Screening and Identification of Thermotolerant and Osmotolerant Bacillus amyloliquefaciens BKHE Isolated from Kinema of Eastern Nepal for Alkaline Protease Production

Alkaline protease is one of the most important industrial enzymes which are excessively used in the detergent industry, food industry, feed industry, pharmaceutical industry, leather industry, etc. 60% of the produced alkaline protease is consumed by the detergent industry alone. In the present study, bacterial isolates that can produce alkaline protease for purpose of bio-detergent were screened among the isolates isolated from kinema (an alkaline fermented food of eastern Nepal). Selected bacterial isolates were further screened for hemolysis activity and the production of other hydrolytic enzymes. Four bacterial isolates selected were tested for their capacity to produce alkaline protease in five different fermentation mediums. Isolate BKHE produces a high amount of alkaline protease (0.4705 ± 0.035 U/mL/min) in fermentation medium M2 (sucrose, 11 g/L; yeast extract, 5 g/L; and KNO3, 5.2 g/l, pH 9). The selected isolate was identified as Bacillus amyloliquefaciens BKHE based on 16S rRNA sequencing and phenotypic features. This bacterial strain was also found to be thermotolerant (confluent growth at 50°C) and salt tolerant up to 10% NaCl concentration. With its versatile ability, bacterial isolate or purified enzymes have potential applications in the food and detergent industry.


Introduction
During the 20th century, also called the age of organic chemistry, coastal environments receive a variety of landderived organic inputs, both natural and synthetic. Among them, detergents are probably the largest class of technical products for domestic use [1]. Ten, with the rise of enzyme technology, the feld of detergent science has been more advanced and the market expanded. Enzymes with a variety of applications in the detergent industry, food industry, feed industry, pharmaceutical industry, leather industry, peptide synthesis, and recovery of silver from used X-ray flms are developed [2]. Among these, detergent industries are the primary consumers of enzymes, in terms of both volume and value [3], consuming about 60% of all the enzymes produced [4]. Among hydrolytic enzymes, microbial alkaline protease dominates commercial applications with a signifcant share of the market captured by subtilisins and/or alkaline protease from Bacillus spp. for laundry detergent applications [5]. Alkaline proteases added to laundry detergents enable the release of proteinaceous material from stains [6]. Te increased usage of these proteases as the detergent additive is mainly due to the cleaning capabilities of these enzymes in environmentally acceptable nonphosphate detergents [2]. Along with alkaline protease; cellulase, lipase, and amylase are the major enzymes incorporated in detergent formulation, amylase being the second most important enzyme used in the detergent industry [7].

Microorganisms and Teir
Maintenance. Te organisms used in this study were isolated from kinema samples on nutrient agar. Tree sundried kinema samples were collected from the hilly area of the Dhankuta District of Nepal. 0.2 gm of kinema samples were homogenized with 10 mL of sterile physiological saline (0.85%). Te kinema homogenate was taken for serial dilution and spread plated on nutrient agar followed by incubation at 37°C for 24 hours under aerobic conditions. Isolates having diferent colony morphology were collected and purifed by sub-culturing in a nutrient agar plate. Among the isolates, Gram-positive and rodshaped bacteria were taken for further research activities.
All the isolated organisms were preserved by lyophilization [27] and stored at −80°C for long-term storage and a bacterial slant was prepared for research activities.

Screening for Hemolysis Activity.
To the sterile blood agar base which has been melted and cooled to 45 to 50°C, 5% (vol/vol) sterile defbrinated blood that has been warmed to room temperature was added. Te media then was poured into Petri plates avoiding any bubbles. Tey were incubated at 37°C for 24 hours. Hemolysis was recorded by the appearance of a zone of clearing around the colonies [28].

