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Isolation, phylogeny and characterization of new [alpha]-amylase producing thermophilic Bacillus sp. from the Jazan region, Saudi Arabia.


Amylases constitute one of the most important groups of industrial enzymes and account for nearly 25% of the total sale of enzymes [1]. They are used in many commercial biotechnological processes including starch degradation, detergent, foodstuff, pharmaceutical, textile, and paper manufacturing [1]. In addition, they play an important role in the biogeochemical cycle of carbon [2]. The amylolytic enzymes can be divided into three groups: [alpha]-amylase, [beta]-amylase, and glucoamylase, which are capable of hydrolyzing starch and glycogen

Amylases are produced by a variety of microorganisms such as bacteria and fungi [3]. Although there are many microbial sources available for producing amylases, only a few such as Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquifaciens are recognized as commercial producers [4]. The capacity of Bacillus strains to produce large quantities of enzymes has placed them among the most important industrial enzyme producers. Indeed, they produce about 60% of commercially available enzymes [1].

Improvement of the yield of [alpha]-amylase and consequent cost reductions depend on the selection of strains, the optimization of the factors affecting biosynthesis, genetic improvements, kinetic studies and the biochemical characterization of the enzyme. Each application of [alpha]-amylase requires unique properties with respect to specificity and stability [5]. The advantages of using thermostable [alpha]-amylase in the starch processing industries include the reduced cost of external cooling, a better solubility of substrates and a lower viscosity, allowing accelerated mixing and pumping [6]. Therefore, there is ongoing interest in the isolation of thermophilic Bacillus strains with higher [alpha]-amylase activity and stability. The purpose of the current investigation was to screen thermophilic Bacillus species isolated from soil in order to study their suitability with regard to [alpha]-amylase production.

Materials and Methods

Isolation and screening of [alpha]-amylase producing Bacillus strains

This study was conducted in October, 2008. Bacillus species were isolated from soil samples collected from the Abha, Regal Almaa and Jazan regions, all located in the south-west of Saudi Arabia. To select an aerobic, rod shaped, gram-positive, thermophilic and spore-forming Bacillus sp., soil samples were incubated at 80[degrees]C for 10 min and placed onto nutrient agar (Difco) plates containing 1% starch [7]. The plates were incubated at 45[degrees]C and examined after 24-48 h. [alpha]-amylase producing colonies were selected by flooding the plates with iodine solution (1% iodine in 2% potassium iodide w/v). The clearance zone-forming ability on starch nutrient agar plates was used for the primary selection of the isolates [1, 4]. The bacterial isolates were screened for their [alpha]-amylase productivity after 48 h in a growth medium containing 1% starch using an incubator shaker at 45[degrees]C and 150 rpm. The cultures were maintained on nutrient agar slants at 4[degrees]C.

DNA Extraction and 16S Ribosomal RNA -PCR Analysis

Bacterial DNA isolates was extracted from 5 ml bacterial cultures grown overnight using QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA) with some modification [8]. Briefly the pellet was suspended in 180 [micro]l of (20 mg/ml lysozyme in 20 mM Tris-HCl, pH 8.0; 2 mM EDTA; 1.2% Triton), followed by incubation for 30 min at 37[degrees]C. Twenty [micro]l of Proteinase K was then mixed by vortexing, and was then incubated at 56[degrees]C for 30 min. The genes encoding the small-subunit rRNA were amplified through the application of primers targeted to universally variable regions [9]. The oligonucleotide primers have the following sequences: 1F (5"AACTGGAGGAAGGTGGGGAT-3") and 1R (5-AGGAGGTGATCCAACCGCA3") were used to amplify bacterial 16S rRNA. The PCR was initiated by incubating the reaction mixture at 95[degrees]C for 3 min, followed by 30 cycles of 50 sec at 95[degrees]C; 1 min at 50[degrees]C; and 2 min at 72[degrees]C. The reaction was terminated with an extension step consisting of 10 min incubation at 72[degrees]C. The PCR products were analyzed on 1.0% agarose gel containing 0.5 [micro]g [ml.sup.-1] ethidium bromide and visualized by BioRad Gel Documentation System 2000.

