VETERINARY RESEARCH INTERNATIONAL Journal homepage: www.jakraya.com/journal/vri
ORIGINAL ARTICLE
Isolation, Molecular Characterization, Purification and Production Optimization of Alpha Galactosidase Enzyme from Bacillus subtilis P. Raja1*, M. Parthiban1 and Ghadevaru Sarathchandra2
1Department of Animal Biotechnology, 2Dean, Faculty of Basic Sciences, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-07. *Corresponding Author: Dr P. Raja Email: [email protected] Received: 18/04/2020 Accepted: 02/05/2020
Abstract The enzymes have great prospective in diverse industrial sectors and
their commercial usage has been increased in the recent years. The enzyme alpha-galactosidase is a glycoside hydrolase enzyme that hydrolyses the terminal α-galactosyl moieties from glycolipids and glycoproteins. These enzymes found to be extensively used in various industries viz. food, feed, sugar, paper and pulp. In the present study, three Bacillus subtilis strain was isolated from soil and their cultural, biochemical and molecular characterization was carried out using 16S rRNA sequencing before used for production of α-galactosidase. The optimum conditions for growth and enzyme induction were determined over the defined period of time. The enzyme was purified by ammonium sulfate precipitation method and the optimum conditions for the enzyme reaction were pH 3 at 37°C. The purified enzyme was stable at 37°C and in buffer at pH 3. The enzyme has been isolated and characterized from microbial sources and ubiquitous nature of this enzyme possesses potential industrial applications. Optimization of the growth conditions and efficient purification strategies can lead to a significant increase in the enzyme production. Keywords: Alpha-galactosidase, Bacillus subtilis, Purification, Characterization.
1. Introduction The α-galactosidase is a reducing disaccharide
that releasing glucose and galactose. It degrades raffinose, a trisaccharide, and stachyose, a tetrasaccharide composed of galactose, fructose and glucose, which are ubiquitously found in a large variety of legumes and vegetables. The ability of α-galactosidase to degrade raffinose and stachyose is highly useful in the food industry for elimination of non-digestible oligosaccharides (NDO) in soy - and legume-derived products (Shibuya et al., 1997).
The enzyme used to improve the nutritive value of feedstuffs by pre-treatment of animal feed with α -galactosidase (Wang et al., 2010). These enzymes also used for medical applications such as treating of fabry disease (mutations of the a-galactosidase gene) using enzyme replacement therapy with α-galactosidase (Fabrazyme (agalsidase beta) and Replagal (agalasidase alpha) produced using recombinant DNA technologies (Pastores, 2007). Another application, which is of great interest in the medical field, is the conversion of B-type blood antigens to O-type antigen (Liu et al., 2007; Olsson and Clausen, 2008). Most of the eukaryotic α -galactosidases including those from plants, animals and fungi (Henrissat, 1991).
Apart from eukaryotic organisms and only very few prokaryotes has been reported including Clostridium josui, Pseudomonas subsp., Streptomyces
spp., Bacillus ssp and Pyrocossus spp (Jindou et al., 2002; Anggraeni et al., 2008). Bacillus spp is considered as ideal candidate for production of protease-resistant enzymes and it produce several proteases such as subtilisin A, alkaline protease, neutral protease, and keratinase.
The growth conditions of Bacillus spp is complex and diverse, with a considerable portion of species existing in soil, animal intestines, and plant tissue, wherein diverse proteases was found. Various proteins from Bacillus have been reported to be protease-resistant (Huang et al., 2017). The potential use of α-galactosidases has accelerated research in α-galactosidases from eukaryotic and microbial sources. Since the microbes provides greater production of α-galactosidases due to its high expression levels, extracellular secretary nature, ease of cultivation and scope for improvement of yield by optimization of culture conditions. Hence, several microorganisms have been exploited for the production of α-galactosidase for use in various biotechnological and medical applications.
