Medium improvement for β-fructofuranosidase production by Aspergillus japonicus

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Process Biochemistry Vol. 33, No. 3, pp. 267-271, 1998 © 1998 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0032-9592/98 $19.00 + 0.00

ELSEVIER PII:

S0032-9592(97)00066-6

Medium improvement for fl-fructofuranosidase production by Aspergillusjaponicus Wen-Chang Chen Department of Bioengineering,Tatung Institute of Technology, Chungshan North Rd., Sec. 3, No. 40, Taipei 10451, Taiwan (Received 14 April 1997; revised version received 27 June 1997; accepted 5 July 1997)

Abstract

The medium composition for the production of intracellular fl-fructofuranosidase (FFase, EC 3.2.1.26) by

Aspergillus japonicus TIT-90076 was improved by using statistical experimental designs and the method of steepest ascent in shake batch cultures. Sucrose and yeast extract were the key nutritional factors affecting enzyme production. Improved media containing 24"05% sucrose and 2"75% yeast extract increased enzyme production by 180%. © 1998 Elsevier Science Ltd

Keywords: fructo-oligosaccharides,fl-fructofuranosidase,Aspergillusjaponicus, experimental design.

affected by process parameters such as medium composition, oxygen tension and/or transfer rate, type of fermentation (submerged or surface), rates of agitation and aeration, type of impeller, pH value and temperature. Among these parameters, the optimal design of culture media is a relevant aspect to be considered in the development of fermentation processes. So far, many authors have reported on the isolation and screening of micro-organisms for FTase or FFase production with high transfructosylating activity and on purifying and characterizing newly found enzymes [7,10,13,15,16]. However, there are few reports concerning medium optimization for enzyme production [7,8]. Although the attainment of optimal conditions for a multivariable fermentation process is often tedious, it is possible to undertake a rational study by using statistical experimental designs. Statistical experimental designs, such as full or fractional factorial designs, can deal with various factors with two levels or more within a single experiment [17]. Recently, these have been successfully applied to the optimization of culture media for the production of metabolites or enzymes in many fermentation processes [18-20]. A mould, Aspergillus japonicus TIT-90076, can produce intracellular FFase with a high transfructosylating activity [21]. In this study, statistical experimental designs and the method of steepest ascent were used to improve the concentrations of medium components for

Introduction

Fructo-oligosaccharides (FOS), in which one to three fructose units are bound to the fl-2,1 position of sucrose, are mainly made up of 1-kestose, nystose, and lZ-fl-fructofuranosyl nystose [1-3]. FOS have recently attracted special attention as food additives because of their favourable functional properties, which include low calorie and non-cariogenic sweeteners, prevention of dental caries, relief of constipation, decrease of total cholesterol and lipid in serum, promotion of animal growth and consecutive improvement of the intestinal microflora, acting as a growth factor for bifidobacteria [1-3]. FOS can be produced by transferring one to three molecules of fructose to the fructose residue in sucrose through the action of fl-D-fructosyltransferase (FTase, EC 2.4.1.9) or fl-fructofuranosidase (FFase, EC 3.2.1.26) with high transfructosylating activities obtained from plants [4] and micro-organisms [5-12]. FOS are now produced commercially through enzymic synthesis from sucrose by microbial FTases or FFases [1,10]. These enzymes have also been found in fungi including Fusarium sp. [13], Aspergillus sp. [7,14, 15], Aureobasidium sp. [8,10], and Penicillium sp. [16], but few have potential for industrial application due to their low transfructosylating activities. It is important to screen for micro-organisms with higher enzyme activities. The productivity of many fermentation processes is 267

W..-C. Chen

268

FFase production by Asp. japonicus TIT-90076 in shake batch cultures. In addition, enzyme production kinetics were compared between the improved and control media in a 5 litre jar fermenter. Materials and methods

Micro-organism and spore suspension preparation A. japonicus TIT-90076, isolated from air in the Taipei area [21], was cultivated on potato dextrose agar (PDA) plates at 30°C for 2-3 days. To prepare spore suspensions, spores were scraped down from the PDA plates and diluted to a concentration of about 4 x 107 spores m l - ~ with sterilized water.

Culture conditions The standard medium for FFase production contained 20% sucrose, 2% yeast extract (Difco, Detroit, Michigan, USA), 2% NaNO3, 0.05% MgSO4-7H20, and 0.5% K2HPO4. Using statistical experimental designs and the method of steepest ascent to assess the effects of culture medium on FFase production, production media were prepared according to Tables 2, 5 and 6, as described later. In shake flask experiments, 1 ml aliquots of the spore suspension were added to Table 1. Definitions and levels of independent variables in 25-2 fractional factorial design Independent variables*

Coded levels

Symbol

-1

+1

Sucrose )(1 10 30 K2HPO4 )(2 0.3 0-7 MgSO4"7H20 X3 0.03 0.07 NaNO3 )(4 1-0 3.0 Yeast extract X5 1-0 3.0 *Concentration of each variable expressed as % (w/v).

Table3. Regression results for 23 2 fractional factorial design Independent variables intercept X1 X2 X3 )(4 )(5

Run No.