16S rDNA sequence analysis

The 16S rRNA gene PCR product amplified from genomic DNA isolated from pure bacterial colonies, was sequenced by MACROGENE, Korea. Homology of the 16S rRNA gene sequence of the isolates with reference to 16S rRNA sequences was analyzed using the BLAST algorithm in GenBank ( Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 [10]. Only the highest-scored BLAST result was considered for phylotype identification.

Growth medium and optimization of culture conditions

The medium employed for growth and enzyme production consisted of (%): 0.1 K[H.sub.2]P[O.sub.4], 0.25 [Na.sub.2]HP[O.sub.4], 0.1 NaCl, 0.2 [(N[H.sub.4]).sub.2]S[O.sub.4], 0.005 MgS[O.sub.4].7[H.sub.2]O, 0.005 Ca[Cl.sub.2], 0.2 tryptone, and 1% soluble starch [11]. The initial pH of the medium was adjusted to 7.0. The inoculum was prepared by growing the bacterial culture in the nutrient broth medium at 45[degrees]C for 24 h. The average cell concentration was adjusted to 0.3 optical density (OD) at 600 nm. Erlenmeyer flasks (250 ml) containin6g 50 ml of growth medium were inoculated with 1% inoculum (approximately 2x[10.sup.6] CFU [ml.sup.-1]) and incubated at 45[degrees]C in an incubator shaker at 150 rpm for 4 d [12]. At constant intervals (12 h), the samples were harvested and the growth was determined by the measurement of optical density ([OD.sub.600nm]). Cells were removed by centrifugation (at 6,000 rpm for 20 min) and supernatants were used for enzyme assay and characterization studies. The factors such as incubation period, pH, temperature, carbon sources 1% [13] and carbon concentration were investigated.

Enzyme assay

[alpha]-amylase was assayed by adding 1 ml of enzyme to 1 ml soluble starch (1%) in an acetate buffer pH 5, and incubated at 50[degrees]C for 15 min. The reaction was stopped by the addition of 2 ml of 3,5-dinitrosalicylic acid reagent [14]. The absorbance was measured using a double beam UV/VIS scanning spectrophotometer (Model: Shimadzu, 1601PC) at 550 nm. One enzyme unit (U ml-1) is defined as the amount of enzyme which releases 1 [micro]mole glucose.

Effect of temperature on the activity and stability

The optimum temperature of the enzyme was evaluated by assaying its activity between 40[degrees]C and 100[degrees]C for 15 min. Thermal stability was investigated by measuring the residual activity after 60 min of pre-incubation at temperatures ranging from 40[degrees]C to 100[degrees]C [11].

Statistical analysis

Parametric testing was performed and the analysis of variance was then used to compare data between different treatments. All analyses were performed at p [less than or equal to] 0.05 using MINITAB, version 13.1.

Results and Discussion

Isolation and screening of [alpha]-amylase producing Bacillus strains

A total of 20 thermophilic, gram-positive and spore-forming bacteria Bacillus sp., capable of producing clear zones on starch nutrient agar plates were obtained from soil samples collected from three regions of Saudi Arabia. Isolates A (1-5) were obtained from Abha, isolates J (1-7) from Jazan and isolates R (1-8) from Regal Almaa. The existence of isolates in the different localities could be affected by the prevailing environmental conditions and nature of the soil. The bacterial isolates were screened for their [alpha]-amylase productivity in a growth medium containing 1% starch (Fig. 1). Although all the isolates were able to produce [alpha]-amylase enzyme, only five strains were found to produce high activities of [alpha]-amylase (J5, J2, R7, A3, and R2). From the morphological and physiological characteristics, isolates were identified as Bacillus sp., according to the description given in Bergys Manual of Systematic Bacteriology [15]. Strain J5 isolated from the Jazan region was selected for further study because it showed the highest [alpha]-amylase productivity (23.7U [ml.sup.-1]).


