Purification and Characterization of an Endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 Mutant

Summary

An extracellular endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 mutant was purified to homogeneity by gel filtration chromatography and showed a specific activity of 119 U/mg. The optimum pH and temperature of the purified enzyme were found to be 6.0 and 50 °C, respectively. The enzyme was stable up to 60 °C, retaining 60% of residual activity for 30 min, but inactivated rapidly above 60 °C. The enzyme was found to be stable at pH=6–9 when it retained 100% of its residual activity. The Lineweaver-Burk plot showed that the apparent K_m and v_max values of the inulinase when using inulin as a substrate were 1.15 mg/mL and 0.15 µM/min, respectively, whereas the k_cat value was found to be 0.145 min^–1. The calculated catalytic efficiency of the enzyme was found to be 0.126 (mg·min)/mL. The purified inulinase can be used in the production of high fructose syrups.

Introduction

Inulin is considered as a renewable raw material in the production of fructose syrup and fructooligosaccharides (FOS) (1) and hence, enzyme inulinases are widely used in food and pharmaceutical industries (2). Microbial inulinases can be classified into exo- and endo-acting enzymes according to their modes of action on inulin. Endoinulinases (2,1-β-d-fructan fructanohydrolase; EC 3.2. 1.7) are specific for inulin and hydrolyse the internal β-2,1-fructofuranosidic linkages to yield inulooligosaccharides such as inulotriose, inulotetraose and inulopentaose as their main products. Exoinulinases (β-d-fructan fructohydrolase; EC 3.2.1.80) successively cleave off terminal fructose units from the non-reducing end of inulin, and also hydrolyse sucrose and raffinose (3, 4). Therefore, inulinases could be used for production of either high fructose syrups by exo-enzymatic hydrolysis of inulin with d-fructose content over 95%, or for production of oligofructoside syrups by endo-enzymatic hydrolysis (5). Inulinases are produced by a few bacteria (Xanthomonas sp., Bacillus sp., Pseudomonas sp., Thermotoga sp., Bifidobacterium sp., Geobacillus sp. and Clostridium sp.), fungi (Aspergillus sp., Penicillium sp. and Fusarium sp.) and yeast (Kluyveromyces sp.) (4). The optimization of the nutritional and growth parameters of X. campestris pv. phaseoli for the production of endoinulinase using the submerged and solid-state cultivations has been reported earlier (6). The rate of endoinulinase and FOS production was further enhanced through ethylmethanesulphonate (EMS) mutagenesis of X. campestris pv. phaseoli and the mutant was named X. campestris pv. phaseoli KM 24 (Xcp KM 24) (7). The present study, therefore, focuses on the purification and characterization of endoinulinase from Xcp KM 24 using gel filtration chromatography.

Materials and Methods

Bacterial strain

Strain Xanthomonas campestris pv. phaseoli was obtained from the culture collection of the Department of Microbiology at the University of KwaZulu-Natal, Durban, South Africa. The strain was improved by chemical mutagenesis using EMS as described earlier (8) with slight modifications. Optimized medium was used to test the mutants for their ability to produce inulinase and FOS. The strain was named Xanthomonas campestris pv. phaseoli KM 24 (Xcp KM 24) (7) and stored as 70% glycerol stocks at –70 °C.

Endoinulinase production by Xanthomonas campestris pv. phaseoli KM 24

For the production of endoinulinase, the mutant Xcp KM 24 was inoculated into 50 mL of the inulinase production medium (IPM) containing (in g/L): inulin 20, yeast extract 20, (NH₄)₂HPO₄ 5, NH₄H₂PO₄ 2, MnCl₂ 0.5, KCl 0.5, MgSO₄ 0.5 and FeSO₄ 0.01 (pH=7.0) (9). Pure inulin prepared from chicory roots was obtained from Sigma-Aldrich, St. Louis, MO, USA. IPM was dispensed into 250- -mL Erlenmeyer flasks and inoculated with 10% (by volume) of a 16-hour-old Xcp KM 24 culture and incubated at 37 °C for 120 h with shaking at 150 rpm. Samples were withdrawn every 12 h, centrifuged at 10 000×g and the supernatant was used for determining inulinase activity (6, 7).