1

2 3 4 5 6 7 8

Coded levels of medium composition

25 - 2

frac-

Enzyme activity (units ml- 1)

X~

X2

X3

X~

X~

-1 +1 -1 +1 -1 +1 -1 +1

-1 -1 +1 +1 -1 -1 +1 +1

-1 -1 -1 -1 +1 +l +1 +1

-1 +1 -1 -1 -1 -1 +1 +1

-1 +1 +1 -1 +1 -1 -1 +1

146 640 410 515 375 570 165 620

t-value for Ho: parameter = 0

430 156.3 - 2.5 2.5 - 37.5 81.3

34.90* 12.70" - 0.20 0.20 3.05 6.60*

*Significant at the 5% level. 100 ml of culture medium in 500 ml Erlenmeyer flasks and cultured at 30°C and 200 rev min -1 (Model 717, Hotech orbital shaker, Taiwan) for 96 h. In fermenter experiments, 30 ml of spore suspension was transferred into a 5 litre fermenter (CMF-5, Mitsuwa Co. Ltd, Japan) containing 3 litre of the culture medium. The culture was carried out for 48 h at 30°C, stirred at 600revmin-I and with an air flow rate of 3.0 litre min -~. After incubation, the whole culture broth was homogenized and examined for transfructosylating activity.

Analysis The reaction mixture used to determine enzyme activity consisted of 9-8 ml of 66% (w/v) sucrose dissolved in 0.1 M citrate buffer (pH 5.5) and 0"2ml of diluted homogenized cell suspension [21]. The enzyme reaction was carried out at 55°C for 1 h and stopped by heating the mixture in boiling water for 15 min. Transfructosylating activity was determined by measuring both the released glucose (G) with a YSI Model 27 glucose analyzer (Yellow Spring Industries, OH, USA) and the released reducing sugars (R) with a DNS method [22]. The following equations were used to calculate concentrations of free fructose (F) and transferred fructose (F') in the reaction mixture: F = R-G

Table 2. Design and experimental results of the tional factorial design

Parameter estimate

and F' = G - F = 2G - R

One unit of the transfructosylating activity is defined as the amount of enzyme required to transfer 1 pmol of fructose per minute [11]. Growth was determined by dry cell weight per volume (mg ml-1). The mycelium was recovered on Whatman No. 1 filter paper, washed thoroughly with distilled water, dried at 80°C overnight and then weighed. The residual sugar in cultured broth was determined by a phenol-sulfuric acid method [23].

Statistical experimental designs Quantitative improvement of the medium was performed with two-step statistical experimental designs and the method of steepest ascent [17]. The first step was a 2 5 - 2 fractional factorial design consisting of eight runs to obtain the first-order model equation

fl-Fructofuranosidase production from Aspergillus japonicus using a multiple regression method in the SAS package (SAS Institute, Rayleigh, NC, USA). Statistical analysis was made to identify those medium variables with a significant effect on FFase production by A. japonicus TIT-90076. The second step was a two-level-two-factor factorial design consisting of four runs to obtain a firstorder model equation. This model was then used to determine the path of steepest ascent to find the improved medium composition.

269

600

450 Cv

"6 300 t~

Results and discussion

E N ¢--

The production medium used as control medium for FFase production was modified from that of Jung et al. [7]. It consisted of 5% sucrose, 2% yeast extract, 2% NaNO3, 0.05% MgSO4"7H20 and 0.5% KzHPO4. The maximal enzyme production obtained was 2 2 6 u n i t s m l - ' after 96h cultivation at 30°C and 200 rev min ' (data not shown). The composition of the culture medium is one of the most important factors influencing cell growth and physiology, and hence formation of bioproducts. To increase enzyme yield, the effect of yeast extract concentration was firstly examined and then two-step statistical experimental designs and the method of steepest ascent were employed.

Effects of yeast extract concentration on FFase production Of the various nitrogen sources such as yeast extract, whey, fish meal, polypeptone, typtone and gelatin used for FFase production from A. japonicus TIT-90076, Liu [21] found that the use of yeast extract produced highest yields. Enzyme yield increased with increased concentrations of yeast extract up to a maximum of about 2.0-3-0% (w/v) (Fig. 1). Since yeast extract contains abundant nitrogen compounds as well as many growth factors, its addition can stimulate FFase production by A. japonicus TIT-90076. Hayashi et al. [6] reported that the optimum concentration of yeast extract for FFase production by A. japonicus MU-2 was between 1-5 and 3% (w/v). A similar result was reported for FFase production from A. japonicus NTU-1249 by Suet al. [12].

