{"id":28497,"date":"2019-09-25T11:52:35","date_gmt":"2019-09-25T11:52:35","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=28497"},"modified":"2020-04-22T08:24:34","modified_gmt":"2020-04-22T08:24:34","slug":"isolation-and-identification-of-amylase-producing-bacteria-from-soil-in-nasinuan-community-forest-maha-sarakham-thailand","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol12no3\/isolation-and-identification-of-amylase-producing-bacteria-from-soil-in-nasinuan-community-forest-maha-sarakham-thailand\/","title":{"rendered":"Isolation and Identification of Amylase-producing Bacteria from Soil in Nasinuan Community Forest, Maha Sarakham, Thailand"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

The starch hydrolytic amylases (\u03b1-amylase, \u03b2-amylase and glucoamylase) are ones of the most widely used enzymes in present-day biotechnology. Glucoamylases (1,4-\u03b1-D-glucan glucanohydrolase; EC 3.2.1.3) or amyloglucosidases, are exo-acting amylases that produce glucose from the non-reducing end of starch and corresponding oligosaccharides. Glucoamylases are implemented commercially for the transformation of malto-oligosaccharides into glucose1<\/sup>. Although amylases are produced from different sources (microorganisms, plants and animals), amylases from microbes are most suitable for industrial production due to microbial short growth period, low cost effective production, eco-friendly behavior, less handling issues for workers, productivity2<\/sup> and easy manipulation of bacterial genes3<\/sup>. Bacteria and fungi tend to secrete amylases outside the cells to perform extra cellular digestion of starch into sugars. Increasing industrial demand for microbial amylases has been observed due to their specificity of reaction, mild conditions prerequisite for the reaction, and less energy consumption than the conventional non-enzymatic chemical methods.<\/p>\n

Extensive application of amylase in food, starch liquefaction, saccharification, brewing, detergent, paper, textile and distilling industries, has brought about a greater attention for the increase in the indigenous production of \u03b1-amylase4<\/sup>. Bacillus <\/em>is a common bacterial source for industrial amylase production. However, different strains have different optimal growth conditions and enzymatic production profile. Reportedly,\u00a0Bacillus<\/em>\u00a0strains have been extensively used industrially to produce \u03b1-amylase including\u00a0B. amyloliquefaciens<\/em>, B. subtilis<\/em>5<\/sup>, B. licheniformis<\/em>6<\/sup>,\u00a0B.<\/em> stearothermophilus<\/em>7<\/sup>, B. megaterium<\/em>8<\/sup> and\u00a0B. circulans<\/em>9<\/sup>.<\/p>\n

To date, a number of amylase-producing bacteria has been documented, however, no study on amylase-producing bacteria from soil in the Nasinuan Community Forest, Kantharawichai District, Mahasarakham Province, Thailand has been reported. This forest seems to be rich in microbial biodiversity that can be useful in the production of industrial enzymes including amylase. This is the first report to identify amylase-producing bacteria isolated from Nasinuan Forest. These bacterial amylases can be locally applied to various industries including pulp and paper, textile, bioethanol, brewery, food processing, animal feed, and agriculture in Thailand in the future.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Soil samples\u00a0<\/strong><\/p>\n

Soil samples were randomly collected below the soil surface 15 cm and kept in polystyrene bags from Nasinuan Community Forest, Kantarawichai District, Mahasarakham Province, Thailand (area of 9.6 hectare; coordinate of 16.340941, 103.210799).<\/p>\n

Isolation of amylase-producing bacteria\u00a0<\/strong><\/p>\n

Soil sample (10 g) was suspended in 90 mL of sterile 0.85% NaCl solution. The suspensions (100 \u00b5L) of serial dilutions were spread on starch agar (g\/L); 3.0 beef extract, 10.0 soluble starch, 15.0 agar pH 7.0 and incubated at 37 \u00baC for 7 days. Next, the plates were flooded with Gram\u2019s iodine. Any colonies with formation of clear zone around the colonies were subcultured in liquid broth and streaked at least five times to obtain pure isolates as confirmed by Gram staining and 1000X light microscopic observation. The pure isolates were point inoculated on starch agar and incubated at 37\u00b0C for 7 days. The diameters of the clear zones over the diameters of the colonies were measured using a ruler as the Halo : Colony ratio.<\/p>\n