Determination of inulinase activity

Inulinase activity was determined by quantifying the amount of reducing sugars released from inulin as described earlier (10). The reaction mixture containing 0.1 mL (10 µM) of crude enzyme extract and 0.9 mL of sodium acetate buffer (100 mM, pH=5.5) was incubated at 50 °C. The reaction was started by adding 1 mL of 2% inulin and allowed to react for 20 min. One inulinase unit (IU) was defined as the amount of enzyme that produces one micromole of fructose equivalent per minute under standard assay conditions.

Concentration and purification of inulinase

One litre of inulinase production culture medium supernatant was sequentially subjected to precipitation with ammonium sulphate from 20 to 100% at intervals of 20% at 4 °C for 16 h. The pellets were collected by centrifugation, dissolved in 5 mL of sodium phosphate buffer (pH=7) and dialysed through the 12-kDa cut-off dialysis membrane from Sigma-Aldrich against the same buffer. The fractions of 40, 60 and 80% showed inulinase activity and they were pooled together. The sample was concentrated using ultrafiltration spin column (Amicon Ultra-15 Centrifugal Filter Unit, molecular mass cut-off 30 kDa, cat. no. UFC903024, Merck KGaA, Darmstadt, Germany) and 1 mL of enzyme sample (1 mg/mL) was loaded into approx. 75-mL Sephadex G-100 column (40 cm×0.75 cm; GE Healthcare Life Sciences, Little Chalfont, UK) and eluted with 50 mM phosphate buffer at a flow rate of 1 mL/min. Fractions of 4 mL were collected and assayed for protein content (at 280 nm) and inulinase activity as described above (data not shown). The fractions showing inulinase activity were pooled together and concentrated again by passing the samples through ultrafiltration spin column in the centrifuge at the speed of 6500×g for 15 min. A small volume of fractions (200 µL) was precipitated with 800 µL of chilled acetone to check the purity and homogeneity of the protein by subjecting it to 12% SDS- -PAGE (11) run on constant potential difference of 100 V. The gel was stained with Coomassie Brilliant Blue R-250 (CBB R-250) and the protein content was determined as previously described by Bradford (12) using bovine serum albumin (BSA) as standard.

Determination of optimum pH and temperature of purified inulinase

In order to determine the optimum pH of the purified inulinase, 0.5% (by mass per volume) inulin substrate solutions were prepared in the following buffers (100 mM): citrate buffer (pH=4–6), sodium phosphate buffer (pH=7), Tris-HCl (pH=8 and 9) and glycine-NaOH (pH=10). The enzyme (1 µM) was incubated with the substrate at 50 °C for 20 min. To determine the optimum temperature, the enzyme (1 µM) was incubated with the substrate prepared in the optimum pH buffer. The assay mixture containing 50 µL (1 µM) of the enzyme solution and 950 µL of the substrate solution in sodium phosphate buffer (100 mM, pH=7) was incubated at temperatures ranging from 25 to 90 °C.

Determination of pH and temperature stability

The pH stability of the enzyme was determined by incubating 1 mL (10 µM) of the enzyme at pH=4–9 at 50 °C. Aliquots of 100 µL were removed at time intervals of 0, 10, 20, 30, 60, 90, 120, 150 and 180 min, and assayed by incubating with 900 µL of inulin substrate as mentioned above. Temperature stability of the enzyme was determined by incubating inulinase (10 µM) at different temperatures i.e. 50–90 °C at the intervals of 10 °C. The aliquots of 100 µL were removed at time intervals of 0, 10, 20, 30, 60, 90, 120, 150 and 180 min, and assayed for inulinase activity. For determination of temperature stability of the purified enzyme, the substrate was prepared in sodium phosphate buffer (100 mM, pH=6). The final concentration of the enzyme in the assay reaction mixture was 1 µM.

