Purification of exo- and endoinulinase from crude inulinase extract for the analysis of fructans

Purification of exo- and endoinulinase f r o m crude inulinase extract for the analysis of fructans* S. Baumgartner and W. Praznik Institute of Chemistry, Universitat fbr Bodenkultur, Gregor-Mendelstr. 33, 1180 Vienna, Austria Received 1 September 1994; revised 29 November 1994

This paper describes a technique for rapid and easy separation of a crude inulinase extract into exo- and endoinulinase from a commercially available inulinase stock (Novozym 230 from Novo Nordisk A/S) by means of non-denaturating polyacrylamide gel electrophores~s. The purified enzymes were tested with inulin. Characterization of the reaction products W~s done by high-performance thin-layer chromatography, size exclusion chromatography and semi-preparative reverse-phase high-performance liquid chromatography.

Keywords: fructans; inulinase; electrophoresis

The structure of fructans is determined by the genetic code and the conditions of biosynthesis in plants and micro-organisms. Native fructans occur as (2--* 1)-linked fl-D-fructofuranosyl residues (inulin type) 1 or as (2-.6)linked fl-D-fructofuranosyl residues (levan type)2 with a terminal glucose. The inulin type occurs as reserve carbohydrate in dicotyledons such as chicory, Jerusalem artichoke or dahlia. The fructans of these plants have been discussed as interesting raw material for industrial utilization in the fields of food, non-food and energy products. The levan type occurs mainly in monocotyledons such as grasses. Inulinases are fructofuranosyl hydrolases, which are produced by bacteria 3'4 and plants 5-8 as well as in moulds 9. Three types of inulinase t° have been described: exoinulinase, which liberates single fructose molecules from the polysaccharide chain; endoinulinase, which hydrolyses inulin to reducing fructo-oligomers; and fructotransferase, which is not a common catabolic carbohydratase. Many analytical procedures have been reported for the purification and characterization of inulinase isoenzymes5'6'11-~5, e.g. affinity to DEAEcellulose or other ion-exchange chromatography methods. In this paper, we describe a non-denaturing separation of inulinase isoenzymes by polyacrylamide gel electrophoresis (PAGE). The different bands were visualized on the gel by means of protein staining, and the active parts of the isoenzymes were identified by an activitystaining procedure. The type of action of the isolated isoenzymes was identified by high-performance thin-layer chromatography (HPTLC). The digestion products were * Paperpresentedat the Ist InternationalConferenceon Polysaccharide Engineering,Trondheim,Norway,6-8 June, 1994.Other papers from this conferencewerepublished in Int. J. Biol. Macromol. 1994, 16(6).

0141-8130/95/$09.50 c-; 1995ElsevierScienceB.V.All rightsreserved

collected and prepared for further use as reducing standards by size exclusion chromatography (SEC) and semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC).

Experimental Materials

Inulinase stock Novozym 230 was a gift from Novo Nordisk A/S (Bioindustrial Group, Vienna, Austria). Chemicals for electrophoresis, TLC and HPLC were obtained from Merck (Labochem, Neuber, Vienna, Austria). Coomassie brilliant blue R-250 and tetrazolium red were obtained from Sigma (Sigma-Aldrich Handels GmbH, Vienna, Austria). HPTLC Whatman pre-coated silica plates LHP-KF were obtained from Whatman (Comesa, Vienna, Austria). Biogel P2 was obtained from Pharmacia (Pharmacia Biotech Europe GmbH, Vienna, Austria). Inulin and non-reducing inulo-oligosaccharides were isolated from Jerusalem artichoke tubers 16'17. Pre-cleaning

The enzyme solution (200 pl) was centrifuged using a filter cartridge (10000#m, from Amicon) for 10min at 5000 rev min-1. The resulting solution was taken up in 500pl acetate buffer (0.01 moll -j, pH5), mixed with saccharose to increase the viscosity, and then applied on the polyacrylamide gel. Electrophoresis

PAGE was carried out using the Mini-Protean II Electrophoresis System from BioRad (BioRad Laboratories GmbH, Vienna, Austria). A separation gel with 15% acrylamide cross-linked 1:75 with bismethyleneacrylamide,

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pH 8.7, was used. The electrophoresis buffer was ~l Tris-glycine system. A voltage of 200 V and a starting current of 100 mA were applied. A 30 #1 aliquot of thc pre-cleaned enzyme solution was separated within I h.

