Inulinase from Kluyveromyces marxianus: culture medium composition and enzyme extraction

Inulinase from Kluyveromyces marxianus: culture medium composition and enzyme extraction

A. PESSOA JR ^* and M. VITOLO

Biochemical and Pharmaceutical Technology Dept./FCF/USP, P.O. Box 66083, CEP 05315-970, São Paulo-SP, Brazil, Phone [phone], Fax: [phone];

E.mail: [e-mail]

(Received: November 16,1998; Accepted: April 19, 1999)

Abstract - K. marxianus DSM 70106 was cultivated for inulinase production in a medium containing 2.0 g/L of yeast extract, 5.0 g/L of peptone and salts. The addition of corn steep liquor did not increase enzyme production. Inulin, as the main carbon source, afforded higher inulinase production than glucose, fructose, sucrose, lactose, maltose and starch. Glucose, fructose and sucrose reduced enzyme production by 46, 58 and 71%, respectively. By using the best culture medium enzyme activity remained stable for 22 months at 4^oC; while at -18^oC it decreased by 10%. Maximal activity was found in the pH range of 3.5 to 5.0 and at temperatures from 50 to 60^oC. Flocculation was used for cell separation. Shifting the pH was more efficient than using polyelectrolytes, CaCl₂, bentonite and Fe₂O₃. Recovery of inulinase by AOT(sodium di-2-ethylhexyl sulfosuccinate)-reversed micelles yielded up to ~20%.

Keywords: Inulinase, kluyveromyces marxianus, culture medium, extraction.

INTRODUCTION

D-Fructose is a sweetener largely used in the food and beverage industries. Furthermore, this cetose is 1.2 to 1.8 times sweeter than sucrose, is well tolerated by diabetics, improves iron absorption by children and favours the removal of ethanol from the blood of alcoholics. These qualities have increased the commercial demand for this sugar. Currently, D-fructose is obtained by enzymatic isomerization of glucose, which, in turn, is obtained by the multienzymatic hydrolysis of starch (Birch et al. 1981, Godfrey and West 1996). An alternative to this process is the hydrolysis of inulin (a linear chain of D-fructofuranosides) by inulinase (2,1 b -D-fructanfructanohydrolyse EC 3.2.1.7) (Gupta et al. 1989). This enzyme is mainly produced by yeasts, filamentous fungi and plants (Bajpai and Margaritis 1985, Claessens et al. 1990, Gupta et al. 1989, Manzoni and Cavazzoni 1992, Wei et al. 1998). According to Manzoni and Cavazzoni (1988) K. marxianus has high inulinase production capability.

Inulinase production is affected by the composition of the medium (yeast extract, peptone, corn steep liquor and malt extract, among others) and by the kind of carbon source employed (sucrose, lactose, glucose and fructose) (Grootwassink and Hewitt 1983).

In developing a new technology for enzyme production, cell separation and enzyme recovery need to be taken into account as important steps in the production process. Cell harvesting or biomass separation, which is usually the first step after fermentation in the downstream processing of biological products, can be substantially aided by flocculation (Hamersveld et al. 1994). Enzyme recovery can be performed in several ways and the use of reversed micelles is particularly interesting. Reversed micelles are aggregates of surfactant molecules containing an inner core of water molecules, dispersed in a continuous organic solvent medium. The considerable biotechnological potential of these systems is derived mainly from the ability of the water droplets to dissolve enzymes, without loss of activity (Pessoa Jr. and Vitolo 1998, Pires and Cabral 1993).

This study illustrates a favourable medium composition not only for cell separation by flocculation but also for enzyme extraction by AOT (anionic surfactant)-reversed micelles and, consequently, for inulinase production.

MATERIAL AND METHODS

Microorganism and Inoculum Preparation

K. marxianus DSM 70106, obtained from "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH," Germany, was employed in all experiments. The stock culture was maintained at 4^oC on malt-extract agar slants. A loopful of the stock culture was transferred to a 100 mL Erlenmeyer flask containing 20 mL of the following medium (g/L): inulin (10.0), peptone (20.0), yeast extract (10.0). The initial pH was adjusted to 5.8. The cultures were incubated on a rotary shaker at 120 rev/min and 30^oC for 48h. Then the cells were separated by centrifugation (1,600g; 20 min) and rinsed with distilled water, and the cell cake was resuspended in an adequate volume of distilled water to attain a final concentration of around 1.0 g_{dry cell} /L.

Growth Conditions

One milliliter of the cell suspension was introduced into a 100 mL Erlenmeyer flask containing 20 mL of inoculum medium with a range of substrates: lactose, maltose, starch and inulin. Culture conditions were the same as those adopted for inoculum preparation. All cell growth tests were performed in duplicate.