Screening for Production of Hydrolytic
Enzymes. An amylolytic test was carried out by culturing bacteria on starch agar (Nutrient Agar with 0.2% starch), incubated at 37°C. After 48 hours the plates were fooded with 1% Lugol's iodine reagent for 10 mins and drained of. Te clear halo region indicated starch hydrolysis [18].
Te bacteria were grown on nutrient agar with 1% CMC for cellulolytic activity. Te CMC agar plates were incubated at 37°C for 24 hours. At the end of the incubation, the agar medium was fooded with an aqueous solution of Congo red (1% w/v) for 15 minutes. Te Congo red solution was then poured of, and the plates were further treated by fooding with 1 M NaCl for 15 minutes. Te formation of a clear zone of hydrolysis indicated cellulose degradation [29].
Te ability of lipase production was carried out in Petri dishes using a medium (composition: peptone 0.5%; yeast extract 0.3%; tributyrin 1% and agar 2% in distilled water) [30]. Te cultured plates were incubated at 30°C for 48 hours.
Skim milk powder, 5 g in 50 ml of distilled water; agar, and 1 g in 50 ml of distilled water were autoclaved separately at 121°C, cooled to 45°C, mixed, and poured into Petri plates. Te cultured plates were incubated at 30°C for 48 hours. Te halo region indicated protease production [18].
Gelatin liquefaction (the formation of a liquid) was tested by stabbing gelatin agar (semisolid with 7.5 g/L agar) in deep tubes. After 48 h of incubation, the cultures were placed in a refrigerator at 4°C until the bottom resolidifes. If gelatin was hydrolyzed, the medium will remain liquid after refrigeration. If gelatin was not hydrolyzed, the medium will resolidify during the time it is in the refrigerator [31].
2.6. Protease Assay. Te reaction mixture in a total volume of 11 ml was composed of 5 ml of 0.65% casein in 50 mM Potassium Phosphate bufer, pH 7.5, and an enzyme solution of 1 ml, 0.75 ml, and 0.5 ml for 3 diferent test sets. After 10 min incubation at 37°C, the reaction was terminated with 5 ml of 110 Mm trichloroacetic acid followed by another incubation of 30 min at 37°C. To 2 ml test fltrate, 5 ml of 500 mM sodium carbonate was added followed by 1 ml 1 M Folin-Ciocalteu's phenol reagent. Te reaction mixture was incubated at 37°C for 30 min and then allowed to cool to room temperature and test fltrate absorbance measured at 660 nm along with blank and standard solution in UV-1800 Shimadzu spectrophotometer. One unit of protease activity was defned as the amount of enzyme which released 1 μmole tyrosine per min under the assay condition [34].

Molecular Characterization of Selected Isolate.
DNA extraction and Sequencing of 16S rRNA Gene: bacterial isolate BKHE was cultured in 10 mL nutrient broth. Incubation was performed at 37°C for 24 hours. Te genomic DNA of the bacteria was isolated according to the procedure of Sambrook and Russel [35].
Raw sequences were assembled and trimmed using the codon code aligner. Te contig sequence generated was subjected to BLASTN and the database "rRNA/ITS databases" was selected. Twelve highly similar sequences (based on blast results) were taken in FASTA format for phylogenetic analysis.

Maximum Parsimony Analysis of Taxa.
Te evolutionary history was inferred using the maximum parsimony method. Te bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed [37]. Te MP tree was obtained using the tree-bisection-regrafting (TBR) algorithm [38] with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). Tis analysis involved 13 nucleotide sequences. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). Evolutionary analysis was conducted in MEGA X [39].

Morphological and Biochemical Characterization.
Te selected protease-producing isolate BKHE was identifed using morphological and biochemical characteristics, according to -Bergey's Manual of Systemic Bacteriology [18] while the sugar utilization test was performed as manufacturer's instruction using Himedia KB009TM HiCarbo Kit (KB009A/KB009B1/KB009C).

Salt and Temperature Tolerance Test for Selected Strain.
For temperature tolerance, the organism was cultured in Nutrient broth and incubated at diferent temperatures (30°C, 40°C, 45°C, 50°C, 60°C) and for salt tolerance, the organism was cultured in Nutrient broth with diferent salt concentrations (2%, 6% and 10% (w/v)) [18]. Te cell density was measured using a DEN-1B Grant bio Densitometer. 18 phi test tubes were used for generating data and data were obtained in McFarland standards.

Statistical
Analysis. Data analysis was performed using OriginPro 9.0, IBM SPSS 15.0, and Microsoft excel. ANOVA was performed for quantitative data where the Tukey test was used to compare the means at a 95% confdence interval.

Isolation and Screening.
Among the purifed isolates, only 10 isolates were found to be Gram-positive and rodshaped bacteria. Only Gram-positive and rod-shaped bacteria were further screened for alkaline protease activity. Figure 1 illustrated three of the Gram-positive and rodshaped bacteria.