Staining After separation, the gel was cut into three identical pieces. One piece was stained with Coomassie brilliant blue R-250 to visualize the protein bands. With a second piece, activity staining was carried out by immersing the gel overnight at 37°C in 100ml of a 1% inulin solution in 0 . 1 m o l l -~ acetate buffer (pH5.0), then in 0.1% triphenyl tetrazolium chloride in 0.1 m o l l -1 sodium hydroxide solution for 15 rain in the dark, and for 15 rain at 100"C for staining. The third piece was prepared for further utilization of the enzymes. It was cut into 5 mm strips and incubated in a 1% inulin solution and 0.1 tool 1- 1 acetate buffer (pH 5.0) at 3 7 C overnight. The products of the enzyme digestion were tested by means of TLC. Figure ! Protein staining of electrophoretically separated crude inulinase extract

TLC and HPTLC T L C was performed on Silicage160 plates from Merck. The solvent was butanol-propanol-ethanol-water (2:3:3:2). The T L C was run twice, dried at I10°C, sprayed with thymol sulfuric acid and again dried at l l0°C to visualize the carbohydrates ~s. The H P T L C was performed under identical conditions. Non-reducing inulo-oligosaccharides were used as standards. SEC The digestion products were applied to a column (1000 × 25 mm internal diameter) filled with Biogel P2. Distilled, filtered and degassed water was used as eluent (pump P I from Pharmacia). For detection, a Waters RI 401 detector and an L K B 2210 flat-bed recorder were used. Fractions (3 ml) were collected using an LKB fraction collector. The separation was carried out at room temperature.

Figure 2 Activity staining with 0.1% TTC of electrophoretically separated crude inulinase extract

Semi-preparative RP-HPLC The column used was a Superformance glass column, 300× 10ram internal diameter, filled with Nucleosil 120-7C18 support iv. Water (deionized, distilled and degassed) with 0 - 5 % methanol was the mobile phase, and a Waters RI 401 mass detector was used at a flow rate of 2.5 ml rain- 1 and a sample volume of 500/~1.

Figure 3 TLC of the gel strips incubated overnight at 37°C in 1% inulin solution: I, inulo-oligosaccharides; bands 5 8, exoinulinase activity; bands 12-14, endoinulinase activity

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Purification of a crude inulinase extract." S. Baumgartner and W. Praznik

Results and discussion The pre-cleaned inulinase was separated into the isoenzymes by n o n - d e n a t u r i n g P A G E . This pre-cleaning was required because the original solution c o n t a i n e d a high c o n c e n t r a t i o n of a m m o n i u m sulfate which caused a b r o a d e n i n g of the different isoenzyme b a n d s a n d made precise cutting impossible. Figure 1 shows the protein staining of the gel after electrophoresis, with a range of different bands, whereas activity staining with tetrazolium red (Figure 2) shows two active regions only. The upper,

Table 1 Fraction numbers for the SEC separation showing the different polysaccharide contents Fraction number

Polysaccharide

50 53 56 6(/ 66 73

Heptasaccharide Hexasaccharide Pentasaccharide Tetrasaccharide Trisaccharide Monosaccharide

more cathodic enzyme band shows exoinulinase activity. The more anodic band shows endoinulinase activity, a fact which is illustrated by TLC of the gel strips (Figure 3) of the third piece. There is activity over the whole gel but there is pure exoinulinase activity from the 6th to the 8th strip. Only monosaccharides are released from inulin. Bands 12 to 14 contained the endoinulinase isoenzymes which hydrolysed inulin into reducing fructo-oligosaccharides. The composition of the degradation products did not change even when the strips were incubated for longer than one day. The vials containing bands 12 to 14 were pooled for SEC separation (Figure 4). The chromatogram shows the elution profile of the SEC separation, where the first peak contains