Cell and Enzyme Production

In the experiments on cell flocculation and inulinase recovery by reversed micelles, K. marxianus DSM 70106 was grown in a 15 L fermenter containing 10 L of the culture medium (g/L) : MgSO₄.6H₂O (0.5), Urea (2.5), KH₂PO₄ (3.0), CaCO₃ (0.1), peptone (5.0), yeast extract (2.0) and inulin (10.0). The culture was grown batchwise at 30^oC, pH 5.0, 1.0 vvm aeration ratio and agitation of 120 rev/min.

Cell Flocculation

The flocculant agents employed were: CaCl₂, bentonite, Fe₂O₃, pH shift and polyelectrolyte (anionic, cationic and non-ionic) (Table 1). Appropriate concentrations (from 0.1 to 100 g/L) of these agents and of the cell suspension were placed in a 200 mL graduated cylinder. The graduated cylinder content (fermented medium containing yeasts cells plus flocculant agents) was stirred with a magnetic stirrer for 30 s. After this period of time, the agitation was interrupted and the volume of the precipitate - expressed as a percentage of the initial volume of the fermented medium (200 mL), which increased continuously, was measured every 10 min during 70 min (Aiba and Nagatani, 1970). The flocculation pH was adjusted with 3.0 M KOH or 3.0 M H₃PO₄.

Medium Yeast Extract (g/L) Peptone (g/L) Presence of salts* Corn-Steep-Liquor (g/L) Inulinase Activity (U/mL) 1 20.0 2.0 Yes - 26.0 2 20.0 2.0 Yes 10.0 25.2 3 2.0 5.0 Yes 10.0 22.9 4 5.0 10.0 Yes - 22.8 5 2.0 5.0 Yes - 21.4 6 10.0 20.0 No - 19.0 7 2.0 2.0 Yes - 17.0 8 5.0 10.0 No 10.0 16.6 9 1.0 1.0 Yes - 12.0 10 - 2.0 Yes - 9.6 11 2.0 - Yes - 7.5 12 0.5 0.5 Yes - 6.5 13 0.2 0.2 Yes - 3.3 14 0.2 - Yes - 2.5 15** 10.0 20.0 No - 0.8

Medium	Yeast Extract (g/L)	Peptone (g/L)	Presence of salts*	Corn-Steep-Liquor (g/L)	Inulinase Activity (U/mL)
1	20.0	2.0	Yes	-	26.0
2	20.0	2.0	Yes	10.0	25.2
3	2.0	5.0	Yes	10.0	22.9
4	5.0	10.0	Yes	-	22.8
5	2.0	5.0	Yes	-	21.4
6	10.0	20.0	No	-	19.0
7	2.0	2.0	Yes	-	17.0
8	5.0	10.0	No	10.0	16.6
9	1.0	1.0	Yes	-	12.0
10	-	2.0	Yes	-	9.6
11	2.0	-	Yes	-	7.5
12	0.5	0.5	Yes	-	6.5
13	0.2	0.2	Yes	-	3.3
14	0.2	-	Yes	-	2.5
15**	10.0	20.0	No	-	0.8

Table 1: Inulinase activity as a function of compositions of the culture media [(salts: MgSO₄.6H₂O (0.5 g/L^-1); (NH₄)₂SO₄ (5.0 g/L^-1); KH₂PO₄ (3.0 g/L^-1); CaCl₂.2H₂O (0.1 g/L^-1); Na₂HPO₄ (4.0 g/L^-1); Carbon source: Inulin (10%)].

**Medium without inulin (control).

Inulinase Activity Studies

Inulinase activity was measured as a function of storage time (months), pH (2.3 to 8.0) and temperature (20 to 70^oC). The cells were harvested by centrifugation and inulinase activity was measured in the supernatant. Samples were stored at 4^oC (refrigerator temperature) and –18^oC (freezer temperature) for 22 months and inulinase activity was measured every two months.

Liquid-Liquid Extraction

The enzyme was extracted from the supernatant by AOT-reversed micelles in isooctan by a two-step procedure. The first step (forward extraction) consisted in mixing 5.0 mL of supernatant with an equal volume of micellar microemulsion (250mM AOT in isooctan). The pH value of the aqueous phase solution (pH = 4.0) was adjusted by adding acetate buffer up to a final concentration of 50 mM. Phase equilibrium was obtained after one minute of intense agitation on a vortex (Type Bender & Hobeins AG, Zürich) followed by centrifugation at 2800xg for 5 min. Afterwards, 4.0 mL of inulinase-AOT-micellar phase was mixed with 4.0 mL of a fresh aqueous phase, so that the second step could be carried out (backward extraction). The enzyme was finally collected after centrifugation (2800xg; 5 min), and the extraction results were reported as total recovered activity (%). Total recovered activity (%) is the percentage of the whole activity [U x volume of the fresh aqueous phase (mL)] obtained after extraction related to the supernatant activity.

