Amyloly c ability of bacteria isolated from termite ( Coptotermes sp . ) gut

BSR 2, BSR 3, BSR 8, and BSR 9, different bacteria isolated from the termite gut, have been shown to possess celluloly c ac vi es, but their amyloly c ability has heretofore been unknown. This study a empted to fill in this knowledge gap. The forma on of a clear zone using the iodine test showed that the bacteria were able to produce and secrete amylase. Based on the results, the best cul va on mes for strains BSR 2, BSR 3, BSR 8, and BSR 9 were 6, 3, 2, and 2 d, respec vely, yielding amylase ac vi es of 2.59 ± 0.13 U/mg, 2.00 ± 0.08 U/mg, 1.67 ± 0.10 U/mg, and 1.55 ± 0.12 U/mg, respec vely. BSR 2 had the highest amylase ac vity compared with the other bacterial isolates. The op mum pH for bacterial amylase ac vity of BSR 2 was 7.0, and the op mum temperature was 40°C. The molecular characteriza on of isolates BSR 2, BSR 3, BSR 8, and BSR 9 was based on 16S rRNA gene sequences. Isolates BSR 8 and BSR 9 were thus iden fied as Brevibacillus parabrevis and Brevibacillus sp. with similari es amoun ng to 92.48% and 95.91%, while the BSR 3 isolate was iden fied as Pseudomonas alcaligenes with a similarity of 94.29%, and the BSR 2 isolate could not be iden fied yet.

In recent years, enzymes have been widely adopted and exploited in various processes.According to Waites et al. (2001), enzymes are preferred in various catalysis processes because they are environmentally friendly, and can also react specifically so as not to cause undesirable side effects.Amylase is one type of enzyme that has played an important role in the industry.Currently, amylase production has reached a high scale that controls about 25% of the enzyme trade (Reddy et al. 2003).A wide-ranging number of industries utilize amylase, including the paper industry, detergent industry, textile industry, medicine industry, and bakery and cake industries.In Indonesia, production of amylase is still not performed locally, so it must be imported.In fact, locally selected microbes can be used as sources of enzyme production.
Termites consume plant litter and are known to be symbiotic with various kinds of cellulolytic bacteria in their digestive organs.Bacteria originating from their gut have been successfully isolated by Janatunaim et al. (2015) and have been shown to possess a cellulolytic capability.The bacteria from termite guts are also thought to have amylolytic potential, since amylose and cellulose are both linear glucose-based polymers frequently found in plant material, which are distinguished by the glycosidic bonds between the glucose units.The bond in amylose is α-1,4-glycosidic, whereas the bond in the cellulose is β-1,4glycosidic.Utilization of amylase derived from the termite gut could become an alternative source of a novel amylase.Therefore, it is necessary to conduct a study to prove that bacteria from the termite gut are able to produce amylase, as well as to perform a more detailed characterization of these enzymes to improve their function in the future.

Rejuvena on of bacteria and isolate prepara on
The bacteria from the termite gut isolated by Janatunaim et al. (2015) were rejuvenated and grown on CMC medium and YPSs medium for up to three days.

Detec on of amyloly c ac vity of bacterial isolates from the termite gut
Detection of bacterial amylolytic activity was done through qualitative and semi-quantitative tests.The qualitative test of amylase activity was carried out by the iodine test, while the semi-quantitative test was done by calculating the ratio of the diameter of the clear zone formed with the diameter of bacterial colonies.The result of this calculation is the value of the relative amylase activity.

Growth of bacteria on liquid YPSs medium
The bacteria from the termite gut were grown in liquid YPSs medium in an incubator shaker at 37°C with a rotation speed of 130 rpm.Furthermore, OD measurements were taken every 24 h for 7 d using a UV-VIS spectrophotometer at 600 nm wavelength.

Measurement of amylase ac vity 2.4.1. Isola on of crude enzyme and measurement of amylase ac vity
Crude supernatant containing amylase of bacteria was isolated by centrifugation.Amylase activity in this supernatant was measured with soluble starch as substrate.
The reducing sugars (maltose) released in this reaction were measured by the Bernfeld method (1951) using 3.5dinitrosalicylic acid (DNS), and its concentration was converted to standard maltose (Bisswanger 2004).One unit of relative amylase activity is the amount of enzymes that can produce 1 μmol maltose per minute per mL of starch solution under test conditions (Bisswanger 2004).