DNA sequence for 16S rRNA gene

Amplicons of 16S rRNA gene (350-bp) for the isolate J5 was subjected to DNA sequencing and the sequence was analyzed using DNA Blast. A phylogenetic tree based on bacterial 16S rDNA sequence showed a close relationship between the isolate J5 and the genus Bacillus (Fig. 2). The isolate 16S rRNA gene alignment with Blastn analysis showed that isolate J5 had the highest homology (81%) with Bacillus subtilis strain EM11 (accretion number, EU571103). It has been proposed that sequence similarity must be below 95% to qualify as evidence of a novel species [16], suggesting that the new isolate may be another strain of Bacillus sp. Fox et al. [17] suggested that Bacillus species whose 16s rRNAs have been sequenced typically differ from one another in many more positions than the B. psychrophilus, B. globisporus pair, whose level of sequence similarity is 99.8%. Indeed, among these Bacillus strains the average species-species pair shares only 88.7% sequence similarity, while the most similar pair (Bacillus rnacerans and Bacillus alvei) exhibits only 94.5% sequence similarity. Therefore, the strain isolated in this study is considered as new specie and named Bacillus sp. AMRI-01.


Effect of time course on [alpha]-amylase production

The activity of [alpha]-amylase was detected after 12 h of incubation, with maximum production (23.7U [ml.sup.-1]) at 48 h (Fig. 3). Extension beyond the optimum time course was generally accompanied by a decrease in the growth rate and enzyme productivity, which gradually declined to 6.8U [ml.sup.-1] after 96 h of incubation. The decreased activity in the later phase of growth was probably due to catabolite repression by glucose released from starch hydrolysis. It can also be observed that the maximum [alpha]-amylase production occurred when cell growth reached the peak at their late exponential and early stationary phases of growth. Similar observations were reported by Asgher et al. [11]. They reported that the effective production of [alpha]-amylase may not occur until the stationary phase has been reached, and the readily available carbon source has been depleted. The short incubation period for Bacillus sp. compared with other bacteria and fungi offers the potential for inexpensive enzyme production [14]. For example, maximum enzyme production was obtained after 24h for Bacillus flavothermus [18] and 72h for Bacillus amyloliquefaciens [12].


Effect of pH on [alpha]-amylase production

The pH of the growth medium plays an important role in terms of inducing enzyme production and morphological changes in the microbes [19]. The production of [alpha]-amylase was investigated at different pH values ranging from 5.0 to 10.0. The results presented in Fig. (4) show that pH 8 was the optimum pH value for [alpha]-amylase production and bacterial growth. The enzyme production at the optimum pH value increased up to 31.3% compared with the production of the enzyme at pH 7. Amoozegar et al. [20] observed the same results in that the maximum [alpha]-amylase production was obtained between pH 7.5 and 8.5. Although Horikoshi [21] reported that Bacillus No.A-40-2 produced a maximum [alpha]-amylase value at pH 10, Asgher et al. [11] found that Bacillus subtilus JS-2004 has the ability to produce an optimum amylase value at pH 7.


Effect of temperature on [alpha]-amylase production

The results illustrated in Fig. (5) show a positive correlation between the growth/enzyme production and the incubation temperature up to 45[degrees]C, followed by a gradual decrease. The effect of temperature on enzyme production was related to the bacterial growth. These results are in accord with the findings of Asgher et al. [11]. In contrast, Saxena et al. [4] reported the maximum enzyme production by Bacillus sp. at 60[degrees]C, whereas Konsula and Liakopoulou-Kyriakides [5] showed that a thermophilic Bacillus subtilis produced the highest [alpha]-amylase production at 40 [degrees]C.