Cell Mass Determination, Protein Analysis and Inulinase Activity Measurement

Cell concentration was measured by optical density (O.D) that was plotted versus dry weight (g/L) on a standard curve. Total protein concentration was measured according to Lowry using BSA as the standard protein. Inulinase activity was measured according to Pessoa Jr et al. (1996). One inulinase unit (U) was defined as the amount of enzyme which catalyses the formation of one micromole of fructose per minute under test conditions.

Chemicals

The chemicals were supplied by Riedel-de-Haen (fermentation nutrients), Hannover, FRG; SIGMA™ (isooctane, bentonite and Fe₂O₃) St. Louis, USA; Merck (CaCl₂ and AOT), São Paulo, Brazil. The flocculants Zetag 43, 47, 48 and 88 (medium cationics) and Zetag 32 (weak cationic) were supplied by Allied Colloids GmbH, Hamburg, Germany; Praestol 2500 (non-ionic), Praestol 2375 (strongly anionic) and Praestol 2530 (medium anionic) were supplied by Stockhausen Chemische Fabrik GmbH, Krefeld, Germany; and CF 802 (strongly cationic) was supplied by Basf, Germany. All the other chemicals were of analytic grade.

RESULTS AND DISCUSSION

Inulinase Production

Several components were added to the culture medium in order to obtain the best conditions for inulinase production. Table 1 shows the composition of the 15 culture media tested and the enzyme activity (U/mL) observed at the end of cultivation. The enzyme activity levels were similar for cultivations in media 1 to 4, as the values varied less than 10%. This indicates that corn steep liquor has no influence on enzyme production. This result can be confirmed by the fact that media 3 and 5 differed only in the amount of corn steep liquor and had similar enzyme activities, respectively, equal to 22.9 and 21.4 U/mL. Nevertheless the presence of yeast extract and peptone affected inulinase production markedly. A tenfold decrease in yeast extract (tests 1 and 7) and a 2.5-fold decrease in peptone (tests 5 and 7) concentrations led to a diminution in inulinase activity of around 34% and 20%, respectively. Taking into account tests 10 and 11 without yeast extract and peptone, respectively, it is clear that both components are necessary for improving inulinase activity. Table 1 shows also that salt plus inulin solution are essential for enzyme production, since inulinase activity in medium 8 (without salts) is 30% lower than medium 4. The effect of carbon source on inulinase production is shown in Figure 1. Inulin was the best carbon source for inulinase production by K. marxianus DSM 70106, while starch, maltose and lactose were inadequate. Similar results were obtained by Yokota et al. (1991) with Arthrobacter sp. These authors concluded that inulin induces inulinase production, whereas cultivation using starch, maltose and lactose as carbon sources restrains the production of this enzyme. Fermentations using glucose, fructose and sucrose produced 43%, 55% and 70% less enzyme than inulin, respectively (Figure 1). According to Looten et al. (1987) and Derycke and Vandamme (1984) this can be ascribed to the inhibitory effect of these sources of sugars. Moreover, in the case of K. marxianus DSM 70106, inulinase is a non-constitutive enzyme, as evidenced by using starch, maltose or lactose as the carbon source (Figure 1). However the degree of inulinase induction depends on the microorganism. Xiao et al. (1988) observed that inulinase produced by Chrysosporium pannorum is inductive, while inulinase from Aspergillus niger is constitutive (Derycke and Vandamme 1984). Provided that inulin was the best carbon source, the other components of the culture medium were changed as shown in Table 1.

Figure 1:
Inulinase activity as a function of carbon source used for K. marxianus cultivation.

Inulinase Activity Studies

Inulinase had a good shelf life of at least 22 months when stored at 4^oC. However, a loss of about 10% of the initial enzyme activity occurred under storage at -18^oC (Figure 2). This could probably be due to the damage caused by the freezing/thawing procedure, like that observed for other enzymes (Breda et al. 1992). Such period of storage is not mentioned in the current literature and shows the high resistance against denaturation of this extracellular enzyme and put in evidence its commercial importance. Inulinase stability for fewer period of storage was evaluated by some authors. Bajpai and Margaritis (1985) evaluated the storage stability of free and immobilized inulinase from Kluyveromyces marxianus at 4^oC for only 32 days without losing activity. Later these authors (Bajpai and Margaritis 1987) verified that after 40 days at room temperature, the loss in activity was found to be only 24%. In addition, Allais et al. (1987) observed no loss in activity for inulinase produced by Bacillus sp after 60 days of storage at 4^oC.