Measurement of protein enzyme levels
Measurement of protein content was done spectrophotometrically with the direct absorbance method at 280 nm wavelength.The standard curve was constructed with BSA, while the blanks used were sterile aqua bidest.

Determina on of op mum bacterial cul va on me
Determination of the optimum cultivation time of bacterial strains from the termite gut for producing amylase was done by measuring amylase activity every 24 h for 7 days.

Determina on of temperature and pH op mum of amylase ac vity
The optimum temperature of amylase activity was determined by measuring the activity at various temperatures of 29°C, 37°C, 50°C, 60°C, 70°C, and 80°C using soluble starch as the substrate at pH 7 and incubation for 5 min.
The optimum pH for activity was determined by dissolving crude enzyme and soluble starch substrate in 0.1 M buffer solution under various pH conditions, i.e. pH 4, 5, 6, 7, 8, 9, and 10, and incubation at the respective optimum temperature.

Iden fica on of bacterial strains
Identification was done by the conventional method of DNA isolation, PCR, and sequencing of 16S rRNA gene.Sequences in alignment with the Bioedit program were used for similarity searches in the NCBI database using BLAST.

Data analysis
Quantitative data, i.e., bacterial amylase activity, incubation time, temperature, and optimum pH were tabulated and then analyzed statistically using ANOVA and DMRT using SPSS v.16.0.

Amyloly c ac vi es of bacterial isolates from the termite gut
It was indicated that the four bacterial isolates, i.e.BSR 2, BSR 3, BSR 8, and BSR 9, when grown on YPSs (Yeast Pepton Starch soluble) medium showed positive results in producing amylase, as indicated by the formation of clear zones around the colony (Table 1).The (+) sign indicates the reac on of hydrolysis of amylose by amylase.The le er a in each image shows the clear zone, and b denotes the colony.

Growth of bacterial isolates from the termite gut
Of the bacterial isolates, BSR 9 grew the fastest, followed by isolates BSR 8, BSR 3, and BSR 2 (Figure 1).Based on the analysis (Table 2), it was found that the bacteria growth of all the isolates showed no significant difference (p > 0.05) from day one to day two.The growth of isolates BSR 2 and BSR 3 also showed results that were not significantly different across measurement days, except for day 5.Likewise, isolates BSR 8 and BSR 9 showed growth that was not significantly different (p > 0.05) to day 7, while the growth of BSR 2 and BSR 9 was significantly different or significant results (p ≤ 0.05) from day 3 to day 7.
There are several things that can cause a decrease in the number of bacterial cells, such as reduced nutrients available so that some cells experience a decrease in growth rate or die.Gaman and Sherrington (1994) argue that the decline in growth rates can be caused by a lack of growth factors such as vitamins and mineral elements.Measurement of cell growth calculated on the basis of OD values does not generally indicate a decrease in cell growth.This is because the measurement of bacterial OD by spectrophotometric technique not only counts living cells but also dead cells, so that even if all the cells have died, the number of cells counted will still show a constant value because there is no cell growth or cell re- duction.However, in the OD measurement results, the data obtained showed a decrease in the number of cells.This may be due to a non-homogeneous sampling, so the number of cells counted from the sample is small.

Op mum me of amylase produc on
From the results of the research, it was analyzed that the optimum incubation time of bacterial isolates BSR 2, BSR 3, BSR 8, and BSR 9 for amylase production were 6, 3, 2, and 2 days, respectively, with the respective values for the specific activity of 2.59 ± 0.13 U/mg, 2.00 ± 0.08 U/mg, 1.67 ± 0.1 U/mg, and 1.55 ± 0.12 U/mg (Table 3).The value of amylase activity of all bacterial isolates increased with cultivation time up to the optimum time point, and began to decrease thereafter (Figure 2).