Table 2 TLC groups for the pooled fractions from the SEC separation showing the different polysaccharide contents TLC group

Pooled fractions of SEC separation

Polysaccharide

I 3 4 5 6 7

71-75 65-69 59-62 55 58 52 54 48-51

Monosaccharide Trisaccharide Tetrasaccharide Pentasaccharide Hexasaccharide Heptasaccharide

J 8.0 50 53 55 60 65 Fraction number

73

Figure 4 SEC separation on Biogel P2 of the pooled bands 12 14: the content of the fractions is as described in 7itble l

2.5

15.3 16.9

Time (min)

Figure 6 Semi-preparative RP-HPLC of the pooled fractions 65-69 of the preceding SEC separation: 8.0 min, no carbohydrates; 12.5rain, reducing frueto-trisaccharide;15.3min, reducing fructo-tetrasaccharide; 16.9 rain, reducing fructo-pentasaccharide

Figure 5 TLC of the separated SEC fractions: TLC groups and the corresponding fractions and oligosaccharide contents are as shown in Table 2. lnulo-oligosaccharides as shown as standards

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Purification of a crude inu/inase extract." S. Baumgartner and W. Praznik

Conclusions

Figure 7 HPTLC of the peaks of the semi-preparativeRP-HPLC: I indicates non-reducing inulo-oligosaccharides as standards, peak at 8.0min contains no carbohydrates. (1) Peak at 12.5min, reducing trisaccharidc; (2) peak at 15.3min, reducing tetrasaccharide; (31 peak at 16.9rain, reducing pentasaccharide

Fructans are the reserve carbohydrates of various plants. There has been a range of investigations on fructans, but little is known about the characteristics of fructan structure. Specifically acting enzymes, such as pure endo- and exoinulinases, are an excellent tool for producing well-defined fragments. These hydrolysation products were preparatively isolated and prepared for further use. Pre-cleaning of the inulinase crude fraction is definitely necessary. After protein staining, a range of different bands can be identified, but only two active regions are evident with activity staining. It is possible to cut the gel and extract the isoenzymes using appropriate buffer systems. Inulin was digested with each of the isoenzymes and the fractions of inulin degradation products were collected. By means of different chromatographic methods, these fructans were purified and will be applied for the analysis of fragments from unknown fructans.

Acknowledgements high-molecular-weight polysaccharides which are not retained on the column. The remaining peaks contain oligosaccharides and monosaccharides as described in

This work was supported by the Austrian Fond zur Wissenschaftlichen Forschung (FWF) (9059-CHE).

Table 1.

The fractions were checked again by T L C (Figure 5). The T L C groups and their corresponding fractions and oligosaccharide content are shown in Table 2. The fractions with the majority of different polysaccharides were pooled and freeze-dried. For further use as reducing standards, the SEC fractions were additionally purified by means of semi-preparative R P - H P L C . Figure 6 shows the separation of the pooled fractions 65-69 from the SEC (corresponding to T L C group 3, Table 2): the peak at 8.0min contains no carbohydrates, peak 2 contains the reducing fructotrisaccharide, peak 3 is the reducing tetrasaccharide, and the peak at 16.9min is the reducing pentasaccharide. A control experiment was performed by H P T L C (Figure 7). Non-reducing inulo-oligosaccharides were used as standards. Inulo-oligosaccharides contain monosaccharides and saccharose at the conditions used, 1-kestose, nystose, pentasaccharide and fructans with increasing degrees of polymerization. The different peaks of the semi-preparative H P L C separation elute in the range of the separated inulo-oligosaccharides, so there is good evidence that they are reducing inulooligosaccharides.

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