Figure 2:
Inulinase storage activity at -18^oC (¾o¾) and 4^oC (¾·¾).

The highest inulinase activity occurred at a pH interval between 3.2 and 5.0 (21.8 and 23.0 U/mL, respectively) (Table 2) and at 50 to 60^oC (23.2 and 24.8 U/mL, respectively) (Figure 3). Different ranges of optimal temperature and pH are found in the literature. The highest inulinase activity produced by Bacillus sp was at pH 6.0 and 45^oC (Allais et al 1987). Ettalibi and Baratti (1990) found pH 4.7 and a temperature of 60^oC as optimal for inulinase from Aspergillus ficuum, and Barthomeuf et al. (1991) verified that a pH range of 5.5 to 5.6 is the best for inulinase obtained from Penicillium rugulosum.

Table 2:
Inulinase Activity as a function of pH

pH	Inulinase Activity (U/mL )
2.3	0
3.2	22.1
4.0	21.8
5.0	23.0
6.0	18.2
7.0	13.6
8.0	12.5

Figure 3:
Inulinase activity as a function of temperature (^oC).

Cell Flocculation

Figure 4 shows the best flocculation results obtained with Fe₂O₃, bentonite and pH shift (flocculation conditions is presented in (Table 3). Preliminary experiments showed that the type (anionic, cationic and non-ionic) and concentration (0.1 to 1.0 g/L) of the polyelectrolytes employed had no effect on cell flocculation/precipitation. These results are not in accordance with the literature, since such compounds are largely employed for yeast separation (Mill, 1964). However, the behaviour of cell flocculation was rapid and very similar for both Fe₂O₃ at 1.5 g/L and bentonite at 5.0 g/L. The problem of using oxides or bentonite for cell flocculation is the aggregation to cell suspension of chemicals that are inappropriate for human consume. Nevertheless, it must be stressed that pH shifting is an efficient flocculation method (Figure 4), because after 10 min flocculation at pH 8.0 a cell free supernatant was attained and inulinase was not denaturated. Besides, through this procedure a non toxic chemical is introduced in the product for humane consume. This type of flocculation presents very low cost since only alkali is necessary. The effect of pH value on yeast flocculation indicates that phosphate groups (pK about 8.0) might exist in the cell surface (Mill, 1964). These results also showed that the flocculation is not absolutely dependent upon the presence of calcium as reported by Mill (1964). According to the results here obtained, the best precipitant agent was pH shifting since the enzyme remained stable, the process is easy to handle and the cheapest alternative by far. This kind of flocculation can also be used to recycle yeasts cells to a semicontinuous ethanol fermentation (Weeks et al. 1983).

Figure 4:
Percentage of precipitate volume as a function of time (min) under the following conditions: 1.5 g/LFe₂O₃ (¾ _*¾ ); 5.0 g/LBentonite (¾ D ¾ ) and a pH shift to 8.0 (¾u ¾ ).

Flocculant Agent pH Flocculant (g/L) CaCl2 (mM) gp.e./g d.m** Cationic, Anionic and Non-Ionic 4.0 to 8.0 0.1 - 1.0 0 - 20.5 0.012 - 0.149 *Fe2O3 5.8 0.5 - 100.0 - *pH shift 8.0 - - *Bentonite 5.8 0.5 - 5.0 - -

Flocculant Agent	pH	Flocculant (g/L)	CaCl2 (mM)	gp.e./g d.m**
Cationic, Anionic and Non-Ionic	4.0 to 8.0	0.1 - 1.0	0 - 20.5	0.012 - 0.149
*Fe2O3	5.8	0.5 - 100.0	-
*pH shift	8.0	-	-
*Bentonite	5.8	0.5 - 5.0	-	-

Table 3: Conditions under which K. marxianus was separated by flocculation

*Conditions under which flocculation occurred. **p.e.= polyelectrolyte **d.m = dry matter

Inulinase Extraction:

