Purification and characterisation of intracellular alpha-galactosidases from Acinetobacter sp.

Two alpha-galactosidases (Ag-I & Ag-II) were purified from Acinetobacter sp. Both the enzymes were monomeric with pH optima of 7.0 and molecular weight of 65 kDa for Ag-I and 37 kDa for Ag-II. The temperature optima for Ag-I was between 50 and 60 °C and that of Ag-II was 40 °C. Both the enzymes were strongly inhibited by metal ions Ag2+ and Hg+, pCMB and SDS (1 %). The enzymes were found to be active on both natural and synthetic substrates. Artificial substrate, pNPGal, has shown more affinity to enzyme than natural substrate raffinose. The half-life (t 1/2) of Ag-I varied from 1.85 h at 90 °C to 7.6 h at 70 °C.


Introduction
In recent years, a substantial amount of interest has been generated on carbohydrate-cleaving enzymes due to their potential industrial and therapeutic applications. a-Galactosidases are enzymes that belong to family of glycosyl hydrolases. Based on aminoacid sequences, alpha-galactosidases are included in four different families-GH4, GH27, GH36 and GH57 (Naumoff 2001). Most of the known bacterial and eukaryotic alpha-galactosidases with confirmed activity belong to GH27 and GH36, which constitute a superfamily (clan GH D) (Naumoff 2004).
a-Galactosidases catalyse hydrolysis of terminal a-1-6 linked galactose residues from different galactooligosaccharides, glycosphingolipids and glycoproteins (Dey and Pridham 1972) and also show transgalactosylation (Kato et al. 1982) and hemagglutination activity (del Campillo and Shannon 1982). Due to its diverse catalytic activity, the enzyme is used in food processing industry for improvement of nutritional value of legumes and animal feeds by removing the flatulence-causing factors (Patil et al. 2010), in beet sugar industry for crystallisation of sugars (Yamane 1971) and a-galactosidase producing organisms as probiotic (Chen and Mustapha 2012;Liu et al. 2014). Recombinant alpha-galactosidases such as Fabrazyme TM and Replagal TM are being currently employed in the treatment of Fabry's disease which is a rare X-linked lysosomal storage disorder (Tsuboi and Yamamoto 2012;Desnick 2004). The enzyme was reported to be applied for the conversion of Type 'B' erythrocytes to Type 'O' erythrocytes (Gao et al. 2011) and also in xenotransplantation (Zeyland et al. 2013). This enzyme is also used in bleaching of softwood in the paper industry (Clarke et al. 2000).
Although a-galactosidases were isolated and purified from various organisms (Singh and Kayastha 2012;Puchart et al. 2001;Saishin et al. 2010;Gao and Arthur 1999), the versatility of this enzyme and the demand for stable a-galactosidase are constantly increasing despite the availability. The present work was taken up to isolate and purify thermostable a-galactosidase from Acinetobacter sp., which will be later applied in degradation of galactooligosaccharides to increase nutritive value of legumes.

Microorganisms
Microorganisms showing a-galactosidase activity were isolated from soil collected from sugarcane industries situated at Sangareddy, Andhra Pradesh. The cultural, morphological and biochemical characteristics of the isolate were identified according to Bergey's Manual of Determinative Bacteriology, 8th edition (Buchanan and Gibbons 1974) and confirmed by 16srRNA sequencing analysis.

Crude enzyme preparation
A loopful of the isolate G1 was inoculated into optimised fermentation medium comprising raffinose 25 g/L, tryptone 10 g/L, K 2 HPO 4 10 g/L, MgSO 4 Á7H 2 O 1 g/L and FeSO 4 Á7H 2 O 1 g/L (pH 7.0). The cultures were grown at 36°C with an agitation of 170 rpm for 12 h. Cells were harvested and suspended in 20 mM Tris lysis buffer (pH 7.0) containing 0.3 g/100 ml lysozyme, 0.1 g/100 ml Triton X 100 and 1 mM PMSF and incubated for 1 h at 30°C. The lysate was centrifuged at 10,000g for 20 min at 4°C. The supernatant thus collected was used to determine intracellular a-galactosidase activity.
Enzyme and total protein assay a-Galactosidase activity was measured according to the method of Dey et al. (1993). A reaction mixture containing 550 ll of 20 mM Tris buffer (pH 7.0), 100 ll of enzyme preparation and 250 ll of 0.2 M pNPGal was incubated at 50°C for 10 min and the reaction was stopped by addition of 0.2 mM Na 2 CO 3 and read at 405 nm. The activity was also measured using 2 mM raffinose as natural substrate in a reaction mixture containing 20 mM Tris buffer (pH 7.0) and enzyme preparation. The resulting amount of reducing sugar was determined by addition of 3,5-dinitrosalicylate reagent (Miller 1959) and the colour read at 540 nm. The concentration of reducing sugar, i.e., galactose, was calculated using standard galactose curve.
One enzyme unit (U) of activity was defined as the amount of enzyme required to produce one lmol of q-nitrophenol or reducing sugars (galactose) per min under the above mentioned assay conditions.
The protein content of the enzyme preparation was determined according to the method of Lowry et al. (1951) using Bovine Serum Albumin (BSA) as standard.

