Characteristics of Raw-Starch Degrading Amylase Bacteria from Natar Hot Spring Lampung H )

Indonesia has a diversity of hot spring as a habitat of bacteria. One of the hot springs is Natar hot spring, Lampung. This study is to report the characteristics of a bacterium called Natl isolate that produces amylase to degrade raw starch from Natar hot spring. Water samples were taken from hot springs with a temperature of 45°C and a pH of 7.0. Natl was isolated by screening on the medium of Starch-Luria Bertani at 37°C. Its amylase-producing bacteria showed an optimum amylolytic activity of a crude enzyme of Natl isolate in soluble starch was 267.2774 U/mL at 6o°C.Genotypic identification results using the16S rRNA gene showed that the Natl isolate is identified as Panninobacter phragmatetus. A crude enzyme of Natl isolate showed a novel amylase ability and could degrade the raw starch substrates, such as corn and sago, with the amount of reducing sugar for each raw starch, 37.0688 pmol/mg, and 24.2697 pmol/mg. In conclusion, Natl amylase is potentially used in industry for its ability to degrade raw starch directly. Article history:

Research, the global enzyme market for the detergent industry is projected to increase to 1.3 billion in 2021 [6]. Amylase production in the world is 30 % of the total production of all enzymes [ 7 ].
Amylase bacteria are used predominantly in industrial applications because their production is more accessible, cheaper, and faster than other amylase microbes [8]. Furthermore, bacterial amylase is easily performed for genetic engineering studies. A large number of amylase bacteria species have been isolated, mostly are Bacillus species [ 9 ]. Amylase, which is involved in the thawing step in the starch processing industry, requires thermostable amylase in a high-temperature process [ 7 ]. The study of thermostable amylaseproducing-thermophilic bacteria from hot springs is one of the trends of screening thermophilic bacteria that produce thermostable amylase. Some of the thermophilic 1

. Introduction
Acid hydrolysis of starch is considered a simple method , as the acid is cheap and quickly obtained [1]. The result of starch hydrolysis by acid are glucose, xylose, arabinose as the main products. On the other hand , furans (furfural and hydroxymethylfurfural) as undesirable byproducts are also obtained [2]. Acid hydrolysis of the starch industry, especially the food and beverage industry, is considered unsafe if consumed [ 3 ]. Therefore, enzymatic hydrolysis was developed using -amylase as an alternative to starch hydrolysis.
-amylase does less production of unwanted byproduct, has specific and higher yield, approximately obtained 95 % more of glucose [ 4 ]. Starch hydrolysis by amylase is also used in the detergent industry for stain removal applications [ 5 ]. Based on Business Communication Company (BCC) Starch hydrolysis using α α α α α α α Jurnal Kimia Sains dan Aplikasi 23 ( 7 ) (2020): 238 -243 239 bacteria produce thermostable amylase have been reported [10, 11, 12, (BIO-25047 ) and purification, then bidirectional PCR product sequencing [ 20]. The resulting fragment of 1.3 kb was compared to other 16S rRNAs in the GenBank database using the NCBI BLAST tool. A phylogenetic tree was constructed using neighbor-joining by NCBI BLAST Tree Method with 1000 bostraps. The morphology and physiology properties of bacteria were determined at the Laboratory of Bacterial Diagnostic, Balai Veteriner Lampung, Bandar Lampung, Indonesia.

. Determination of Amylolytic Activity on Soluble Starch
-amylase is widely known for degrading various substrates [16], which could be a soluble substrate and a raw substrate. Based on a recent study of amylase, there are some raw starch degradingamylases; -amylase from Bacillus aquimaris MKSC 6.2 (BaqA) [ 17 ], Geobacillus thermoleovorans (GTA) [18], and Geobacillus thermoleovorans (Gt-amy II) [ 19 ] have been reported. The ability of some -amylases, as mentioned above, that can directly hydrolyze a raw starch is considered useful in the starch-processing industry. The initial step causes gelatinization is to introduce amylose and amylopectin to be easily degraded by amylase. This step requires high temperature and can be neglected if using raw starch degrading -amylases to reduce the industrial cost.