16S rRNA gene sequencing and phylogenetic analysis<\/strong><\/p>\n

Pure bacterial isolates were identified using genomic DNAs obtained from the above method and universal primers: forward primer 27F 5′-GAGAGTTTGATYCTGGCTCAG-3′ and reverse primer 1492R 5’AAGGAGGTGATCCARCCGCA -3′. In 25 \u00b5L PCR mixture, it was composed of genomic DNA 0.5 ng, 2X Master Mix (One PCR) of 100 mM Tris-HCl (pH 9.1), 0.1% TritonTMX-100, 200 mM dNTP, 1.5 mM MgCl2<\/sub>, 0.005 U Taq DNA Polymerase and 0.2 \u00b5M forward and reverse primer with volume adjustment with nuclease-free water. PCR thermocycler (Thermo Scientific Hybaid Px2) was programmed as follows: (1) initial denaturation for 2 min at 94 \u00baC for 1 cycle; (2) denaturation at 94 \u00baC for 45 s; annealing at 54 \u00baC for 45 s, and extension at 72 \u00baC for 1 min for 32 cycles; (3) final extension at 72 \u00baC for 7 min. Samples were held at 4 \u00baC till further analysis. The PCR products of 16S rRNAs (~ 1,500 bp) were detected on 0.8% agarose gel, purified using the PCR product purification kit (Vivantis, Malaysia), sent to First Base Co. Ltd. (Malaysia) for DNA sequencing. The 16S rRNA gene sequences were then compared with others available in GenBank using BLASTN program (Basic Local Alignment Search Tools)10<\/sup>. The Phylogenetic tree was constructed using Muscle method for sequence alignment and maximum likelihood method using MEGA X with 1,000 replicates of bootstrap values11<\/sup>. All 16S rRNA partial sequences of our amylase-producing isolates were deposited on NCBI database.<\/p>\n

Determination of amylase enzyme activity<\/strong><\/p>\n

The method followed Bhaskara et al. (2011)12<\/sup><\/strong>. The three bacteria with the highest Halo: Colony ratios were chosen for determination of amylase enzyme activity. Each isolate was subcultured in starch liquid broth (g\/L): 10.0 soluble starch, 10.0 peptone, 20.0 yeast extract, 0.05 KH2<\/sub>PO4<\/sub>, 0.015 MnCl2<\/sub>.4H2<\/sub>O, 0.25 MgSO4<\/sub>.7H2<\/sub>O, 0.05 CaCl2<\/sub>.2H2<\/sub>O, 0.01 FeSO4<\/sub>.7H2<\/sub>O and incubated at 37 \u00baC, 150 rpm for 1, 2 and 3 days. The clear supernatant (crude extracellular amylase enzyme) was obtained after centrifugation at 10,000g for 15 min at 4 \u00baC. The crude extract was concentrated using MWCO 10 kDa ultracentrifuge protein concentrator (Vivaspin, Sartorius, UK).<\/p>\n

The DNS method13<\/sup><\/strong> was used to determine the amylase activity of each bacterial isolate at each enzyme induction days. One mL of crude enzyme was mixed with 1 mL of 1% starch solution in 1 mL 0.05 M sodium phosphate buffer pH 7.0. The samples were incubated at 37\u00b0C for 30 min. After incubation, 0.5 mL DNS solution was added to each sample to stop the reaction and then boiled at 100\u00b0C for 5 min in the water bath. The colour intensity of the solution was observed by measuring the optical density (OD) using a spectrophotometer at 575 nm. The reading was compared to a prepared blank solution (without crude enzyme). The OD values of samples at T30min<\/sub> were subtracted from those of samples at T0min<\/sub> since glucose still remained in the T0min<\/sub> samples after enzyme induction process at 1, 2, 3 days. The process was carried out in triplicates. The concentration of glucose produced for each solution was obtained from the glucose standard curve. The activity of amylase was calculated. One unit of amylase activity is defined as the amount of amylase required to catalyze the formation of reducing sugar which is equal to 1 \u00b5mol of glucose per min under assay conditions14<\/sup>.
\nThe crude enzyme of the bacterial isolate having the highest activity was chosen for further work.<\/p>\n