Statistical experimental design No. 1 (SED 1) The first-step SED 1 was a fractional factorial design (FFD), 25 2. A Student's t-test, based on the hypothesis that the true parameter is zero, was employed in a multiple regression to elucidate the significance of the medium components [17]. Liu [21] reported that sucrose was the best inducer for FFase production from A. japonicus TIT-90076 and enzyme yield was suppressed at initial sucrose concentrations greater than 25.0%. Based on previous results, sucrose and yeast extract were finally selected as

UJ

150

0

0

I

I

I

I

I

I

1

2

3

4

5

6

Yeast extract (%(w/v))

Fig. 1. Effect of yeast extract concentration on FFase production.

carbon and nitrogen sources, respectively. Moreover, the concentration ranges tested in SED 1 were 10-30% and 1.0-3-0%, respectively, for sucrose and yeast extract. In addition, concentration ranges of the inorganic salts were chosen according to the standard production medium and were transformed to the coded level as shown in Table 1. The design consists of eight runs and in each run the concentration of each constituent at level - 1 and level +1 is given by the rules of FFD (Table 2). Enzyme activity was determined for each run and a multiple regression firstorder model equation obtained from the results is described as follows: FFase production (units ml ') = 430+ 156.3X, - 2"53(2+ 2"5X3 - 37"5X4 +81.3X5 and R 2 = 0.991 According to the parametric coefficients obtained, it may be concluded that sucrose (X 0, MgSOa'7H20 (X3) and yeast extract (Xs) have a positive effect on enzyme production, while KaHPO4 0(2) and NaNO3 (X4) show a negative effect. In addition, sucrose and yeast extract are found to be the most important components for

Table 4. Definitions and levels of independent variables in 22 factorial design Independent variables*

Sucrose Yeast extract

Symbol

X, X2

Coded levels -1

+1

15 1

25 3

*Concentration of each variable expressed as % (w/v).

W.-C. Chen

270

Table 5. Design and experimental results of the 22 factorial design Run No.

Coded levels of medium composition

1 2 3 4

Xl

X2

- 1 - 1 +1 +1

- 1 +1 - 1 +1

Enzyme activity (units ml- l)

355 525 535 635

FFase production of each different step are shown in Table 6. When A. japonicus TIT-90076 was cultivated in step 3 medium (24.05% sucrose and 2.75% yeast extract), maximum FFase production, 640 units m1-1, was obtained after 96 h cultivation. In conclusion, the improved medium, obtained by the combination of statistical experimental designs and the method of steepest ascent, showed a 180% increase in FFase production, compared with the control medium.

FFase production in fermenter experiments FFase production from the results of statistical analysis (Table 3). It had been reported that addition of adequate amounts of KzHPO4, MgSO4"7H20 and NaNO3 in the culture medium would have a positive effect on enzyme production [7,8,12]. However, sodium nitrate was found to have no influence on FFase production from A. japonicus TIT-90076 in the preliminary experiments. Liu [21] also reported that addition of K2HPO4 and MgSOa'7H20 might shift the morphology of fungal growth from filamentous to pellet form, but had no significant effect on FFase production. Accordingly, all mineral salts were eliminated from the medium and only sucrose and yeast extract were selected as main factors in the next step of medium improvement.

Statistical experimental design No. 2 (SED 2) and the steepest ascent method The second-step SED 2 was a full factorial design consisting of a two-factor-two-level pattern with four design points [17]. The concentrations of sucrose and yeast extract were transformed to the coded level as shown in Table 4, and enzyme activity was analyzed for each run to fit the first-order model equation (Table 5). The equation is described as follows:

Time courses of residual sugar and FFase production by A. japonicus TIT-90076 in a 5 litre fermenter were also observed for 48 h using both the improved and control media at 30°C and at 3.01itremin 1, 600 rev min -1 (Fig. 2). Maximum enzyme production with the improved medium was 810 units ml - I which was about 180% higher than that with the control medium (290units m l - l ) , but maximum growth was almost the same (about 13.8-14.0mgm1-1) in both media (data not shown). Moreover, sucrose was nearly exhausted after 4 8 h cultivation with the control medium, while there was still a large amount of residual sugar remaining with the improved medium (Fig. 2). Sucrose appears to be the best inducer for FTases or FFases production by various microorganisms [7,8,10]. Liu [21] concluded that one of the most important factors in FFase production from A. japonicus TIT-90076 was sucrose concentration. Similar results can also be observed in this study. Moreover, maximum enzyme production and maximum growth reached in both media were higher in fermenter 30 - 800 25

FFase production (units ml 1) 600

20-

= 512.5 +72"5X1+67"5X2 and R 2 = 0.970

4" E

According to the equation, the path of steepest ascent was determined to find the optimal concentration of each component [17]. The medium composition and Table6. Results of FFase production by A. japonicus TIT-90076 along the path of steepest ascent of the 22 factorial design Component

15"~

10

400

._~ => o E

-

n,"

E

200

w

5I

Concentration (% (w/v)) Step0 Step1 Step2 Step3 Step4 Step5

0 0

Sucrose 20-0 21"35 22"70 24-05 25'40 26"75 Yeast extract 2"0 2.25 2-5 2"75 3'0 3"25 FFase (units 574 590 610 640 630 600 m1-1)

10

i

i

I

20

30

40

0 50

Cultivation time (h)

Fig. 2. Comparative kinetics of FFase production by A. japonicus TIT-90076 with control (D, residual sugar; I, enzyme activity) and improved (©, residual sugar; e, enzyme activity) media in a 5 litre fermenter.

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