Furthermore
Indonesia has a diversity of natural hot spring as a habitat for thermophilic bacteria. One of the hot springs is Natar hot spring in Lampung. Limited information about the identification of thermophilic bacteria that can produce -amylase from Natar is the basis of research on the screening of thermophilic bacteria that produce amylase in Natar. Based on the previous study, one bacterium ( Natl isolate) with amylolytic activity has been screened from Natar hot spring in Lampung. The purpose of this study is to determine the characteristics of Natl isolate.
Natl isolate was grown in 50 mL Luria Bertani (1% w / v tryptone, 0.5 % w / v yeast extract, 1% w / v NaCl) on shaker at temperature of 37°C and 150 rpm for 24 hours. The amylolytic activity was determined by measuring the amount of reducing sugar using the DNS method [21]. Amylase assay was performed from a mixture of 25 pLamylase bacterial crude and 25 pL 1% w / v of soluble starch. A 50 pL DNS solution stopped the activity. The mixture was incubated in the water bath at 50°C for 10 minutes, and then the reaction stopped in the boiling water for 10 minutes. As a control reaction, DNS reagent was added before adding amylase. All reaction was performed triplicates. The absorbance was measured at 500 nm. The protein concentration was determined using the Bradford method with the absorbance measured at 595 nm [22]. One unit of amylase activity is defined as the amount of amylase needed to produce 1 pmol reducing sugar under specified conditions.

. Determination of Amylolytic Activity on Raw Starch
The amylolytic activity of Natl isolate on corn and sago raw starch granule was determined by incubating1% w / v of each raw starch with 1 mL of crude in a shaker at a temperature of 37°C and 150 rpm for 24 hours. Followed by 5000 rpm centrifugation for 10 min, the supernatant ' s amylolytic activity was measured by the DNS method as it was in line with the amylolytic activity ' s determination of soluble starch [ 21]. The activity was performed triplicates. Meanwhile, the crude pellets were sent into Laboratorium Terpadu dan Sentra Inovasi Teknologi, Universitas Lampung, Bandar Lampung, Indonesia, for scanning electron microscopy of hydrolysis amylase of the raw starch granule.

. Results and Discussion
Based on the previous study, one Natl isolate was screened for their amylase activity when growing in selective media containing soluble starch. The appearance of the clear zone after staining with KI / I2

Methodology
This research was divided into three steps to determine the characteristics of Natl isolates. Bacterial characteristics were determined in the form of (i) morphology and physiology of Natl isolates, (ii) identification of Natl isolate genotypes based on 16S ribosomal RNA analysis, and (iii) determination of amylolytic activity in soluble and raw starches and (iv) scanning electron microscopy of raw starch granules treated by Natl isolates.