Screening for Hemolytic Activity and Production of Hydrolytic Enzymes.
Further screening of bacterial isolate was based on its capabilities to hydrolyze blood cells and produce other hydrolytic enzymes. Results are shown in Table 1, isolates BKTB, BKTC, BKTD, BKRK, and BKHE were found to show β-hemolysis. Along with hemolysis property, these isolates were able to produce other hydrolytic enzymes which are widely used in detergent formulation after protease. Figure 3 illustrated the hydrolytic enzymes produced by one of the isolated BKHE.

Alkaline protease production in diferent medium
compositions. Te one-waybetween-groups analysis was performed to observe the enzymatic activity of diferent bacterial isolates (Figure 4(a)), and Figure 4(b) illustrated the enzymatic activity in diferent production mediums. While comparing means of enzymatic activity between diferent medium compositions produced by diferent bacterial strains, cell-free fermentation broth of isolates BKHE (0.4705 U/mL/ min, SE � 0.035) and BKTC (0.3252 U/mL/min, SE � 0.031) showed signifcantly high enzymatic activity in production medium M2 at p < 0.05 confdence level; cell-free fermentation broth of BKTD (0.155 U/mL/min, SE � 0.059) and BKRK (0.193 U/mL/min, SE � 0.015) showed signifcantly high enzymatic activity in production medium M3 at p < 0.05 confdence level, as shown in Figure 4(a).
But while comparing means of enzymatic activity between diferent bacterial isolates, cell-free fermentation broth of isolate BKHE in production medium M2 showed International Journal of Microbiology signifcantly high enzymatic activity than other bacterial isolates at p < 0.05 confdence level. So, a high enzymeproducing isolate was found to be isolate BKHE and the best production medium among the compared ones was production medium M2.   Bacillus ( Figure 5)

Salt Tolerance and Temperature Tolerance Test for Selected
Strain. Selected strain B. amyloliquefciens BKHE was grown in diferent salt concentrations. Cell density was not signifcantly diferent among the medium with diferent salt concentrations at p < 0.05 confdence level as illustrated in Figure 6(a). Cell density at 10% sodium chloride concentration was 2.05 ± 0.07 MF. It was also cultured in nutrient broth and incubated at a diferent temperature, high cell density was observed at 45°C (4.55 MF, SE � 0.21) which is signifcantly higher than cell densities when incubated at 30°C, 40°C, 50°C, and 60°C at p < 0.05 confdence level as shown in Figure 6(b).