Determination of K_m, v_max and k_cat values

The initial rate of enzymatic activity was measured to determine kinetic parameters for the substrate hydrolysis. The Michaelis-Menten constant (K_m) was determined from the Lineweaver-Burk plot by applying the Michaelis- -Menten equation (Eq. 1). The activity of inulinase was measured using Lineweaver-Burk plot analysis, by incubating it at substrate concentrations from 0.2–15 µg/mL in sodium phosphate buffer (pH=6 and 50 °C). The reciprocal values of the rate of substrate hydrolysis (1/v) were plotted against the reciprocal values of the substrate concentrations (1/[S]), and the K_m values were determined by fitting the resulting data using ORIGIN v. 8 Pro software (OriginLab Corporation, Northampton, MA, USA). The v_max was also determined from the Lineweaver-Burk plot. The catalytic constant of the enzyme substrate reaction (k_cat), also referred to as the turnover number, represents the number of reactions catalysed by each active site per unit time and was determined by Eq. 2, while the catalytic efficiency of the enzyme was calculated by using Eq. 3:

where [S] is the substrate concentration, v₀ is the initial velocity, v_max is the maximum velocity, K_m is the Michaelis- -Menten constant and [E]_t is the total enzyme concentration (1 µM).

Thin layer chromatography to determine the catalytic nature of the inulinase

The products of inulin hydrolysis with purified inulinase from Xcp KM 24 strain were determined by performing the thin layer chromatography (TLC). The enzyme reaction (0.5 mL of purified inulinase (1 µM) and 0.5 mL of inulin (0.5%, by mass per volume)) was carried out at 50 °C and pH=6.0 for 24 h. Precoated TLC plates (Silica gel 60, Merck, Darmstadt, Germany) spotted with samples were developed with the solvent system, ethyl acetate/acetic acid/2-propanol/formic acid/water (25:10:5: 1:15, by volume). Sucrose (Sigma-Aldrich), glucose (Sigma--Aldrich), fructose (Merck), 1-kestose, 1,1-kestotetraose and 1,1,1-kestopentaose (Megazyme, Wicklow, Ireland) were used as standards. The TLC plates were developed using detection reagent containing 1% (by mass per volume) orcinol and 10% (by volume) sulphuric acid in absolute ethanol and heating them at 100 °C for 5 min to detect sugars (7).

Statistical analysis

All the kinetic parameters were determined by fitting the data using ORIGIN v. 8 Pro software (OriginLab Corporation). The assays for the kinetic analysis and rate constant determinations were carried out in triplicate, and the average value was considered throughout. The p-value lower than 0.05 was considered statistically significant.