Besides several sources, α-galactosidase from bacteria, especially probiotic bacteria have been reported since they are used as ‘‘live cultures’’ in fermented soymilk (Farzadi et al., 2011) and α-galactosidases from extremophilic bacteria have been the source for extremely thermostable enzymes that can
Raja et al...Isolation, Molecular Characterization, Purification and Production Optimization of Alpha Galactosidase Enzyme from Bacillus subtilis
withstand the high-processing temperatures in pasteurization of soymilk (Brouns et al., 2006). The aim of the present study is to assess the ability of the Bacillus spp for the production of α-galactosidase and its optimization for the maximum enzyme production and their activity. 2. Materials and Methods 2.1 Sample Collections
Total eight (8) soil samples (10 grams each) were collected from different locations in and around Chennai. Each sample was packaged in a sterile bottle and labelled appropriately. The soil samples were brought to the Department of Animal Biotechnology, Madras Veterinary College, Chennai for further analyses. 2.2 Isolation of Bacillus subtilis
Ten grams (10g) of each soil sample was suspended in 90 ml of sterile distilled water. The soil suspension was heat shocked at 60oC for one hour in a water-bath to kill non-spore forming organisms (Ubalua, 2014).
A loopful each of the soil suspension was inoculated by streaking on nutrient agar medium. The inoculated plates were incubated aerobically at 37oC for 24 hrs and examined for the appearance of colonies. The colonies that exhibited cultural characteristics typical of Bacillus species i.e. round or irregular; thick and opaque; cream-colored colonies were sub-cultured onto nutrient agar slants for subsequent identification. 2.3 Identification of Bacillus subtilis
Bacillus subtilis isolates were primarily identified on the basis of taxonomic properties and microgen identification system. The characteristic morphological, cultural and biochemical properties were observed (Bergey, 2004; Cowan and Steel, 2003). 2.3.1 Cultural Characterization
Isolates on nutrient agar plates were examined for size, pigmentation, form, margin and elevation of the colonies. 2.3.2 Morphological Characterization
Morphological characteristics such as the cell shape, cell arrangement as well as the Gram’s reaction of the organism were determined by Gram staining technique. Endospore-staining technique was also carried out to morphologically characterize the isolates. 2.3.3 Biochemical Characterization
Bacillus subtilis isolates were further subjected to HiBacillus identification system following standard procedures (HiMedia). Biochemical tests such as catalase, motility, citrate, urease, indole, Methyl red, Voges Proskauer, nitrate reduction, starch hydrolysis as
well as sugar fermentation were carried out according to standard procedures. 2.4 Genomic DNA Extraction and PCR Assay
The genomic DNA was extracted as per method described by Ausubel et al. (1995). Briefly, 2 ml of overnight grown cultures in nutrient broth was used. A 1.5 ml of the culture was centrifuged at 12 000 g for 10 min and the resultant pellet was re-suspended in 500 µl 1 × TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Proteinase K and SDS were added to final concentrations of 100 µg/ml and 0.5% respectively, and incubated at 37ºC for 1 h. After incubation, NaCl (5 M) and CTAB/NaCl (10% w/v cetyl trimethyl ammonium bromide in 0.7 M NaCl) were added and incubated at 65ºC for 10 min. The mixture was extracted once each with an equal volume of chloroform-isoamyl alcohol (24:1) and phenol-chloroform-isoamyl alcohol (25:24:1). DNA was precipitated from the aqueous phase using 0.6 volumes of isopropanol and washed once with 70% ethanol.
The DNA pellet obtained after final centrifugation was air dried and dissolved in 30 µl 1 × TE buffer and then stored at -20°C for further use. The PCR reaction was carried out for total volume of 50 µl containing 25 µl of Redtaq PCR master mix, 1 ml each of forward primer (FP-5’-AGAGTTTGATCCTGGCTCAG-3’) and reverse primer (RR- 5’-AGGAGGTGATCCAACCGCA-3’) for amplification of 16S rRNA universal sequence, 5 ml of template, and 18 ml of nuclease-free water. 2.5 Optimization of PCR
The PCR amplification was carried out for 16S r-RNA as following conditions: An initial denaturation step at 94°C for 4 min followed by a second denaturation step at 94°C for 1 min, annealing for 1 min at 62°C, an extension at 72°C for 90 s, and a final extension step of 72°C for 10 min. A total of 35 serial cycles of amplification reaction was performed. 2.6 Sequence Analysis
The PCR amplified products were purified using PCR gel purification kit (Bio Basic Inc, Canada) and sequencing was performed at M/s. Shrimpex Biotech, Chennai-600019 (India).