In order to determine the specific enzyme activity of the selected isolates, the Folin-Lowry method for total protein estimation was used15<\/sup>.\u00a0 The specific enzyme activity was measured using the following formula.<\/p>\n

Specific activity (U\/mg) = Enzyme activity (U\/ml)\/Extracellular protein concentration (mg\/ml)<\/p>\n

Optimal pH and temperature of amylase enzyme activity<\/strong><\/p>\n

Soluble starch solutions (1%) in different pH from 3 to 10 were tested. One mL of different 1% substrate solution was added along with 1 mL of the respective buffers; 0.05 M citrate buffer (pH 3 to 5), 0.05 M sodium phosphate buffer (pH 6 and 7), 0.05 M Tris-HCl (pH 8 and 9) and 0.05 M glycine NaOH (pH 10). One mL of crude enzyme was added to these buffers as well. The samples were incubated at 37\u00b0C for 30 min. The specific amylase activity was calculated. The pH at which the highest activity was observed was noted. Likewise, different substrate solutions were made by dissolving 1% soluble starch in pH 7.0 solutions One mL of 1% soluble starch was added along with 1 mL of 0.05 M sodium phosphate buffer (pH 7). One mL of crude enzyme was added to the buffers as well. The samples were incubated at 20, 30, 40, 50, 60, 70, and 80\u00b0C for 30 min. The specific amylase activity was calculated. The temperature at which the highest activity was observed was noted. Both optimal pH and temperature were used to determine the final specific amylase enzyme activity.<\/p>\n

Statistical analysis<\/strong><\/p>\n

One-Way Analysis Of Variance (One-way ANOVA) was used with Duncan Multiple Range\u2019s Test on SPSS Statistics Ver. 17.0. Results were expressed as means \u00b1 SD with statistical difference when p < 0.05.<\/p>\n

Results<\/strong><\/p>\n

Isolation of amylase-producing bacteria<\/strong><\/p>\n

In this study, 13 amylase-positive isolates showed clear zones on 1% starch agar with different Halo : Colony ratios. The colonies showing clear zones of iodine solution were taken as positive starch-degrading bacterial colonies. All bacterial isolates showed similar colony morphologies and appeared as Gram-positive and rod-shaped bacteria (Table 1). The result showed Halo : Colony ratios ranging from 1.18 to 1.71 (Table 1). Bacterial isolates were then used to determine the optimal amylase enzyme induction duration and only the 3 isolates with highest specific activities were shown in 3.4.<\/p>\n

Strain identification of amylase-producing bacteria<\/strong><\/p>\n

All 13 amylase-positive bacterial strains were subjected to 16S rRNA gene sequencing for strain identification. The BLAST results displayed that all amylase-positive isolates belong to the genus Bacillus<\/em>, except for one isolate belonging to Enterobacter<\/em> (Table 2). Their closest relatives were found in India, China, Korea, Indonesia, Argentina, Italy, Israel, USA, Argentina and South Africa with a range of 96-100% identity.<\/p>\n

Phylogenetic analysis<\/strong><\/p>\n

The phylogenetic tree of 13 amylase-positive bacterial strains and 2 reference strains with putative amylase enzymes using MEGA 7.0 showed that E. <\/em>cloacae 2.1AL2 was evolutionarily different from the other Bacillus <\/em>strains (Fig. 1). B. thuringiensis<\/em> serovar konkukian (NCBI accession no. AB617494.1) reference strain evolved differently to our Bacillus <\/em>bacteria. However, another reference strain B. cereus <\/em>ATCC 14579 (NCBI accession no. MG708176.1) isolated from safflower leaf, Eqypt showed similar evolution to our bacteria (Fig. 1).<\/p>\n\n\n\n
\"Table<\/td>\nTable 1: Characteristics and Halo : Colony ratios of 13 amylase-producing bacterial strains<\/strong><\/p>\n