Results and Discussion

Purification of inulinase

The present study reports the purification and characterization of an endoinulinase from Xcp KM 24, a mutant strain of Xanthomonas campestris pv. phaseoli. The endoinulinase produced by Xcp KM 24 was purified to homogeneity in two steps, ammonium sulphate precipitation and Sephadex G-100 column chromatography with 77% yield, and had a specific activity of 174.74 U/mg. Table 1 shows the summary of the enzyme purification and the total yield. The final enzyme preparation was homogeneous on SDS-PAGE, with a molecular mass of 55 kDa (Fig. 1). A considerable variation in molecular mass of inulinases has been reported earlier, e.g. Arthrobacter sp. (75 kDa), Bacillus stearothermophilus KP1289 (54 kDa), Aspergillus candidus (54 kDa), Penicillium sp. TN-88 (68 kDa), Kluyveromyces marxianus var. bulgaricus (57 kDa) and Streptomyces sp. (45 kDa) (13–18). Five exoinulinases from Aspergillus ficuum showed the same molecular mass of 74 kDa and three endoinulinases had a molecular mass of 64 kDa (19). A new thermophilic inulinase-producing strain Bacillus smithii T7, which grows optimally at 60 °C, was isolated from soil samples with a medium containing inulin as a sole carbon source. Maximum inulinase yield of 135.2 IU/mL was achieved with medium pH=7.0, containing 2.0% inulin. The purified inulinase from the extracellular extract shows endoinulinolytic activity (20). A. ficuum JNSP5-06 produces five enzymes with molecular masses of 70, 40, 46, 34 and 31 kDa (21). A. ficuum JNSP5-06 endoinulinase expressed in Escherichia coli exhibited M of 60 kDa (22), which is in contrast to the above study. The crystal structural analysis of inulinase from A. ficuum JNSP5-06 at 1.5 Ĺ and its comparison with other glycoside hydrolase family 32 (GH32) enzymes reveal the presence of an extra pocket in the INU2 catalytic site, formed by two loops and the conserved motif W-M(I)-N-D(E)P-N-G. This cavity could explain the endo-activity of the enzyme, the critical role of Trp40, and particularly the cleavage at the third unit of the inulin (-like) substrates (23). Most of the inulinases from fungi have been reported to have a molecular mass above 50 kDa (24). The molecular mass of the purified inulinase from the supernatant of the cell culture of the marine yeast Cryptococcus aureus G7a was estimated to be 60 kDa (25), while the molecular mass of the purified inulinase from Pichia guilliermondii strain 1 was estimated to be 50 kDa (26). However, it has been reported that the M of extracellular inulinase from the terrestrial yeast Kluyveromyces fragilis is 250 kDa. The molecular mass of the purified exoinulinase from bacteria was estimated to be approx. 54 kDa (27, 28). This suggests that molecular mass of the exoinulinases from bacteria is almost the same as of the exoinulinases from yeasts. K. marxianus CBS 6556 inulinase (rKmINU) gene expressed in methylotrophic host Pichia pastoris showed a specific activity of 2714 U/mg (29), which is 12-fold higher than those of other inulinases described previously. It displayed excellent stability from 30 to 50 °C and pH=3.0–5.0, and its half-life was over 96 h under these conditions. Moreover, rKmINU saccharified Jerusalem artichoke tuber juice effectively (29). A 79.8-kDa endoinulinase gene (enIA) from Arthrobacter sp. S37 overexpressed in Yarrowia lipolytica Po1h showed endoinulinase and specific endoinulinase activities of 16.7 U/mL and 93.4 U/mg, respectively (30). From Lactobacillus casei IAM1045, levH1 gene encoding an inulinase was cloned and sequenced, and structure-function relationship was investigated by site- -directed mutagenesis (31). This gene product belongs to GH32 enzyme group, and is composed of four domains. From the catalytic domain of levH1 gene, the 8th motif was newly found in the β-sandwich module, and the necessity of its D683 residue for catalysis was confirmed. LevH1 was found to be an exo-type inulinase producing exclusively fructose, and the knockout of levH1 resulted in the loss of the bacterial ability to catabolize inulin for growth (31). An inulinase of M=66 kDa from a marine bacterium Bacillus cereus MU-31 is also reported to have an activity of 96 U/mL (32).

Table 1. Summary of the purification and yield of endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 supernatant.

Purification step	γ(protein) mg/mL	Endoinulinase activity U/mL	Yield %	Specific activity U/mg	Purification factor
Crude culture filtrate	0.217	22.12	100	102	1
(NH4)2SO4 precipitation	0.179	18.76	85.2	104.8	1.02
Sephadex G-100	0.097	16.95	77	174.74	1.74

Fig. 1.

SDS-PAGE of purified inulinase. Lane 1: molecular mass marker, lane 2: purified endoinulinase

Optimum temperature and thermostability of inulinase

The inulinase activity measured as a function of temperature from 30 to 90 °C shows that the activity of the enzyme was the highest at 50 °C (Fig. 2). The enzyme is stable at up to 60 °C, retaining over 60% activity for 30 min, but inactivated rapidly at higher temperatures. At 90 °C, the enzyme lost its complete activity within 10 min (Fig. 3). A novel inulinolytic strain of Xanthomonas sp. has been reported that produces an endoinulinase optimally active at 45 °C and pH=6 (33). The optimal temperature of the purified enzyme from the marine yeast C. aureus G7a is reported to be 50 °C and the enzyme is very stable at up to 65 °C (25). Therefore, inulinase produced by Xcp KM 24 seems to have considerable thermostability as compared to others reported in literature. However, the inulinase activity produced by P. guilliermondii strain 1 is the highest at 60 °C and the enzyme is very stable at up to 60 °C (26). Inulinase from terrestrial microorganisms in general shows the highest activity below 50 °C, whereas optimum temperature is mostly between 30 and 55 °C (16, 24, 34, 35). The optimum temperature for an endoinulinase from B. smithii T7 was 70 °C, the t_1/2 of the endoinulinase was 9 h and 2.5 h at 70 and 80 °C, respectively (20).

Fig. 2.