The nucleotide Sequence data was subjected to BLAST analysis (www.ncbi.nlm.nih.gov), assembled and analyzed using Seqman and MegAlign programs of Lasergene package (version 7.1.0) (DNA Star Inc. Madison, WI). Nucleotide sequence alignment was performed by ClustalW method with MegAlignTM program (DNA Star Inc), and the predicted amino acid sequence was analyzed by ProteanTM program of Lasergene (DNA Star Inc). Phylogenetic analysis of 16S rRNA sequence was performed using maximum likelihood method with 1000 bootstrap replication in the MEGA software version 6.
Raja et al...Isolation, Molecular Characterization, Purification and Production Optimization of Alpha Galactosidase Enzyme from Bacillus
2.7 Organic Solvent PrecipitationThe collected supernatant is used to enzyme
assay by chilled acetone: methanolratio) method at 4°C. Further, the precipitate is collected by centrifuging at 8000 rpm for 10 mins at 4°C and same is dissolved in 0.02M citrate phosphate buffer (pH 7) and used for enzyme assay. 2.8 Ammonium Sulphate P
Purification of α-GalactosidaseThe centrifuged cell supernatant is precipitated
using 70% ammonium sulphate method. The precipitate is collected by centrifuging at 8000 rpm for 15 mins4°C and dissolved and dialyzed in 0.05 M citrate phosphate (pH. 7) buffer. 2.9 α-Galactosidase Enzyme Assay
The α-Galactosidase enzyme assay is performed using ρ-nitrophenyl α-D-galactopyranoside (as substrate. Briefly, 200 µL of 100 mM sodibuffer (pH 5.0), 250 µL of 2 mM ρNPGal solution and 50 µL of enzyme preparation is used.
The reaction was carried out for 15 min at 50and the reaction is terminated by the addition of 1 ml of 0.5 M sodium carbonate. The amount of (ρNP) released was determined at 410 nm. The unit of enzyme production is assessed as the galactosidase which liberates 1 µmol of under optimized assay conditions. 2.10 Effect of pH and Temperature on Enzyme
Production To estimate the optimum pH and to assess the
effect of temperature for α-galactosidase activity were studied. For this, range of pH (citrate phosphate buffer, range of pH: 2.5 to 7.5) and the effect of temperature (30-80̊C), different incubation temperatures were studied. 3. Results and Discussion 3.1 Isolation and Identification of
subtilis Out of eight isolates, three isolates were
confirmed to be B. subtilis based on cultural, microscopic, biochemical as well as molecular characterization using 16S rRNA sequencing (Table 1).
All the three isolates are Gram shaped short chains and produced sub terminal endospores. The motile colonies are pale yellowpigmented, flat and translucent, aerobic, growth occurs in 37°C (Fig 1). Briefly, they are positive for catalase, citrate utilization and negative for methyl red. They also reduced the nitrate and hydrolyzed the starch. 3.2 PCR Amplification and Sequence Analysis
of 16S rRNA
Raja et al...Isolation, Molecular Characterization, Purification and Production Optimization of Alpha zyme from Bacillus subtilis
Veterinary Research International | April-June, 2020 | Volume 08 | Issue 02 | Pages 88-93
90
Organic Solvent Precipitation The collected supernatant is used to enzyme
ne: methanol precipitation (1:1 ratio) method at 4°C. Further, the precipitate is collected by centrifuging at 8000 rpm for 10 mins at 4°C and same is dissolved in 0.02M citrate phosphate buffer (pH 7) and used for enzyme assay.