Effect of temperature on amylase ac vity
Temperature is an important factor in the enzymatic activity of amylase.In general, the rate of chemical reactions of both enzyme-catalyzed and noncatalyzed reactions will increase with higher temperatures until reaching an optimum.Temperature changes above or below the optimum temperature may result in an increase or decrease in amylase activity (Hmidet et al. 2008).Increasing the temperature to the optimum value will increase the activity of FIGURE 2 Forma on of amylase ac vity during cul va on of bacterial isolates from the termite gut.Standard devia on is calculated from 3 repe ons.(♦) BSR 2, (■) BSR 3, (▲) BSR 8, (X) BSR 9. enzymes in forming the product, while a temperature exceeding the optimum may cause damage or denaturation of the enzyme.As shown in Figure 3 and Table 4, the optimum temperature of amylase activity was at 40°C with an activity value of 2.59 ± 0.13 U/mg, which was significantly different (p ≤ 0.05) from the amylase activity at other incubation temperatures.Based on the analysis, it was found that amylase activity at 50°C to 80°C did not show any significant difference (p > 0.05), meaning that a temperature change in the range of 50-80°C did not greatly disturb enzyme activity.
The results of the research showed data in accordance with Mishra and Behera (2008), who reported that bacterial isolates exhibiting amylase activity have high amylase activity at 40-45°C, especially Bacillus species.The results also concurred with a study by Gebreselema (2015), which found the optimum temperature for amylase activity to be 40°C in Bacillus and 37°C in Streptomyces.

Effect of pH on amylase ac vity
The pH value greatly affects the shape of the enzyme ion structure which can be positive ions, negative ions or double charged ions (zwitter ion).With the change of pH conditions it will affect the effectiveness of the active site of the enzyme in forming the enzyme-substrate complex.When the pH conditions are too acidic or too alkaline from the optimum pH conditions of the enzyme, enzyme denaturation may result in a decrease of enzyme activity, even it may completely be lost (Poedjiadi 1994).
The amylase of bacterial isolate BSR 2 showed its optimum activity at pH 7. As Figure 4 and Table 5 show, amylase activity continued to increase as the pH increased from acidic to neutral, i.e. pH 4 to 7, and then showed a decrease when going into the alkaline region, i.e. pH 8 to 10.These results suggest that the optimum pH to obtain the best amylase activity of bacterial isolate BSR 2 from the termite gut is pH 7.That is, amylase from BSR 2 bacteria will perform optimally at a neutral pH condition.
The amylase activity calculated at pH 7 was significantly different (p ≤ 0.05) compared with the amylase activity at other pH levels, except at pH 6, which places the acidic atmosphere close to neutral.This is in accordance with Baker (1983), who stated that amylase derived from insects is generally active in neutral to slightly acidic pH conditions (Terra et al. 1996), since bacterial isolates used in this study also come from insects, namely termites.Vihinen and Mantsiila (1989) also confirmed that the optimum pH of amylase varied from 2 to 10.5, but most were well active at pH 5-8.The results of this study were

FIGURE 4
The results of the measurement of bacterial isola on acvity of BSR 2 on the pH varia on.Standard devia on is calculated from 3 repe ons.also supported by the results of Mishra and Behera (2008) and Alariya et al. (2013), both of whom reported that the best amylase activity of E. coli, Pseudomonas fluorescens, Bacillus subtilis, and Serratia marscens bacteria occurred at pH 7.
In pH conditions that are too acidic or too alkaline, the activity of amylase will generally be slower and decrease, as stated by Pujawati (2012), that the pH of the environment affects the speed of enzyme activity in catalyzing a reaction (Abdel-Fattah et al. 2013).The results of Hmidet et al. (2008) also showed that enzymes that are given a pH treatment that is too alkaline (pH 10) could result in protein denaturation and total loss of activity.However, from the results of this study it was found that the amylase of isolate BSR2 had activity that was not significantly different (p > 0.05) when it was treated at an acidic pH level (i.e. 4 and 5) as well as the pH bases of 8, 9, and 10.These data suggest that amylase from BSR 2 is able to tolerate a wide range of pH.