Bacterial Identification
Natl isolate was inoculated on Luria Bertani agar plates (1% w / v tryptone, 0.5 % w / v yeast extract, 1% w / v NaCl, 2% w / v agar, 1% w / v soluble starch). Natl isolate was grown at a temperature of 37°C for 24 hours and then α α α α γ Jurnal Kimia Sains dan Aplikasi 23 ( 7 ) (2020): 238 -243 240 ( Figure 2) was aligned and compared to the 16S rRNA of various bacteria from GenBank data. The phylogenetic tree was constructed and showed that Natl isolate belongs to Panninobacter phragmatetus (Figure 3 ). It is suggested that amylase bacterial from Natar hot spring in Lampung, be identified as Panninobacter phragmatetus with strain name Natl. solution around the Natl colony indicating the ability to hydrolyze soluble starch. Natl isolate has a crude amylolytic activity of 240.7267 U / mL in a 1% soluble starch substrate using DNS reagents at a temperature of 50°C . Hot springs are a common source of bacteria that produce enzymes, one of which is amylase. Several hot springs in Indonesia can be the source of bacterial growth and have potential applications in the starch-processing industry. Bacillus megaterium has been isolated from Hatuasa hot spring in Tuhelu Village Ambon, which excreted amylase after being screened on a starch agar plate [10]. Bacillus licheniformis BT 5.9 has been identified as a potential bacterium producing a thermostableamylase from Changar hot springs in Malang [11]. Two isolates (BR 002 and BR 015 ) of thermostable -amylase were isolated from Bora Hot Springs, Central Sulawesi [12]. Anoxybacillus flavithermus AE 3 was isolated as anamylase-producing bacterium from Bukit Kili hot spring in Solok, West Sumatera [ 13 ]. Amylase producing bacteria have been isolated from Dondang hot spring in Muara Jawa Sub District, Kalimantan [ 14 ]. One colony was identified as Thermoactinomyces saachari based on morphology and physiology identification as -amylase bacteria from Singgahan hot spring, Tuban, East Java [ 15 ].  CGGTTAGCGCACCACCTTCGGGTAACCCAACTCCCATGGT  GTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACC  GCGTCATGCTGTTACGCGATTACTAGCGATTCCAACTTCA  TGCTCTCGAGTTGCAGAGAACAATCCGAACTGAGACGGCT  TTTGGAGATTAGCTCCGGGTCGCCCCTTCGCTGCCCACTG  TCACCGCCATTGTAGCACGTGTGTAGCCCAGCCCGTAAGG  GCCATGAGGACTTGACGTCATCCCCACCTTCCTCTCGGCTT  ATCACCGGCAGTCCCCCTAGAGTGCCCAACTCAATGCTGG  CAACTAAGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACC  CAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACC  TGTCCTGGCGTCCCCGAAGGGAACCCACGGTCTCCCGTGG  TAGCACCAAATGTCAAGGGCTGGTAAGGTTCTGCGCGTTG  CTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCC  CGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCC  AGGCGGGAAGCTTAATGCGTTAACTGCGCCACCAAGATGC  ATGCACCCTGACGGCTAGCTTCCATCGTTTACGGCGTGGA  CTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGC  ACCTCAGCGTCAGTACCGGGCCAGTGAGCCGCCTTCGCCA  CTGGTGTTCTTCCGAATATCTACGAATTTCACCTCTACACT  CGGAGTTCCACTCACCTCTCCCGGACTCCAGACTCCCAGTA  TCAAAGGCAGTTCCGAGGTTGAGCCCCGGGATTTCACCCC  TGACTTAAAAGTCCGCCTACGTGCGCTTTACGCCCAGTGA  TTCCGAACAACGCTAGCCCCCTTCGTATTACCGCGGCTGCT  GGCACGAAGTTAGCCGGGGCTTCTTCTGCGAGTAACGTCA  TTATCCTCCTCGCTGAAAGAGCTTTACAACCCTAGGGCCT  TCATCACTCACGCGGCATGGCTGGATCAGGCTTGCGCCCA  TTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTG  GGCCGTGTCTCAGTCCCAGTGTGGCTGATCATCCTCTCAG  ACCAGCTACTGATCGTCGCCTTGGTGAGCCATTACCCCACC  AACTAGCTAATCAGACGCGGGCCAATCCTTCGGCGATAAA  TCTTTCCCCCGTAGGGCGCATGCGGTATTAGCAGCCGTTT  CCAGCTGTTGTTCCGCACCAAAGGGTATGTTCCCACGTGT  TACTCACCCGTCTGCCACTCCCCGTTACCAGGG The morphological analysis results showed that the Natl isolate produced circular, flat, light yellow colonies on agar plates. It has pink-red rods, coccobacilli, Gramnegative bacteria, positive for catalase and oxidase production, and -hemolytic on blood agar plates. The physiological tests for Natl isolate were carried out with Microbact GNB 12A / B / E, 24 E., The results of the Natl isolate physiological test, are shown in Table1.