Liquid-liquid extraction by reversed micelles requires low ionic strength to be well performed (Pessoa and Vitolo, 1998). To decrease this ionic strength, the (NH₄)₂SO₄ and CaCl₂.2H₂O, used in the inulinase fermentation medium, were replaced by urea and CaCO₃, respectively. This replacement did not affect enzyme production. The inulinase and total protein contents in the supernatant of the fermented medium used in the extraction process were in the range of 30 to 40 U/mL and 4 to 5 mg/L, respectively. Extraction by reversed micelles is divided in two steps: forward and backward extraction. In the forward extraction the inulinase, which is a protein with high molecular weight (230 kDa), can be extracted from an aqueous solution to a reversed-micelle phase of 250 mM AOT (surfactant) in isooctane (solvent). As this type of extraction generally is due to an electrostatic interaction between the protein and the reversed micelle, a strong dependence on the pH, ionic strength and temperature of the aqueous solution should be expected (Wolbert et al, 1989; Pires and Cabral, 1993). By manipulating these three parameters, it was possible to observe a shift in the solubilization of inulinase in the micelle phase. The forward extraction of inulinase was performed using a pH range of 3.0 to 5.0 and 0.05 M NaCl ionic strength. Different types of salt (NaCl and KCl – data not shown) were tested for this extraction and at pH 3.0 and 5.0, the enzyme recovery was zero. This shows that the extraction is not governed by an electrostatic interaction between the enzyme and the anionic surfactant. However, in the extraction performed at pH 4.0 (with 0.50 M acetate buffer, 250 mM AOT and 21^oC), which is near the pI value, about 20% extraction was achieved. The driving force responsible for the extraction of inulinase under these conditions is a hydrophobic interaction of the inulinase with the surfactant. Considering the hydrophobic interaction between the enzyme and the surfactant at pH 4.0 and the existence of hydrophobic forces, which are strongly dependent on increase in temperature, the extraction of inulinase was performed under several temperatures between 5 and 37^oC (Figure 5). When the temperatures of forward and backward extractions are lowered, total inulinase recovery decreases. This happens because the driving force for the extraction is hydrophobic in nature and the pH and pI values are close (Pires and Cabral 1993). In the backward-extraction, enzymes are usually back-extracted by interacting the organic phase loaded with enzyme with a new aqueous phase at a high ionic strength (Pessoa Jr. and Vitolo 1998). In order to verify if increased ionic strength could improve the back extraction of the inulinase, different buffer and NaCl concentrations were tested. However, no change was detected (data not shown) because the hydrophobic interactions between enzyme and reversed micelles are not influenced by variations on ionic strength. The low total enzyme recovered can be a consequence of either enzyme denaturation during extraction or the impossibility of obtaining a greater interaction between the enzyme and the micelles.

Figure 5:
Effect of extraction temperatures on inulinase recovery by AOT-reversed micelles. Forward extraction conditions: pH 4.0, 0.05 M acetate buffer and 250 mM AOT. Backward-extraction conditions: pH 7.0, 0.10 phosphate buffer and 1.0 M NaCl. Temperatures: ¾O ¾ (5ºC), ¾· ¾ (21ºC), ¾¨ ¾ (30ºC), ¾n ¾ (37ºC).

CONCLUDING REMARKS

Inulinase production by K. marxianus DSM 70106 depends on the composition of the culture medium, which must contain at least 5.0 g/Lyeast extract and 2.0 g/Lpeptone, as well as the following salts (g/L): MgSO₄ (0.5); (NH₄)₂SO₄ (5.0); KH₂PO₄ (3.0); CaCl₂.2H₂O (0.1); and Na₂HPO₄ (4.0); plus Inulin (10.0). Inulinase production was not affected by corn steep liquor (10.0 g/L), but the enzyme was induced by inulin and inhibited by other sugars tested. Catabolic repression by the sugars tested also occurred. The high inulinase stability verified after 22 months of storage increases its feasibility for practical applications. Cell flocculation using pH shift to 8.0 was shown to be the most appropriate flocculation agent. AOT-reversed micelles allowed the recovery of 20.6% of the total initial enzyme. Maximum enzyme recovery by extraction was determined by the pH value. It is also important to emphasise that inulinase extraction by reversed micelles is a promising technique since no enzyme inactivation was observed in the presence of organic solvente (isooctane) and surfactant (AOT).

ACKNOWLEDGMENTS

Adalberto Pessoa Jr acknowledges receipt of a Doctor of Science fellowship from CNPq/RHAE (Brazil). The authors also acknowledge Maria Eunice M. Coelho’s assistance in reviewing this text.

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*

To whom correspondence should be addressed

Publication Dates

• Publication in this collection
  16 Dec 1999
• Date of issue
  Sept 1999

History

• Accepted
  19 Apr 1999
• Received
  16 Nov 1998