Iden fica on of the bacterial isolates
Amplification of the 16S rRNA genes of bacterial isolates BSR 2, BSR 3, BSR 8, and BSR 9 was performed (Figure 5).The sequences of the 16S rRNA genes of bacterial isolates BSR 8 and BSR 9 showed the highest level of similarity with those of Brevibacillus sp.AB938197.1 (Table 6).Brevibacillus spp.are Gram-positive bacteria (Thomas 2006), and most of the Brevibacillus bacteria are isolated from the environment, especially from the soil, and are also commonly found in milk and cheese (Vos et al. 2009).The bacterium isolated from the termite gut, BSR 3, showed the highest similarity level with Pseudomonas alcaligenes, at 94.29%.Pseudomonas spp.are generally facultative aerobic but do not form spores. Common features of the genus Pseudomonas are Gram negative, obligate, motile, negative fermentative oxidation (Cornelis 2008).Pseudomonas can be isolated from soil or water.Silaban (1999) successfully cultivated Pseudomonas alcaligenes in CMC (carboxymethyl cellulose) media which showed that the bacteria had the ability to degrade cellulose.Nevertheless, no studies have shown that Pseudomonas, especially Pseudomonas alcaligenes, can be isolated from termite gut.As for the BSR 2 isolate, it only shows a low similarity level with the existing data in gen-Bank so it can be said the identification of the type of bacterial isolate is not yet possible.The highest value of conformity is with Pseudomonas sp. and Stenotrophomonas maltophilia but only below 60% with a 36% query cover score.
Based on various studies of cellulolytic bacteria in termite digestive tracts, the bacteria identified were Bacillus megaterium and Paracoccus yeei from a study conducted by Ferbiyanto et al. (2015).The study was conducted on cellulolytic bacteria isolated from the digestive tract of Macrotermus gilvus.A similar study was conducted by Wenzel et al. (2002), who successfully isolated cellulolytic bacteria from the digestive tract of Zootermopsis angusticollis.The cellulolytic bacteria were identified as Cellulomonas, Bacillus cereus, Bacillus megaterium, and Paenibacillus species.

Conclusions
Bacterial isolates BSR 2, BSR 3, BSR 8, and BSR 9 from the termite gut can produce amylase, and best amylolytic bacterial isolate is BSR 2. The optimum bacterial cultivation time of BSR 2, BSR 3, BSR 8, and BSR 9 for producing amylase were 6 d, 3 d, 2 d, and 2 d; respective amylase activity values were 2.59 ± 0.13 U/mg, 2.00 ± 0.08 U/mg, 1.67 ± 0.1 U/mg, and 1.55 ± 0.12 U/mg.The optimum conditions for the amylase activity of bacterial isolate BSR 2 from the termite gut was at 40°C and pH 7. BSR 8 and BSR 9 were identified as Brevibacillus parabrevis and Brevibacillus sp.based on similarities of the sequence of the 16S rRNA genes (92.48% and 95.91%), BSR 3 was identified as Pseudomonas alcaligenes with similarities amounting to 94.29%, while the BSR 2 isolate could not be identified yet due to low sequence similarities.

FIGURE 3
FIGURE 3 Results of measurement of amylase ac vity of bacterial isolate BSR 2 on temperature varia on.Standard devia on is calculated from 3 repe ons.

Temperature
.08 ab a-c The difference in nota on on the values in the same column shows a significant difference at the 5% significance level (p ≤ 0.05).
difference in nota on on the values in the same column shows a significant difference at the 5% significance level (p ≤ 0.05).

FIGURE 5
FIGURE 5Results of amplifica on of the 16S rRNA genes of bacterial isolates BSR 2, BSR 3, BSR 8, and BSR 9, which a er purifica on gave a single band at 1500 bp.

TABLE 2
Average OD values of bacterial isolates from the termite gut every 24 hours for 7 days.Standard devia on is calculated from 2 repe ons.
a-c Different nota ons on values in the same line indicate a significant difference at the level of significance 5% (p ≤ 0,05).w-y Different nota ons on values in the same column indicate a no ceable difference at the level of significance 5% (p ≤ 0,05).

TABLE 4
Mean of amylase ac vity of bacterial isolate BSR 2 on temperature varia on.Standard devia on is calculated from 3 repe ons.

TABLE 5
The mean of amylase ac vity of bacterial isolate BSR 2 on pH varia on.Standard devia on is calculated from 3 repe ons.

TABLE 6
Results of BLASTN of the sequence of the16S rRNA genes and similarity analysis.