Purification and characterization of enzyme a-galactosidase
Step 1: ammonium sulphate precipitation The precipitate obtained at 60 % (NH 4 ) 2 SO 4 saturation was harvested by centrifugation at 10,000g for 30 min, 4°C. The pellet obtained was dissolved in 20 mM Tris-HCl buffer (pH 7.2) and dialysed at 4°C with three changes of buffer at 4°C.
Step 2: ion exchange chromatography The dialyzed sample was loaded on to Q-Sepharose fast flow column with a bed height of 9 cm and internal diameter of 0.5 cm which was previously equilibrated with 20 mM Tris-HCl, pH 7.2 and a flow rate of 1 ml/min. The column was washed with three bed volumes of 20 mM Tris-HCl, pH 7.2 to remove unbound protein. The bound proteins were eluted using NaCl gradient from 0.1 to 1 M. Both unbound and bound fractions were concentrated and analysed for a-galactosidase activity using qNPGal as substrate.
Step3: gel filtration chromatography The fractions showing a-galactosidase activity were concentrated and loaded on to Sephacryl S-300 gel filtration column (1 9 90 cm) pre-equilibrated with 20 mM Tris-HCl, pH 7.2. 1 ml fractions were collected at a flow rate of 0.5 ml/min. Fractions exhibiting activity were concentrated and analysed for purity by PAGE.
Native and SDS-PAGE electrophoresis SDS Polyacrylamide gel electrophoresis was performed in 10 % gels to determine molecular mass and purity of the enzyme (Laemmli 1970).
The enzyme was also electrophoresed in Native 10 % gels to check for purity (Davis 1964). The protein bands were visualised by staining with Coomassie Brilliant Blue 250.

pH optimum
The pH optima for activity of the enzyme was assessed by monitoring the enzyme activity in different buffers with pH ranging from 5.0 to 11.0-citrate phosphate buffer (pH 5.0-7.0), Tris-HCl buffer (pH 7.0-9.0) and glycine-NaOH buffer (pH 9.2-11.0). The pH stability of the purified enzymes Ag-I & II was analysed by incubating them indifferent buffers with pH values ranging from 5 to 11 for a time period of 1 to 24 h at 36°C.

Temperature optimum, stability and half-life
The temperature optima for maximal activity of the pure enzyme was determined by incubating the reaction mixture at different temperatures ranging from 30 to 90°C in Tris-HCl buffer (pH 7.0). The half-life (t 1/2 min) of the enzyme Ag-I was determined by incubating the enzyme at different temperatures ranging from 70 to 90°C for 5 h.

Determination of kinetic constants
The kinetic parameters, K m , V max and K cat values of the purified a-galactosidase enzyme Ag-I and Ag-II were determined from Lineweaver-Burk plot. The rate of hydrolysis was determined with concentration ranging from 0.4 to 2.8 mM for both the substrates-p-nitrophenyla-D-galactopyranoside (pNPGal) and raffinose against enzyme activity. The reaction was carried out at 50°C in 20 mM tris buffer, pH 7.0. A graph was plotted with 1/v versus 1/s. K m and V max values were calculated based on linear regression slope. Further K cat value was calculated using formula where E t is the total enzyme concentration.
Influence of metal ions, inhibitors and surfactants on a-galactosidase activity The influence of different metal ions such as Mg 2? , K ? , Ca 2? , Cu 2? , Co 2? , Ag 2? , Hg ? , Zn 2? and chelator like EDTA were studied by preincubating the enzymes Ag-I & II with 5 mM concentration of ions in 20 mM Tris buffer at pH 7.0 for 1 h at 36°C and then checking for enzyme activity. The activity was also monitored after pre incubation with sugars like glucose, galactose, lactose, stachyose and other inhibitors like urea, PCMB (q-Chloro-mercuric benzoic acid), b-mercaptoethanol and EDTA at a concentration of 5 mM for 1 h at 36°C and also with detergents like SDS, Triton X-100 and Tween-20 at a concentration of 1 % for 4 h.

Results and discussion
Identification of the isolate A microorganism with the ability to produce alpha galactosidase enzyme was isolated from sugarcane waste and was identified as Acinetobacter sp., from its morphological features, biochemical characteristics and 16srRNA sequencing analysis. Industrially important enzymes-pectinases, lipases and biodegradable enzymes were reported to be synthesised by Acinetobacter species (Shafqat et al. 2015; Das and Chandran 2011).

a-Galactosidase purification and molecular weight
The enzyme was purified by (NH 4 ) 2 SO 4 precipitation, ion exchange chromatography and gel-filtration. Two forms of a-galactosidases were resolved in gel filtration chromatography with molecular weights of 65 kDa for Ag-I and 37 kDa for Ag-II (Fig. 1). Both the enzymes were monomeric (Fig. 2a, b). The enzyme recovery from the crude is shown in Table 1. Multiple forms of a-galactosidase have been reported in both prokaryotes and eukaryotes (Pederson and Goodman 1980;Gherardini et al. 1985;Alani et al. 1989).

pH optimum and stability
Both Ag-I and Ag-II were found optimally active and stable at neutral pH. Enzyme Ag-I was highly active and stable over broad pH range showing more than 60 % activity up to pH 9.0 with low activities at acidic pH. After 24 h of incubation at 36°C, Ag-I retained 85 % enzyme activity at pH 7.0, whereas Ag-II retained 52 % activity (Fig. 3a, b). Most of the thermostable a-galactosidases were reported to be stable at neutral pH (Pederson and Goodman 1980;Frank et al. 1985;Alani et al. 1989;Gote et al. 2006).

