected based on their appearance on culture plates from the soil sample. All isolates were screened for amylase production. Among all of these, one was found to be the most promising starch degrader. This isolate was designated as MI-1 and was selected for further characterization. After the 3rd day of incubation, white colonies appeared (Fig. 1a) which produced brown pigmentation after the 5th day of incubation (Fig. 1b). Microscopic observation and staining properties of the selected strain (MI-1) revealed that the strain exhibited a filamentous structure (Fig. 1c). The present isolate was found to be gram positive and non-acid fast, which is the most important criteria of the Streptomyces species.

Fig. 1. Colony morphology of the MI-2 isolate on casein-KNO3 agar medium at 28 °C: (A) after 3 d of incubation; (B) after 5 d of incubation; and (C) microscopic view of the MI-2 isolate at 10X magnification

The DNA extracted from Streptomyces MI-1 was run on 0.8% agarose gel for confirmation. The 16S rRNA was amplified through PCR using a Streptomyces specific forward and reverse primer. The amplified PCR product was run on 0.8% agarose gel, and the amplified product (1.5 Kb) was confirmed 16S rDNA amplification. To identify the strain up to the species level, sequencing of the 16S rRNA was performed. A phylogenetic tree was constructed. The length of the sequence was 1470 bp (as shown in Fig. 2).

Fig. 2. The PCR amplified 16S rRNA fragment (1.4 Kb) and its nucleotide sequence

Fig. 3. Phylogenetic tree of the newly isolated Streptomyces strain MI-1

A tree based on the 16SrRNA gene sequences showed that the isolate occupied a new phylogenetic position within the sub cluster. Therefore, MI-1 was determined to belong to the Streptomyces species (Fig. 3).

Screening of the Amylase Activity

The screening of the isolated bacterial strain for amylase activity was performed on a blue color agar plate. Conversion of the blue color to white indicated the hydrolysis of starch in the media. Among the 12 isolates under study, MI-1 exhibited positive starch degrading activity on starch agar plate with a clearing zone of 10 mm ± 0.24 mm (Fig. 4).

Fig. 4. Starch hydrolysis via the MI-1 Streptomyces strain on an agar plate

Cultural Characteristics

The amylase activity positive strain MI-1 was studied for certain morphological parameters from its culture plates. The culture of the selected strain MI-1 on a caesin-KNO3-agar medium and starch-casein-agar medium indicated it as strain of the Streptomyces species (Table 1).

Table 1. Cultural Characteristics of the Selected Isolate MI-1 on Different Media

Effect of the pH and Temperature on the Growth

The effect of changing the media pH and temperature on the growth of the bacterial strain was checked by monitoring the OD of culture at 540 nm. The results showed that the MI-1 strain had a growth pattern between 25 and 40 °C, with maximum growth at a temperature of 35 °C (Fig. 5a). It also exhibited good growth under a wide range of pHs, from 3 to 9, with the maximum growth at a pH of 7 (Fig. 5b). A wide growth range of pHs and temperatures make the bacterial strain and its extracellular enzyme suitable for various industrial processes taking place under these conditions.

Characterization for the Hydrolysis of the Carbon Sources

Different carbon sources, e.g., starch, maltose, glucose, and fructose were added to the nutrient medium from 1% to 5% for the growth optimization of Streptomyces MI-1. The maximum cell density was recorded when 3% starch (Fig. 5c) and glucose (Fig. 5d) were added to the nutrient media. Maltose and fructose were also used as carbon sources in the nutrient media. The maximum growth of MI-1 was recorded with maltose and fructose concentrations of 4% (Fig. 5e) and 5% (Fig. 5f), respectively. These findings indicated that the isolated strain has the ability to hydrolyze a wide range of carbon sources via its hydrolytic enzymes secreted in the media.

Such a bacterial strain could be desirable as a microbial factory for various hydrolytic enzymes, including alpha amylase.

Alpha Amylase Production in the Culture Supernatant

In the culture supernatant, the α-amylase production was measured after every 24 h until 192 h was reached. After 24 h of growth, the α-amylase production was found to be 0.202 IU/mL, which gradually increased, and the maximum production was found to be 2.136 IU/mL after 144 h (Fig. 6).

Fig. 5. Effect of the physical and nutritional parameters on the growth of Streptomyces MI-1

Enzymatic activity decreased after 144 h (day 6), which was possibly due to the depletion of nutrients and the accumulation of wastes as a result of metabolic activities.

Fig. 6. Effect of the fermentation time on the amylase activity of the culture supernatant

Optimization Conditions for Amylase Activity

To find the effect of different physical parameters, e.g., assay temperature, pH, substrate concentration, and metals ions, on the alpha amylase activity, enzyme assays were performed at different values (as shown in Fig. 7).