. Identification of Natl Isolate
Fragments of 1322 base pairs of Natl isolate DNA were amplified and determined (Figure1). The nucleotide sequence of 16S rRNA gene fragment Natl isolates Figure 2. The nucleotide sequence of the 16S rRNA gene from Natl isolate α starch by enzyme needed glucoamylase, which can optimally be hydrolyzed at a temperature of 50°C and 6o°C [ 23 ]. The specific characteristic of moderate thermostable microorganism used in industrial has optimal amylase activity at 50°C and above [ 24 ]. Natl has the potential to be used in the starch industry as a moderate thermostable bacterium, which has optimal amylolytic activity at a temperature of 6o°C and still has amylolytic activity around 8% at a temperature of 8o°C than the optimal activity.

. Amylolytic Activity on Raw Starch
The determination of Natl amylase activity in raw starch has been carried out by measuring the supernatant adsorption after incubating a mixture of the Natl crude enzyme with corn and sago starch for 24 hours at 37°C .
The amylolytic activities of Natl on corn and sago raw starch granule are 37.0688 pmol / mg and 24.2697 pmol / mg, respectively, determined by the DNS method [ 21]. The results of scanning electron microscopy of amylase treated corn granules by the Natl crude enzyme show that corn granules have small pores on the surface (Figure 5 A), compared to corn granules starch, which had not been applied to amylase hydrolysis (Figure 5 B).

. Amylolytic Activity on Soluble Starch
The crude enzyme of Panninobacter phragmatetus strain Natl shows amylase activity in soluble starch and raw starch. The crude enzyme of Natl isolate can hydrolyze soluble starch at a temperature range 40 -8o°C ( Figure 4 ). Natl isolate displayed an optimum amylase activity in soluble starch at a temperature of 6o°C, with the amylolytic activity of 267.2774 U / mL determined by the DNS method [21]. Natl shows the amylolytic activities in soluble starch at a temperature of 40°C , and 50°C is 131.435 U / mL and 240.7267 U / mL, respectively. Natl can still actively hydrolyze soluble starch at temperature 70°C and 8o°C, in which the amylase activities are 114.0236 U / mL and 23.5817 U / mL, respectively. Previous studies reported that several hot spring bacteria produce amylase, each of which had optimum amylolytic activity at 70°C [10], 50°C [11], 50°C and 70°C [12]. Amylase treated corn granules were incubated with Natl crude enzyme. Small pores on the surface of amylase treated corn granules by Natl show that Natl crude enzyme can hydrolyze a corn raw starch granule. The results compared to the untreated corn granules which have no small pores on the surface. This indicates that Natl crude amylase can directly hydrolyze raw corn starch. Previous studies reported that -Amylase from Bacillus aquimaris MKSC 6.2 and B. amyloliquifaciens ABBD showed the same hydrolyze pattern to raw corn starch, that holes were found on the surface of corn raw starch [ 17 , 25 ]. The crude enzyme of Panninobacter phragmatetus strain Natl can be a novel amylase because of its ability to hydrolyze raw starch directly along with other strains that had been reported previously [26]. The study reported that Bacillus aquimaris MKSC 6.2 could degrade various raw starch granules, such as corn, cassava, sago, potato, and rice [ 17 ]. Geobacillus thermoleovorans (Gt-amy II) had the optimum ability to directly hydrolyze a corn raw starch granule [ 19 ]. Amylase from Anoxybacillus strains SK 3 -4 (ASKA) and Anoxybacillus strains DT 3 -1 could degrade sago and potato granule starch [ 27 ]. The gelatinization step in the starch industry needs high temperature and could be abolished if using -amylases to directly degrade raw starch [ [ 17 ]. The advantage of using raw starch degrading -amylases can save the cost. Natl isolate has the ability as a raw starch degrading -amylases so that it becomes promising and potentially used as an enzyme in the starch industry.

. Conclusion
The result shows that Natl isolate as a moderate thermostable bacterium from Natar hot springs in Lampung Province produces amylase that degrades soluble starch and raw starch granules, such as corn and sago. A novel amylase from Natl isolate has the potential to be use in the starch-processing industry.