Puri fi cation and Characterization of an Endoinulinase from Xanthomonas campestris pv . phaseoli KM 24 Mutant

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 classifi ed into exoand endo-acting enzymes according to their modes of action on inulin. Endoinulinases (2,1-β-d-fructan fructanohydrolase; EC 3.2. 1.7) are specifi c 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 raffi nose (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., Bifi dobacterium 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 purifi cation and characterization of endoinulinase from Xcp KM 24 using gel fi ltration chromatography. ISSN 1330-9862 original scientifi c paper


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 classifi ed 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 specifi c 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 raffi nose (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., Bifi dobacterium 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 purifi cation and characterization of endoinulinase from Xcp KM 24 using gel fi ltration chromatography.

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 modi fi cations.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.

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 buff er (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 defi ned as the amount of enzyme that produces one micromole of fructose equivalent per minute under standard assay conditions.

Concentration and purifi cation 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 buff er (pH=7) and dialysed through the 12-kDa cut-off dialysis membrane from Sigma-Aldrich against the same buff er.The fractions of 40, 60 and 80 % showed inulinase activity and they were pooled together.The sample was concentrated using ultrafi ltration 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, Litt le Chalfont, UK) and eluted with 50 mM phosphate buff er at a fl ow 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 ultrafi ltration 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 diff erence 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 purifi ed inulinase
In order to determine the optimum pH of the purifi ed inulinase, 0.5 % (by mass per volume) inulin substrate solutions were prepared in the following buff ers (100 mM): citrate buff er (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 buff er.The assay mixture containing 50 μL (1 μM) of the enzyme solution and 950 μL of the substrate solution in sodium phosphate buff er (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 diff erent 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 purifi ed enzyme, the substrate was prepared in sodium phosphate buff er (100 mM, pH=6).The fi nal 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 buff er (pH=6 and 50 °C).The reciprocal values of the rate of substrate hydrolysis (1/v) were plott ed against the reciprocal values of the substrate concentrations (1/[S]), and the K m values were determined by fi tt ing the resulting data using ORIGIN v. 8 Pro soft ware (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 effi ciency of the enzyme was calculated by using Eq.3: where [S] is the substrate concentration, v 0 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).

Statistical analysis
All the kinetic parameters were determined by fitt ing the data using ORIGIN v. 8 Pro soft ware (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.

Purifi cation of inulinase
The present study reports the purifi cation 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 purifi ed to homogeneity in two steps, ammonium sulphate precipi-tation and Sephadex G-100 column chromatography with 77 % yield, and had a specifi c activity of 174.74 U/mg.Table 1 shows the summary of the enzyme purifi cation and the total yield.The fi nal 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)(14)(15)(16)(17)(18).Five exoinulinases from Aspergillus fi cuum 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 purifi ed inulinase from the extracellular extract shows endoinulinolytic activity (20).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 purifi ed 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 purifi ed 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 purifi ed 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 specifi c 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 saccharifi ed Jerusalem artichoke tuber juice eff ectively (29).A 79.8-kDa endoinulinase gene (enIA) from Arthrobacter sp.S37 overexpressed in Yarrowia lipolytica Po1h showed endoinulinase and specifi c 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 confi rmed.
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).

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 purifi ed 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).

Optimum pH and pH stability of inulinase
The inulinase activity was measured in the pH range of 4-10 in the buff ers with the same ionic concentrations.
The results indicate the enzyme to be optimally active at pH=6.0 (Fig. 4).The activity of the purifi ed enzyme was stable between pH=6.0-9.0 (Fig. 5).Aft er 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 purifi ed 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 purifi ed 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 thermo autotrophicum was maximally active at 60 °C and neutral pH (38).The d-fructofuranosidase of Bifi dobacterium infantis is a monomeric protein of 70 kDa and possesses both inulinase and invertase activities (39), and the purifi ed 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 aft er 5 h at 80 °C and pH=7.0, was cloned from Thermotoga maritima (40).The optimum pH values of the purifi ed inulinases from fungi and yeast are in the range of 4.5-6.0(2,26,34,35).

Kinetic properties of inulinase
Enzymes are characterized by measuring their reactions with regard to their substrate affi nities and maximal velocity rates.By measuring the rate of substrate utilization (v) at diff erent 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 effi ciency 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.
fi cuum JNSP5-06 were 14.8 and 25.6 mg/mL, respectively (21). A. fi cuum 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 affi nity for inulin substrate (20).Debaryomyces cantarelli (15 mM) (44), Candida salmenticensis (17 mM) (45) and A. fi cuum (10-15 mM) (19) showed higher K m than the inulinase reported here, which makes it a bett er 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.

Product of inulin hydrolysis by purifi ed 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 aft er hydrolysis for 10 and 30 min or 2 h and this hydrolysis patt ern 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 aft er 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).Aft er 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).

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 ethylmeth ane sulpho nate mutagenesis of the organism and named it X.campestris pv.phaseoli, mutant KM 24 (Xcp KM 24).The present study, therefore, focused on the purifi cation and characterization of endoinulinase from Xcp KM 24 using gel fi ltration 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.
ISSN 1330-9862 original scientifi c paper doi: 10.17113/ft b.53.02.15.3902 A. fi cuum JNSP5-06 produces fi ve enzymes with molecular masses of 70, 40, 46, 34 and 31 kDa (21). A. fi cuum 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. fi cuum JNSP5-06 at 1.5 Å 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,

Fig. 2 .Fig. 3 .
Fig. 2. Optimum temperature for the purifi ed endoinulinase determined by performing the standard enzyme assays at diff erent temperatures in 100 mM citrate buff er (pH=6)

Fig. 4 .Fig. 6 .Fig. 5 .
Fig. 4. Optimum pH for the purifi ed endoinulinase determined by performing the standard enzyme assays at diff erent pH values and 50 °C

Table 1 .
Summary of the purifi cation and yield of endoinulinase from Xanthomonas campestris pv.phaseoliKM 24 supernatant