Fig. 7. Study of the effect of different parameters on the amylase activity for determination of the (a) optimum temperature; (b) optimum pH; (c) effect of substrate concentration; and (d) effect of metal ions

The results showed that the enzyme had good activity in the temperature range of 30 °C to 45 °C, with optimum activity at 40 °C (Fig. 7a). Alpha amylase activity reached zero as the temperature rose from 45 to 50 °C. The optimum pH for the production of alpha amylase by Streptomyces MI-1 was 7 (Fig. 7b). There was zero activity at a pH of 3 and at pH 10, indicating denaturation of the enzyme under extreme acidic and alkaline conditions.

To study the effect of substrate concentration, the enzyme activity was evaluated with 0.01 to 0.1 mg/mL starch solution as substrate. The results showed that there was gradual increase in activity up to 0.04 mg/mL starch, and the graphed line reached a plateau in the range 0.04 to 0.1 mg/mL, indicating saturation of active sites by substrate molecules (Fig. 7c). There was positive effect of magnesium (Mg), calcium (Ca), and no effect of mercury (Hg) metal ions (10 mM conc. of each). There were inhibitory effects shown by zinc (Zn), copper (Cu), and iron (Fe) metals ions on amylase by MI-1 strain compared to the control, i.e. without metal ions (0.29 IU/mL) (Fig. 7d).


As an unexplored ecological niche that may produce novel and unique metabolites, Streptomyces species are found under different environmental conditions (Ball et al. 1989; Sarrouh et al. 2012). The Streptomyces strain was isolated on selective media by applying physical and chemical treatments to soil samples and suitable culture conditions, as suggested by Wachinger et al. (1989) and Hayakawa et al. (2004). The Streptomyces strains can be easily distinguished from other bacteria due to their unique growth pattern, rough and embedded colonies, and color of the substrate, as well as aerial mycelia and diffused pigments on the media (Hayakawa et al. 2004).

The selected isolate was characterized on the basis of the colony morphology on starch casein agar plates and microscopic morphology via the cover slip and slide culture technique (Anderson and Wellington 2001). The criteria for the physiological characterization and utilization of different carbon sources and results of other physiological characteristics, e.g., the effect of temperature and the effect of pH on the growth of Streptomyces, give further confirmation and identification of the selected isolate MI-2 up to the genus level (Shirling and Gottlib 1996; Vijayakumar et al. 2007). After 7 d of incubation, the selected isolate MI-1 produced a brownish colour on the reverse side of the starch casein agar media at a temperature of 28 °C. Similar results have been revealed by Vijayakumar et al. (2007). The selected colony of the MI-1 strain exhibited a leathery and chalky texture, a branching and filamentous substrate, as well as aerial mycelia (Lechevalier 1989). As such, the aerial spore colour and reverse side of the Streptomyces, which are grown on culture media, could vary depending on the nutrients that are provided to the medium.

Microscopic studies and staining properties of the selected isolate MI-1 showed that the species was Gram positive and non-acid fast, which is an important criterion of the Streptomyces species (Oskay et al. 2004). Different biochemical characteristics of Streptomyces bacteria are used for identification (Islam et al. 2014). In the present study, some biochemical tests were performed. Positive results were observed for catalase, casein, and starch hydrolysis in the case of the Streptomyces MI-1 in the present study. Apart from a few, most of the isolates of actinobacteria are efficient in casein and starch hydrolyzing, and all the isolates were found to be positive in the catalase test. Comparatively, Stack et al. (1969) characterized many strains of actinobacteria and none of them were found to be positive for the catalase test. Based on the present and previous studies, it was concluded that the biochemical characteristics of actinobacteria varied and depend on the nutrients that are supplemented into the biochemical media and biochemistry of the microbes. Therefore, this aspect can be potentially used as taxonomic criteria for identification at a genus level.

The present investigation revealed that Streptomyces MI-1 showed its optimum growth at a temperature of 35 °C. Moderate growth of the isolate was recorded from a temperature of 25 to 40 °C, which means that the isolates are mesophilic. Similarly selected Streptomyces species grew well at a pH of 6 to 7 and behaved neutrophilic in the culture. There are different reports categories for amylases. Not all of them are thermotolerant; rather, some are very sensitive, and some bacteria produce thermotolerant amylase which could tolerate higher range of temperature. The one that was isolated in this work did not belong within this class and would potentially fall into a different category, which is thermosensitive. It became denatured at higher temperatures, as can be seen in Fig 7, where the optimum temperature appears to be 40 °C. In the case of pH, the best performance was at 7. Similar findings have been reported by Islam et al. (2014). Growth of Streptomyces MI-2 occurred in all the carbon sources tested in the present study. These findings enhanced the applicability of the isolated strain in various industrial processes. According to the morphological, cultural, physiological, biochemical, and molecular level characterization, the present Streptomyces species, MI-1 is closely related to the better-known species Streptomyces bacillaris.