Optimum temperature for the purified endoinulinase determined by performing the standard enzyme assays at different temperatures in 100 mM citrate buffer (pH=6)

Fig. 3.

Temperature stability of endoinulinase (1 µM) of Xcp KM 24 mutant determined by incubating the enzyme at different temperatures (50–90 °C)

Optimum pH and pH stability of inulinase

The inulinase activity was measured in the pH range of 4–10 in the buffers with the same ionic concentrations. The results indicate the enzyme to be optimally active at pH=6.0 (Fig. 4). The activity of the purified enzyme was stable between pH=6.0–9.0 (Fig. 5). After 2 h at 50 °C, more than 40 and 45% of the residual activity remained at pH=6.0 and 9.0, respectively. An extracellular endoinulinase purified from X. oryzae (9) was optimally active at pH=7.5 and 50 °C and stable over a pH range of 6.0–9.0. An exoinulinase of 83 kDa was purified from Streptococcus salivarius with optimum pH=7.0 (36). The inulin-inducible inulinase from Clostridium acetobutylicum was reported to be produced both extra- and intracellularly with the pH and temperature optima of 5.5 and 47 °C, respectively (37). The endoinulinase from Clostridium thermoautotrophicum was maximally active at 60 °C and neutral pH (38). The d-fructofuranosidase of Bifidobacterium infantis is a monomeric protein of 70 kDa and possesses both inulinase and invertase activities (39), and the purified endoinulinase showed the optimum pH and temperature of 6.0 and 37 °C, respectively. The optimum pH for an endoinulinase from B. smithii T7 was 4.5 and the enzyme was stable at pH=4.0–8.0 (20). The gene encoding for one of the most thermostable bacterial inulinases, which retained 85% of its initial activity after 5 h at 80 °C and pH=7.0, was cloned from Thermotoga maritima (40). The optimum pH values of the purified inulinases from fungi and yeast are in the range of 4.5–6.0 (2, 26, 34, 35).

Fig. 4.

Optimum pH for the purified endoinulinase determined by performing the standard enzyme assays at different pH values and 50 °C

Fig. 5.

pH stability of endoinulinase determined by incubating the enzyme at pH=4–9 and 50 °C and performing the enzyme assay at pH=6.0

Kinetic properties of inulinase

Enzymes are characterized by measuring their reactions with regard to their substrate affinities and maximal velocity rates. By measuring the rate of substrate utilization (v) at different substrate concentrations [S], K_m and v_max can be calculated using Lineweaver-Burk, Eadie/Hofstee or Wilkinson methods (41, 42). In this study, the Lineweaver–Burk plots showed that the apparent K_m and v_max values of the inulinase when using inulin were 1.15 mg/mL and 0.0000261 mg/(mL·min), respectively (Fig. 6). The k_cat value was found to be 0.145 min^–1. The calculated catalytic efficiency of the enzyme was found to be 0.126 mg/(mL·min). The K_m=1.15 mg/mL for this enzyme is lower than that of other reported inulinases, e.g. K_m values of the two endoinulinases, Endo-I and Endo-II, produced by A. ficuum JNSP5-06 were 14.8 and 25.6 mg/mL, respectively (21). A. ficuum JNSP5-06 endoinulinase was expressed in E. coli and the K_m and v_max values with inulin as the substrate were found to be (67.4±4.2) mg/mL and (349.2±13.7) mg/(mL·min), respectively (22). The K_m value of inulinase from P. guilliermondii strain 1 using inulin as substrate was 21.1 mg/mL (43). Streptomyces sp. ALKC4 endoinulinase showed K_m (1.63 mM) and v_max (450 IU/mg) using inulin as substrate, which is almost the same as the enzyme reported in this study (18). An endoinulinase from B. smithii T7 exhibited comparatively lower K_m (4.17 mM) and higher v_max (833 IU per mg of protein), which demonstrated that this endoinulinase has greater affinity for inulin substrate (20). Debaryomyces cantarelli (15 mM) (44), Candida salmenticensis (17 mM) (45) and A. ficuum (10–15 mM) (19) showed higher K_m than the inulinase reported here, which makes it a better candidate for inulin hydrolysis. Because of lower K_m value and high thermal stability, Xcp KM 24 endoinulinase can be used for commercial applications like for large scale FOS (alternative sweetener) production, and can help reducing the overall production cost.