Optimum temperature and thermal stability
The purified Ag-I enzyme showed an optimum temperature between 50 and 60°C and was able to retain more than 80 % activity at 70°C and thereafter a sharp decrease was observed at temperatures above 70°C. Ag-II displayed an optimum temperature at 40°C but markedly decreased at higher temperatures above 60°C (Fig. 4). The half-life of Ag-I at 70°C was 7.69 hours and thereby decreased rapidly ( Fig. 5; Table 2). a-Galactosidases from Bifidobacterium  breve (Xiao et al. 2000) and Lactobacillus fermentum (Carrera-Silva et al. 2006) were reported to have a temperature of 50°C. a-Galactosidases isolated from few hyperthermophilic bacteria were reported to be stable with half-life ranging from 6 to 9 h at 85°C (Miller et al. 2001;Duffaud et al. 1997) and 17 h at 80°C (Giuseppin et al. 1993). Even in the present study the enzyme Ag-I had shown high thermal stability which can be further exploited for processing of legumes at higher temperatures.

Effect of metal ions on enzyme activity
Assay of enzyme activity in the presence of different metal ions, sugar and non-sugar inhibitors and detergents indicated that both the enzymes (Ag I & II) were completely inhibited by Ag 2? , Hg ? and partially in presence of Cu 2? . Mg 2? and Co 2? stimulated activity whereas other metal ions showed greater than or equal to 90 % activity (Fig. 6).
With respect to Ag II the activity was found to be reduced between 75 and 60 % in the presence of metal ions-K ? , Ca 2? and Zn 2? . Ag-I & II were neither not strongly stimulated by metal ions nor inhibited by EDTA, indicating that it does not require any cofactor for enzyme activity. a-Galactosidases isolated from different bacterial species  were reported to be strongly inhibited by Ag 2? , Hg ? ions and Cu 2? (Fridjonsson et al. 1999;Zhao et al. 2008;Akiba and Horikoshi 1976). Chinen et al. (1981) suggested that the inhibition may be due to reaction with amino, carboxyl and imidazolium group of histidine present at the enzyme active site.

Effect of inhibitors and surfactants
The activity of enzymes (Ag-I & II) was decreased from 20 to 30 % in the presence of sugar inhibitors-glucose, galactose, lactose and stachyose ( Table 3). The observed decrease in activity may be due to competitive inhibition by sugars (Zapater et al. 1990). Similar observation of substrate/product inhibition by various sugars was reported in other a-galactosidases from bacterial and fungal species (Gote et al. 2006;Falkoski et al. 2006). Triton X-100 and Tween-20 enhanced the activity of Ag I. A slight decrease in activity was observed in the presence of Beta-mercaptoethanol. Both the enzymes were also strongly inhibited by pCMB, a thiol modifier and SDS. Inhibition by pCMB may be due to reaction with thiol groups at active site (Dey and Pridham 1972;Gote et al. 2006) and that of SDS may be due to strong affinity to proteins and its denaturing property (Falkoski et al. 2006).

Determination of kinetic constants for soluble enzyme Ag-I & II
The kinetic constants for both the forms of a-galactosidases are shown in Table 4. Both enzymes Ag-I and Ag-II  Tween-20 128 100 Sugar and non-sugar inhibitors are incubated with purified enzymes at a concentration of 5 mM for 1 h at 36°C. Detergents are incubated at a concentration of 1 % for 4 h Alpha-galactosidase I (Ag-I) qNPGal 14.51 0.33 4.45 9 10 2 13.48 9 10 2 Raffinose 27.24 3.26 1.1 9 10 2 0.33 9 10 2 Alpha-galactosidase II (Ag-II) qNPGal 6.73 0.68 1.52 9 10 2 2.23 9 10 2 Raffinose 5.44 0.74 0.6 9 10 2 0.81 9 10 2 have a higher affinity to synthetic substrate pNPGal (low K m value) as compared with natural substrate raffinose. The rate of hydrolysis is high for raffinose (Ag-I) although the K m value is high compared to synthetic substrate indicating that although the affinity is low, the catalytic processing is higher for the substrates.

Conclusion
Multiple forms of intracellular a-galactosidases (Ag I & II) isolated from Acinetobacter sp. were purified to homogeneity. Of both the enzymes, Ag I was found to be thermostable up to 70°C. This enzyme has potential applications of these enzyme preparations in high temperature catalytic processes.