Effect of carbon sources on [alpha]-amylase production

A number of carbon sources (1% w/v) were tested in order to determine their effect on growth and [alpha]-amylase production. The results suggest that [alpha]-amylase was an inducible enzyme and generally induced in the presence of carbon sources (Fig. 6). Potato starch (29.1U [ml.sup.-1]) and corn starch (28.5U ml-1) were the best inducers for [alpha]-amylase production followed by maltose (23.0U ml-1). Growth and enzyme production did not alter when potato and corn starch were used as carbon sources. The same findings were reported by Oliveira et al. [3]. They mentioned that the induction of [alpha]-amylase requires substrates having [alpha]-1,4 glucoside bond, including starch and maltose. Furthermore, the results obtained indicated that glucose, mannitol, cellobiose and lactose repressed [alpha]-amylase production (Fig. 5). The biosynthesis of [alpha]-amylase in most species of the genus Bacillus is repressed by readily metabolizable substrates, especially glucose, by a mechanism of catabolite repression [22]. This repression is mediated by the protein encoded by the CreA gene as mentioned by Kato et al. [23]. On the other hand, Sarikaya and Gurgun [12] showed that the highest [alpha]-amylase yield was obtained by the addition of Na-citrate and sucrose for the strains of Bacillus subtilis and Bacillus amyloliquefaciens, respectively.


Effect of starch concentration on [alpha]-amylase production

Different concentrations of starch were used to elucidate the best concentration for maximum [alpha]-amylase production. The results illustrated in Fig. (7) reveal that the enzyme production increased in proportion to starch concentrations, giving the maximum amylase production (48.7U [ml.sup.-1]) at 3% of starch concentration. The enzyme production was increased 1.68 fold when starch concentration increased to 3% compared with the production at 1%. However, the [alpha]-amylase production rapidly declined when the starch concentrations increased beyond 3% (Fig. 7). The reduction in enzyme productivity at a high substrate concentration is more likely due to the high viscosity of the medium affecting the availability of oxygen concentration required for the microbial growth. This observation was in harmony with the findings of Agger et al. [24].


Effect of temperature on [alpha]-amylase activity and stability

The results illustrated in Fig. (8) indicate that the optimum temperature for [alpha]-amylase activity was 60[degrees]C, which maintained about 96.6% of the maximal enzyme activity at 70[degrees]C. The enzyme sharply lost about 44.6% of its activity at 100[degrees]C. Similarly, [alpha]-amylase produced by Bacillus megaterium and Bacillus sp. PN5 exhibited an optimum activity at 60[degrees]C [25]. Moreover, the highest [alpha]-amylase activity produced by Bacillus sp. TS-23 and Bacillus subtilis JS-2004 was achieved at 70[degrees]C [11, 22]. On the other hand, Mamo and Gessesse [26] reported that optimal temperature for a maximum [alpha]-amylase production by Bacillus sp, WN11 was at 75-80[degrees]C.

Thermal stability was also investigated by measuring the residual activity after 60 min of pre-incubation at different temperatures ranging from 40[degrees]C to 100[degrees]C. The enzyme was completely active up to 80[degrees]C, while 95.5% and 88.7% of the original activities remained after pre-incubation at 90 and 100[degrees]C, respectively. It was reported that the 100% enzyme activity was retained at 90[degrees]C for 1 h [27]. Furthermore, Asgher et al. [11] found that the [alpha]-amylase was highly stable for 1 h at 60[degrees]C and 70[degrees]C, while at 80[degrees]C and 90[degrees]C, 12% and 48% of the original activities were lost, respectively. The stability of the enzyme could be due to its genetic adaptability to carry out its biological activities at a higher temperature. This thermo stability is an important factor for the use of amylolytic enzymes in starch-processing industries. Therefore, the enzyme produced in this study is quite suitable for the liquefaction of starch.



Based upon the 16S rDNA sequence data, isolate J5 is considered as a new species of Bacillus and therefore named Bacillus sp. AMRI-01. The results of this investigation demonstrate that Bacillus sp. AMRI-01 is a potential source of thermophilic [alpha]-amylase production. The temperature properties for enzyme activity and stability make the enzyme quite suitable for biotechnological applications, especially in starch liquefaction.

Accession: The GenBank accession number for the 16S rDNA sequence of the strain J5, Bacillus sp. AMRI-01 is GU271225."


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Saad A. Alamri

Department of Biology, College of Science, King Khalid University, P.O. Box 10255, Abha 61321, Saudi Arabia

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Author:Alamri, Saad A.
Publication:International Journal of Biotechnology & Biochemistry
Date:Oct 1, 2010
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