Click here to View table<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Optimal amylase enzyme induction duration<\/strong><\/p>\n

It was shown that the incubation duration that induced amylase production with the highest specific activity was 2-3 days among all 3 isolates without statistic differences. Bacillus<\/em> sp. 3.5AL2 seemed to produce the highest specific activity at 0.91 \u00b1 0.12 U\/mg at day 2 at 37\u00b0C (Table 3). Thus, this strain and this condition was used for inducing amylase production for further analysis.<\/p>\n

Optimal pH and temperature of amylase enzyme activity<\/strong><\/p>\n

Bacillus sp. 3.5AL2 showed the highest specific activity at pH 7.0 when 37\u00b0C was fixed and at 60\u00b0C when pH 7.0 was fixed (Fig. 2). Thus, both optimal conditions were used to determine specific amylase activity and 1.97 \u00b1 0.41 U\/mg (Table 4) was obtained.<\/p>\n

Table 2: Thirteen amylase-positive bacterial strains identified by 16S rRNA analysis<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Isolate<\/strong><\/td>\nAccession<\/strong><\/p>\n

no. a<\/sup><\/strong><\/td>\n

Closest relativeb<\/sup><\/strong><\/td>\nAccession no.c<\/sup><\/strong><\/td>\n% Identityd<\/sup><\/strong><\/td>\nOrigine<\/sup><\/strong><\/td>\n<\/tr>\n
1.1AL1<\/strong><\/td>\nMK578206<\/td>\nBacillus pseudomycoides<\/em> FJAT-hcl-17<\/td>\nKY653098.1<\/td>\n99%<\/td>\nHost Paris polyphylla <\/em>var. chinensis<\/em>, China<\/td>\n<\/tr>\n
1.1AL2<\/strong><\/td>\nMK578207<\/td>\nBacillus<\/em> sp. 188Cu-As<\/td>\nKM349197.1<\/td>\n99%<\/td>\nBiofilms, Argentina<\/td>\n<\/tr>\n
1.2AL3<\/strong><\/td>\nMK578208<\/td>\nBacillus anthracis<\/em> SAK4<\/td>\nMG706137.1<\/td>\n100%<\/td>\nSoil, Republic of Korea<\/td>\n<\/tr>\n
1.4AL1<\/strong><\/td>\nMK578209<\/td>\nBacillus <\/em>sp. CNJ732 PL04<\/td>\nDQ448749.1<\/td>\n99%<\/td>\nMarine sediment, USA<\/td>\n<\/tr>\n
1.4AL3<\/strong><\/td>\nMK578210<\/td>\nBacillus cereus <\/em>B19<\/td>\nMK229038.1<\/td>\n99%<\/td>\nWheat grain, Isarael<\/td>\n<\/tr>\n
2.1AL2<\/strong><\/td>\nMK578211<\/td>\nEnterobacter cloacae <\/em>HNXY160623<\/td>\nKX431213.1<\/td>\n98%<\/td>\nAvian embryo, China<\/td>\n<\/tr>\n
2.3AL1<\/strong><\/td>\nMK578212<\/td>\nBacillus thuringiensis<\/em> BD17-E12<\/td>\nHF584771.1<\/td>\n99%<\/td>\nGrapevine root system, Italy<\/td>\n<\/tr>\n
2.3AL8<\/strong><\/td>\nMK578213<\/td>\nBacillus cereus<\/em> DFT-1<\/td>\nKY750685.1<\/td>\n98%<\/td>\nSeawater of industrial area, Indonesia<\/td>\n<\/tr>\n
2.3AL9<\/strong><\/td>\nMK578214<\/td>\nBacillus <\/em>sp. PTP1<\/td>\nKY910137.1<\/td>\n96%<\/td>\nPapaya mealybug gut, India<\/td>\n<\/tr>\n
2.4AL2<\/strong><\/td>\nMK578215<\/td>\nBacillus pseudomycoides<\/em> 74<\/td>\nMH910178.1<\/td>\n99%<\/td>\nIndia<\/td>\n<\/tr>\n
3.2AL1<\/strong><\/td>\nMK578216<\/td>\nBacillus thuringiensis <\/em><\/p>\n