Precipitation for Galactosidase
The centrifuged cell supernatant is precipitated using 70% ammonium sulphate method. The precipitate is collected by centrifuging at 8000 rpm for 15 mins at 4°C and dissolved and dialyzed in 0.05 M citrate
Galactosidase Enzyme Assay Galactosidase enzyme assay is performed
galactopyranoside (ρNPGal) as substrate. Briefly, 200 µL of 100 mM sodium acetate buffer (pH 5.0), 250 µL of 2 mM ρNPGal solution and 50 µL of enzyme preparation is used.
as carried out for 15 min at 50°C and the reaction is terminated by the addition of 1 ml of 0.5 M sodium carbonate. The amount of ρ-nitrophenol
NP) released was determined at 410 nm. The unit of oduction is assessed as the amount of α-
galactosidase which liberates 1 µmol of ρNP per min
Temperature on Enzyme
te the optimum pH and to assess the alactosidase activity were
studied. For this, range of pH (citrate phosphate buffer, range of pH: 2.5 to 7.5) and the effect of temperature
˚C), different incubation temperatures were
Isolation and Identification of Bacillus
Out of eight isolates, three isolates were based on cultural,
microscopic, biochemical as well as molecular rRNA sequencing (Table 1).
are Gram positive; rod shaped short chains and produced sub terminal
olonies are pale yellow-pigmented, flat and translucent, aerobic, growth occurs
. Briefly, they are positive for catalase, citrate utilization and negative for urease, indole and
ed. They also reduced the nitrate and
Sequence Analysis
In the present study, theamplified using the specific primers for all three different isolates of Bacillus subtilis soils of different locations in and around Chennai (Fig 2) and further their sequence has been determined by sequencing.
Using BLAST search analysis, all the three strains are belongs to species identities of these three Bacillus97% to 100%.
Fig 1: Colonies of
Fig 2: PCR amplification of 16s rRNA gene of subtilis.
M-100 bp marker, L1-L3-Isolates of Bacillus subtilis, L4Negative Control 3.3 Phylogenetic Analysis of Three Isolates of
Bacillus subtilis The phylogenetic tree was constructed by using
the Maximum Likelihood method based on the Tamura-Nei model (Tamura The 16S rRNA gene is used it is highly conserved between different species of bacteria. It is suggested thatused as a reliable molecular clock becausesequences from distantly related bacterial lineages are shown to have similar functionalities.
Most of the strains of the present study, while constructing the phylogenetic tree these strains are formed the distinct cluster. The closest relationship was found between and B. huizhouensis. In similar context Earl (2007) reported that analysis using 16S rRNA gene sequences from each strain failed to distinguish the subtilis subspecies or B. vallismortis
Raja et al...Isolation, Molecular Characterization, Purification and Production Optimization of Alpha
In the present study, the 16S rRNA gene was amplified using the specific primers for all three
Bacillus subtilis collected from s in and around Chennai (Fig
2) and further their sequence has been determined by
arch analysis, all the three strains are belongs to species Bacillus subtilis. The
Bacillus isolates were ranged
Fig 1: Colonies of Bacillus subtilis.
Fig 2: PCR amplification of 16s rRNA gene of B.
Isolates of Bacillus subtilis, L4-
3.3 Phylogenetic Analysis of Three Isolates of
The phylogenetic tree was constructed by using the Maximum Likelihood method based on the
Nei model (Tamura and Nei, 1993) (Fig 3). for phylogenetic studies as
it is highly conserved between different species of bacteria. It is suggested that 16S rRNA gene can be
as a reliable molecular clock because 16S rRNA ly related bacterial lineages are
shown to have similar functionalities. Most of the strains of Bacillus spp included in
the present study, while constructing the phylogenetic tree these strains are formed the distinct cluster. The
found between B. aryabhattai . In similar context Earl et al.
(2007) reported that analysis using 16S rRNA gene sequences from each strain failed to distinguish the B.
B. vallismortis as –
Raja et al...Isolation, Molecular Characterization, Purification and Production Optimization of Alpha Galactosidase Enzyme from Bacillus subtilis
Fig 3: Phylogenetic analysis of Bacillus subtilis.
The phylogenetic tree reveals all the three strains of B. subtilis are formed the distinct cluster along with other Bacillus spp obtained from NCBI. The closest relationship was found between B. aryabhattai and B. huizhouensis.
phylogenetically distinct taxa due to the limited number of informative sites at these loci. Moreover, other researchers (Freitas et al., 2008; LimaBittencourt et al., 2007; Pontes et al., 2007) reported that analysis of 16S rRNA gene sequences alone is not sufficient to identify Bacillus species.