Fig. 6.

Lineweaver-Burk plot for inulinase activity. Enzyme (1 µM) was incubated with inulin (0.2–15 µg/mL) at 50 °C and pH=6.0 for 20 min. The reciprocal values of enzyme units (1/v) were plotted against the reciprocal values of the substrate concentrations (1/[S]), and the K_m values were determined by fitting the resulting data using ORIGIN v. 8 Pro. IU=inulinase unit (the amount of enzyme that produces 1 µmol of fructose equivalent per min)

Product of inulin hydrolysis by purified inulinase

Thin layer chromatography analysis of products of inulin hydrolysis showed that oligosaccharides were the predominant end product during hydrolysis for 10 min to 2 h (Fig. 7). Oligosaccharides with various degrees of polymerization were observed in the samples after hydrolysis for 10 and 30 min or 2 h and this hydrolysis pattern suggests the presence of an active endoinulinase. The prolonged enzyme hydrolysis (24 h) did not result in any kind of mono- (glucose and fructose) or disaccharides (sucrose) in the hydrolysates, which pointed to the absence of exoinulinase activity of the enzyme (data not shown). However, the prolonged enzyme hydrolysis (24 h) by the crude extract reported in our previous study with this organism (7) revealed the production of mono- (glucose and fructose) or disaccharides (sucrose) in the hydrolysates, which pointed to the presence of exoinulinase in the extract at low or undetectable levels, despite ammonium sulphate precipitation, with only endoinulinase activity observed. Inulin hydrolysis by an extracellular inulinase of Rhizopus sp. resulted in the production of fructose and oligosaccharides after 24 h of incubation (46). Fructose formation was completely absent when inulin was hydrolysed with crude endoinulinase of X. oryzae No. 5 (47). The native endoinulinase produced by Arthrobacter sp. S37 hydrolysed inulin at optimal pH=7.5 and 50 °C mainly into inulotriose (F3), inulotetraose (F4) and inulopentaose (F5) (12). Inulobiose was the major product of the activity of immobilized endoinulinase produced by Pseudomonas sp. No. 65 or immobilized recombinant E. coli, possessing endoinulinase gene (48). In soluble form, the endoinulinase produced by Pseudomonas sp. No. 65 gave two major components, inulobiose and DP3 oligosaccharides (49). When inulooligosaccharide (IOS) from chicory juice was hydrolysed by an endoinulinase from Pseudomonas sp, the major reaction products were DP2, DP3 and DP4 (32). After the endoinulinase gene (inul) of Pseudomonas sp. was expressed in E. coli HB101, the intact cells of the recombinant E. coli and the native enzyme from Pseudomonas sp. were used to produce IOS. It was found that higher levels of inulobiose (the smallest molecule in the product) were observed when intact cells were used (49).

Fig. 7.

Thin layer chromatography of reaction products formed during hydrolysis of inulin by purified inulinase from X. campestris KM 24. L1=sucrose, L2=glucose, L3=fructose, L4=inulin, L5=1-kestose, L6=1,1-kestotetraose, L7=1,1,1-kestopentaose, L8= enzyme+inulin (0 h), L9=enzyme+inulin (10 min), L10=enzyme+ inulin (30 min), L11=enzyme+inulin (2 h)

Conclusions

Inulin-hydrolysing enzymes (inulinases) are widely used in food and pharmaceutical industries. Endoinulinases hydrolyse the internal β-2,1-fructofuranosidic linkages to yield inulooligosaccharides such as inulotriose, inulotetraose and inulopentaose as their main products. We have reported previously the optimization of the nutritional and growth parameters for Xanthomonas campestris pv. phaseoli to enhance the endoinulinase and fructooligosaccharide (FOS) production through ethylmethanesulphonate mutagenesis of the organism and named it X. campestris pv. phaseoli, mutant KM 24 (Xcp KM 24). The present study, therefore, focused on the purification and characterization of endoinulinase from Xcp KM 24 using gel filtration chromatography. Since the importance and potential applications of FOS is growing globally, this endoinulinase could be used for commercial applications like for large scale FOS (alternative sweetener) production.