The plate agar results of Streptomyces MI-1 showed that the strain MI-1 could produce amylolytic enzymes as a transparent zone formed around the bacterial colony (as shown in Fig. 4). This simple method is useful in the preliminary screening of amylase producing bacteria and agreed with previous reports. The maximum amylase activity of Streptomyces MI-1 after the 6th day (144 h) of growth was 2.136 U/mL, which was higher than the amylase activity of Streptomyces griseoflavus P4 on the 7th day (168 h) of growth, which was 1.66 IU/mL, as reported by Julaluck and Hataichanoke (2012) and Kim and Goodfellow (2002). Furthermore, Mahon et al. (1997) and Julaluck and Hataichanoke (2012) stated that the maximum amylase production occurred after the 3rd day (72 h) of incubation, while Wang et al. (2015) similarly reported the highest α-amylase yield occurred after 48 h of incubation. The differences in the production time were attributable to strain variation or a difference in the growth parameters of that strain.

The Streptomyces MI-1 exhibited maximum amylase activity at a temperature of 40 °C temperature. Similar findings have been reported by Julaluck and Hataichanoke (2012) for Streptomyces griseoflavus P4, which showed a maximum amylase activity at a temperature of 40 °C. In the same way, the α-amylase activity of Streptomyces PDS1 reached its maximum at a temperature of 40 °C (Ragunathan and Padhmadas 2013). Many researchers reported the maximum alpha amylase activity in Streptomyces at a temperature range of 40 °C to 60 °C (Fogarty 1983; Ammar et al. 2002; Deutch 2002; Hogue et al. 2006). In the present study, among the substrate concentrations tested, the highest amylase activity was observed at a 4% starch concentration. Beyond 4% starch, the amylase activity did not increase and a further increase in the substrate concentration does not increase the enzyme activity. These results were also in agreement with the previous reports of Oyeleke and Oduwole (2009), who determined the highest amylase activity at 4% starch concentration for Bacillus species.

In the present study, the effects of different metal ions on the enzyme activity were evaluated. The maximum amylase activity in the case of Streptomyces MI-1 was recorded in the presence of Mg2+ followed by Ca2+. These results were in confirmation with the results of Lonsane and Ramesh (1990), who reported the maximum amylase activity with Mg2+, followed by Ca2+. In the present finding, the lowest alpha amylase activity was recorded with Hg2+ and Zn2+, while Cu2+ and Fe2+ showed an inhibitory effect. These results agreed with the report by Gupta et al. (2008), who reported enzyme inactivation with Cu2+. However, they were contrary to the results of Mayzaud (1980), who reported enzyme inactivation with Mg2+ and enhanced activity with Cu2+. The inhibitory effect of some metal may be related to some pH changes, which are associated with their use in the reaction mixture. In the present study, the effect of chelating agent EDTA on the amylase activity was also evaluated. The chelating agent EDTA inhibits the α-amylase activity of Streptomyces MI-1. These results were corroborated with the results of Chakraborty et al. (2009). Thus, the present study has unveiled a locally isolated strain that is capable of producing enzyme and that could potentially find many uses in industrial applications. In principle, protein engineering could open new vistas, including the discovery of interesting domains in the enzyme from the soil-borne bacteria.


  1. The current investigation showed promise for exploitation of soil for the discovery of new species that could potentially be major source for new enzymes to be used in industry.
  2. Under optimized conditions, Streptomyces MI-1 yielded a considerable amount of catalytically active α-amylase and that could provide option of a relatively cheaper indigenous source for amylase production.
  3. For the first time, the present work here reports on the alpha amylase production of a Streptomyces strain from the lands of Kotli Azad Kashmir.
  4. Moreover, this study also revealed the values as well as the microbial wealth of amylase producing bacteria which can be helpful for the development of biotechnological processes, especially at industrial level.


The authors extend their appreciation to the Ministry of Education in KSA for funding this research work through the project number IFP-KKU-2020/14.


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Article submitted: May 16, 2022; Peer review completed: August 11, 2022; Revised version received: October 24, 2022; Accepted: October 25, 2022; Published: October 31, 2022.

DOI: 10.15376/biores.18.1.6-18