Fig 4: Production of alpha-galactosidase from both curde and
partially purified B. subtilis culture. The maximum alpha galactosidase activity was reported at 8th day in purified B. subtilis. 3.4 Production of α-Galactosidase Enzyme
The overnight grown fresh cultures of Bacillus subtilis was inoculated in 200 ml of nutrient broth and
kept it at 200 rpm in shaker incubator at 37˚C. The 70% ammonium sulphate partially purified B. subtilis and to assess the α-galactosidase enzyme activity by using p-nitrophenyl α-D galactopyranoside as substrate. However, maximum alpha galactosidase activity was reported at 8th day in partially purified B. subtilis (Fig 4). There are several reports for the production of α-galacosidase from A. niger (Adya and Elbein, 1977), T. reesei (Zeilinger et al., 1993), Mortierella vinacea (Shibuya et al., 1995) and Thermotoga neaplolitana (Duffaud et al., 1997). 3.5 Characterization and Optimization of
Purified Enzyme The α-Galactosidase was observed to have
maximum enzyme activity at pH 3 with incubation temperate of 37-40°C (Fig 5). Gajdhane and Dandge (2016) reported maximum α-Galactosidase activity at the pH 5 with incubation temperature 50°C produced from Fusarium moniliforme. Rezende et al. (2004) reported production of α-galactosidase purified at the temperature range of 45-65°C and at the pH range of 4.0-5.0. Shibuya et al. (1995) also reported maximal α-galactosidase production at 55°C and pH 4.5 from P. Purpurogenum. Guimaraes et al. (2001) reported
Raja et al...Isolation, Molecular Characterization, Purification and Production Optimization of Alpha Galactosidase Enzyme from Bacillus subtilis
Fig 5: Temperature and pH optimization of α-galactosidase
activity. The α-Galactosidase was observed to have maximum enzyme activity at pH 3 with incubation temperate of 37-40°C. 4. Conclusion
At present study, the Bacillus subtilis was isolated from the soil and their cultural, morphological, biochemical and molecular characterization was carried out. The ability of the isolated strains for production of α-galactosidase was assessed using partially purified B. subtilis strain. In recent years, the production of industrially important microbial enzymes is the potential area of research since they are highly efficient and easy available nature. Hence, the present study emphasizes the production of α-Galactosidase from Bacillus subtilis. Acknowledgements
This work was supported by Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) in the form of Sub project On “Novel sources of alpha galactosidase for enzyme lysis of galactose-alpha-1,3-galactose structure from eukaryotic cell membrane” (USO No. 50168/G5/2019) to the Department of Animal Biotechnology, Faculty of Basic Sciences, Madras Veterinary College, Chennai-07.
References Adya S and Elbein AD (1977). Glycoprotein enzymes
secreted by Aspergillus niger: Purification and properties of a-galactosidase. Journal of Bacteriology, 129: 850-856.
Anggraeni AA, Sakka M and Kimura T (2008). Characterization of Bacillus halodurans a-galactosidase Mel4A encoded by the mel4A gene (BH2228). Bioscience, Biotechnology and Biochemistry, 72: 2459-62.
Ausubel FM, Brent R, Kingsten RE, Moore DD, Seidman JG, Smith JA and Struhl K (1995). Short protocols in molecular biology. John Wiley and Sons, New York.
Bergey’s Manual of Determinative Bacteriology (2004). Eds., John G. Holt et al. (9th Edn.).
Brouns SJ, Smits N and Wu H (2006). Identification of a novel a-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. Journal of Bacteriology, 88: 2392-9.
Cowan ST and Steel KJ (2003). Manual for the identification of medical bacteria (3rd Ed) / edited and rev. by G.I.
Barrow and R.K.A. Feltham. Cambridge University Press. London. pp. 188-238.
Duffaud GD, McCutchen MC, Leduc P, Parker KN and Kelly RM (1997). Purification and characterization of extremely thermostable b-mannosidase, and a-galactosidase from the hyperthermophilic Eubacterium Thermotoganeapolitana 5068. Applied and Environmental Microbiology, 63: 169-177.