IARI-IIWP-38<\/td>\n

KF054891.1<\/td>\n99%<\/td>\nWheat rhizospere, India<\/td>\n<\/tr>\n
3.4AL1<\/strong><\/td>\nMK578217<\/td>\nBacillus cereus <\/em>F4a<\/td>\nMK088302.1<\/td>\n99%<\/td>\nTea rhizosphere soil, India<\/td>\n<\/tr>\n
3.5AL2<\/strong><\/td>\nMK578218<\/td>\nBacillus cereus <\/em>SP1-AB4<\/td>\nMH013307.1<\/td>\n99%<\/td>\nMarine sponge, South Africa<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

a<\/sup>GenBank accession no. of our strains deposited on NCBI website (http:\/\/www.ncbi.nlm.nih.gov\/pubmed)<\/p>\n

b<\/sup>Closest species with highest % identity and highest Max score on BLAST search<\/p>\n

c<\/sup>GenBank accession no. of closest relative strains on NCBI website<\/p>\n

d<\/sup>Based on BLAST search results, identity (%) of strains compared to the
\nclosest relatives.<\/p>\n

e<\/sup>Based on BLAST search results, origin of the closest relatives.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1: Phylogenetic tree of 13 amylase-positive bacterial strains and 2 Bacillus<\/em> reference strains<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 2: Optimal pH (A) and temperature (B) of amylase enzyme activity from Bacillus<\/em> sp. 3.5AL2<\/strong><\/p>\n

\u00a0Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Table 3:\u00a0Optimal amylase enzyme induction duration<\/strong><\/p>\n\n\n\n\n\n\n\n
\u00a0<\/strong><\/p>\n

Isolate<\/strong><\/td>\n

Specific amylase activity<\/strong><\/p>\n

(U\/mg protein)<\/strong><\/td>\n<\/tr>\n

24 h<\/strong><\/td>\n48 h<\/strong><\/td>\n72 h<\/strong><\/td>\n<\/tr>\n
Bacillus <\/em>sp. 1.1AL2<\/td>\n0.21 \u00b1 0.04Ab<\/sup><\/td>\n0.40 \u00b1 0.04Ca<\/sup><\/td>\n0.41 \u00b1 0.04Ba<\/sup><\/td>\n<\/tr>\n
B. cereus<\/em> 2.3AL8<\/td>\n0.45 \u00b1 0.06Ab<\/sup><\/td>\n0.62 \u00b1 0.04Ba<\/sup><\/td>\n0.65 \u00b1 0.06Aa<\/sup><\/td>\n<\/tr>\n
Bacillus<\/em> sp. 3.5AL2<\/td>\n0.32 \u00b1 0.11Ab<\/sup><\/td>\n0.91 \u00b1 0.12Aa<\/sup><\/td>\n0.86 \u00b1 0.11Aa<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are means \u00b1 SD in triplicate.<\/p>\n

Capital and small letters indicate statistic differences (p < 0.05) in the column and row, respectively.<\/p>\n

Table 4:\u00a0Specific amylase enzyme activity at optimal pH and temperature<\/strong><\/p>\n\n\n\n\n
Strain<\/strong><\/td>\nActivity (U\/mg)<\/strong><\/td>\nOptimal Temp. (\u00b0C)<\/strong><\/td>\nOptimal pH<\/strong><\/td>\n<\/tr>\n
Bacillus<\/em> sp. 3.5AL2<\/td>\n1.97 \u00b1 0.41<\/td>\n60<\/td>\n7.0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n