Discussion
We have identifed alkaline protease, amylase, lipase, and surfactant-producingB. amyloliquefaciens strain BKHE, which was isolated from the kinema sample from eastern Nepal. Only isolate with viscous colony texture, Grampositive and, rod-shaped were screened for alkaline protease production, as these are the basic features of Bacillus spp [18]. Among nine alkaline protease-positive strains, fve of them (BKTB, BKTC, BKTD, BKRK, and BKHE) were able to hydrolyze blood cells. But the result of screening for alkaline protease showed that isolate BKTD produced a signifcantly smaller halo zone when compared with other hemolysis-positive isolates. So, only isolates BKTC, BKTD, BKRK, and BKHE were further compared quantitatively for the production of alkaline protease in diferent production  Figure 5: Maximum parsimony tree generated from 16S rRNA sequence data. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates are collapsed. Te percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to branches. mediums. A hemolytic activity might indicate the bacterial isolate is a biosurfactant producer, although other various lytic enzymes produced by strain may cause hemolysis [41]. Biosurfactants, along with their wide applications, are antimicrobials too [42]. So, the product from hemolytic bacteria along with protease activity can be efectively used in hospitals to remove blood spills and clots and kill infectious microbes. Another screening parameter was the capability to produce other hydrolytic enzymes. Since other enzymes such as amylase, lipase, and cellulase are also used in detergent formulations, so the capacity to produce these will be of great signifcance. Among four selected isolates, BKTC and BKTD were found to produce all sets of hydrolytic enzymes, while BKHE and BKRK were cellulase negative (from Table 1). Since the production cost and downstream process cost of the enzyme are high, it is important to reduce the cost to make these enzymes sellable to detergent industries. Te beneft of producing multiple hydrolytic enzymes lies here, as multiple enzymes can be coproduced in the same fermentation media by the same bacterial strain. As these enzymes are used together not only in detergent industries, these are used together in the food and pharmaceutical industries too. Coproduction of cocktail enzymes is one way to reduce the cost of production and these approaches were found to be practical by other researchers [4,7,43]. Among the four isolates selected, the highest alkaline protease was produced by BKHE in medium M2 (sucrose, 11 g/L; yeast extract, 5 g/L; and KNO3, 5.2 g/l, pH 9). While comparing diferent media compositions, M2 was found to be best among the medium compared. BKTC also showed high production (signifcantly high when compared with BKTD and BKRK but signifcantly low when compared with BKHE at a confdence level of p < 0.05 ) and this paper only deals with the preliminary screening and media selection for the production of alkaline protease. Optimum conditions are yet to be identifed for the production of alkaline protease production. Te optimum condition depends on temperature, pH, media composition, oxygen transfer rate, and diferent bacterial strains [13,32,[44][45][46][47][48][49]. So, there are many factors to be considered for optimal production from the selected isolate. In our study, medium M2 has been identifed as the best medium among the selected ones for protease production. Sucrose in the medium induces protease production [32] and nitrate salt helps to accelerate the production [50]. While in some, yeast extract gave high protease production than nitrate [13], and in some gelatin induced protease production [11]. Tere has also been a report of better alkaline production in the presence of glucose as a carbon source and yeast extract and peptone as an organic nitrogen source [12,33]. Te results shown by our isolates are also diferent for diferent isolates. BKHE produced high alkaline protease in presence of yeast extract, sucrose, and potassium nitrate and low production in gelatin-based and glucose-based mediums. So, the selection of appropriate carbon and nitrogen source is highly dependent upon the bacterial strain and needs to be optimized as per the requirement of the strain.
16S rRNA sequencing showed that the isolate is "Bacillus amyloliquefaciens BKHE" and further phenotypic features of the strain were studied to conclude the taxonomy with the polyphasic approach [18,51]. Phenotypic features of the selected strain were compared with phenotypic features of B. amyloliquefaciens [18] B. velezensis [40] and B. valismortis [18] obtained from the database. Since BLAST result and phylogenetic analysis ( Figure 6) suggest the strain belongs to B. amyloliquefaciens but diferentiating margins with other species were little. While comparing the phenotypic features in Table 2, BKHE was found to match most of the features with B. amyloliquefciens, the only diferences observed were in the fermentation of rafnose and salicin. B. amyloliquefciens and BKHE are oxidases negative while B. velezensis and B. valismortis are positive. Phenotypic features of BKHE that difer from B. velezensis were H2S production and utilization of lactose, xylose, rafnose, melibiose, L-arabinose, mannose, salicin, inositol, α-methyl-D-glucoside, and ONPG as shown in the table. Diferences between BKHE and B. valismortis also can be observed in Table 2. On basis of both 16S rRNA sequencing and phenotypic features of the strain BKHE, it was identifed as "B. amyloliquefciens BKHE."

International Journal of Microbiology
International Journal of Microbiology Alkaline protease with desirable properties such as activity at high pH, salinity, and temperature is suitable for commercial application in detergent industries [44,52]. Proteases from hyperthermophiles and thermophiles are the natural choice for exploring inherent heat stability [53]. So, exploring ecological niches such as extreme or high temperature, pH, salinity, pressure, and toxicity is one of the promising ways to isolate microbes with required properties [19,53]. Kinema, also being alkaline and dried, makes a suitable niche for isolating alkaline protease-positive isolates. B. amyloliquefaciens BKHE was found to have confuent growth at 50°C as the kinema sample was sundried and thermotolerant strains were able to cope with the drying process. Also, this strain was found to be tolerant to high salt concentration and high pH, so alkaline protease produced from this isolate could be used in formulations of biodetergent, although further screening in purifed enzymes is yet to be done. Also, this strain could be used for the coproduction of protease, amylase, and lipase, which are also used in detergent formulations.

Conclusion
A bacterial isolate with high alkaline protease-producing ability was identifed as B. amyloliquefaciens BKHE based on 16S rRNA sequencing and phenotypic features. Te strain was found to be cellulase negative and was positive for protease, amylase, and lipase. Also, its ability to withstand high temperatures and osmolarity opens the door for versatile applications of bacteria or enzymes in the food, feed, and detergent industry. Since our strain produces multiple enzymes, it can be used for the coproduction of multiple enzymes which could be used in the detergent and food industry.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this paper.

References
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