Earl AM, Losick R and Kolter R (2007). Bacillus subtilis Genome Diversity. Journal of Bacteriology, 189: 1163-1170.
Farzadi M, Khatami S, Mousavi M and Amirmozafari N (2011). Purification and characterization of a-galactosidase from Lactobacillus acidophilus. African Journal of Biotechnology, 10: 1873-9.
Freitas DB, Mariana P, Reis MP, Lima-Bittencourt CI, Costa PS, Assis PS, Chartone Souza E and Nascimento AMA (2008). Genotypic and phenotypic diversity of Bacillus spp. isolated from steel plant waste. BMC Research Notes, 1: 92-103.
Raja et al...Isolation, Molecular Characterization, Purification and Production Optimization of Alpha Galactosidase Enzyme from Bacillus subtilis
Gajdhane and Dandge (2016). Production optimization, purification and characterization of the α-galactosidase from fusarium moniliforme NCIM 1099. Research Journal of Life Sciences, Bioinformatics, Pharmaceutical and Chemical Sciences, 2(1): 21-32.
Guimaraes VM, Rezende ST, Moreira MA, Barros EB and Felix CR (2001). Characterization of a-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides. Phytochemistry, 58: 67-63.
Henrissat B (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 280: 309-16.
Huang Y, Zhang H, Ben P, Duan Y, Lu M and Li Z (2017). Characterization of a novel GH36 alpha-galactosidase from Bacillus megaterium and its application in degradation of raffinose family oligosaccharides. International Journal of Biological Macromolecules, 108: 98-104.
Jindou S, Karita S and Fujino E (2002). A-Galactosidase Aga27A, an enzymatic component of the Clostridium josui cellulosome. Journal of Bacteriology, 184: 600-4.
Lima-Bittencourt CI, Astolfi-Filho S, Chartone-Souza E, Santos FR and Nascimento AMA (2007). Analysis of Chromobacterium sp. natural isolates from different Brazilian ecosystems. BMC Microbiology, 7: 1-9.
Liu QP, Sulzenbacher G and Yuan H (2007). Bacterial glycosidases for the production of universal red blood cells. Nature Biotechnology, 25: 454-64.
Olsson ML and Clausen H (2008). Modifying the red cell surface: Towards an ABO-universal blood supply. British Journal of Haematology, 140: 3-12.
Pastores GM (2007). Agalsidase (Replagal) in the treatment of Anderson-Fabry disease. Biologics, 1: 291-300.
Pontes DS, Lima-Bittencourt CI, Chartone-Souza E and Nascimento AMA (2007). Molecular approaches: Advantages and artifacts in assessing bacterial diversity. Journal of Industrial Microbiology and Biotechnology, 34: 463-473.
Rezende, Sebastião Tavares de, Guimarães, Valéria Monteze, Rodrigues, Marília de Castro and Felix, Carlos Roberto (2005). Purification and characterization of an alpha-galactosidase from Aspergillus fumigatus. Brazilian Archives of Biology and Technology, 48(2): 195-202.
Shibuya H, Kobayashi H and Sato T (1997). Purification, characterization and cDNA cloning of a novel a-galactosidase from Mortierella vinacea. Bioscience, Biotechnology and Biochemistry, 61: 592-8.
Shibuya H, Kobayashi H, Park GG, Komatsu Y, Sato T, Kaneko R, Nagasaki H, Yoshida S, Kasamo K and Kusakabe I (1995). Purification and some properties of a-galactosidases from Penicillium purpurogenum. Bioscience, Biotechnology and Biochemistry, 59: 2333-2335.
Ubalua AO (2014). The use of corn starch for growth and production of α-amylase from Bacillus subtilis. Journal of Microbiology Research, 4(4): 153-160.
Wang H, Luo H and Li J (2010). An a-galactosidase from an acidophilic Bispora sp. EY-1 strain acts synergistically with b-mannanase. Bio-Resource Technology, 101: 8376-82.
Zeilinger S, Kristufek D, Arissan-Atac I, Hodits R and Kubicek CP (1993). Condition of formation, purification, and characterization of an a-galactosidase of Trichodrema reesei RUT C-30. Applied Environment Microbiology, 59: 1347-1353.
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