\nDiscussion<\/strong><\/p>\n

Thai customs (2017) data showed that Thailand has imported industrial enzymes from other countries with the value of 2,625 million Bahts in 2016 and the import rate was increased by 9.40% per year. One of the most widely used enzymes in Thailand, amylase, has been applied in garments, textile and food industries. However, there is a lack of local production of commercial amylase and thus this work aimed to isolate soil bacteria from local forest with the capacity to produce amylase for industrial uses.<\/p>\n

Starch-rich residues may be a better potential source where amylase-positive bacteria can be isolated16<\/sup>. In addition, these could be isolated from places such as cassava farms, soil, and processing flour factories17<\/sup>. In this work, 13 amylase-producing bacteria were isolated and identified as Bacillus<\/em> spp. and Enterobacter<\/em> sp. which is similar to the previous reports18, 19, 20<\/sup> \u00a0where Bacillus<\/em> spp. were mostly found as amylase producers.<\/p>\n

Our results showed that Bacillus<\/em> sp. 3.5AL2, the isolate with the highest specific activity among 13 isolates, produced the highest specific activity at 0.91 \u00b1 0.12 U\/mg at 2-day incubation at 37\u00b0C. The optimal pH and temperature for amylase activity were 7.0 and 60\u00b0C, respectively. Both optimal conditions gave the specific amylase activity from Bacillus<\/em> sp. 3.5AL2 of 1.97 \u00b1 0.41 U\/mg. It is known that the enzymes that can work at high temperature are considered as having an advantage in industrial processes such as starch liquefaction21<\/sup>. Thus, amylase from Bacillus<\/em> sp. 3.5AL2 has a potential use in such industries.<\/p>\n

Similarly, Bacillus <\/em>sp. GM890 showed the optimal temperature for amylase at 60\u00b0C20<\/sup>, Bacillus licheniformis<\/em> AI20 show the highest activity between the range of 60-80\u00b0C22<\/sup> and Bacillus <\/em>sp. WA21 had a lower optimal temperature of 55\u00b0C for amylase19<\/sup>.<\/p>\n

Most of the starch – degrading bacterial strain revealed a pH range between 6.0 and 7.0 for normal growth and enzyme activity4<\/sup>. Likewise, in this work, Bacillus<\/em> sp. 3.5AL2 had an optimal pH at 7.0 which does not require any addition of acid or alkali. Our finding was in accordance with Bacillus <\/em>sp. isolated from dhal industry waste exhibiting an optimal temperature of 60\u00b0C23<\/sup> and B.<\/em> amyloliquefaciens<\/em> with an optimal pH of 7.024<\/sup>. Nevertheless, Bacillus <\/em>sp. WA21 showed the optimal pH of the amylase at 6.0 which is less than that found in this study19<\/sup>.<\/p>\n

This kind of a study is very important for the starting point of commercial amylase production in Thailand. However, additional research investigation is essential to make amylase production cost-effective. There is a need for thorough enzyme characterization, further determination of the thermostability, pH stability, influence of different metal ions and different substrates, especially agricultural wastes in Thailand, and their concentrations.<\/p>\n

After obtaining all the data needed as mentioned above, it is thought that the production of commercial amylase in Thailand enzyme industries will be increasing and widespread in every region and in turn will be benefiting the country\u2019s economy due to self-reliance on its own resources to produce amylase enzyme.<\/p>\n

Conclusion<\/strong><\/p>\n

This is the first report of identifying 13 amylase-producing bacterial isolates from soil in Nasinuan Community Forest, Maha Sarakham. All bacteria were identified as Bacillus<\/em> spp. except for one from Enterobacter<\/em> genus. These bacteria can be used for amylase production and applied locally and nationally in agriculture, food processing and textile industries in the future. Thus, this will reduce the cost of industrial enzyme import from other countries, offer sustainability of local enzyme production and enhance the economy of the nation.<\/p>\n

Acknowledgments<\/strong><\/p>\n

The authors would like to thank Mahasarakham University (Grant Year 2018) for financial support awarded to SD, and Faculty of Technology, Mahasarakham University (Grant Year 2017) awarded to VL.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

Authors declare no conflict of interest.<\/p>\n

References<\/strong><\/p>\n

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