Research Article

Received: 18 November 2008 Revised: 19 December 2008 Accepted: 20 December 2008 Published online in Wiley Interscience: 18 February 2009

(www.interscience.wiley.com) DOI 10.1002/jctb.2134

Inulinase bio-production using agroindustrial residues: screening of microorganisms and process parameters optimization
Yemiko Makino,a Helen Treichel,b Marcio Antonio Mazutti,a Francisco Maugeria and Maria Isabel Rodriguesa
Abstract
BACKGROUND: The use of agroindustrial residues as substrate for inulinase bio-production is a good choice to reduce production costs, since enzyme activity will be improved and the downstream step of the process will be viable technically and economically. In addition, the screening of microorganisms that are able to overproduce inulinase using these substrates is fundamental to guarantee successful medium substitution. Based on these considerations, the objective of this present work was to select different strains of yeasts of the genus Kluyveromyces to produce the inulinase. RESULTS: Initially, 10 strains were tested in synthetic medium and six of these selected to perform tests in agroindustrial medium. Screening showed that the strains NCYC 587 and NRRL Y-7571 were able to produce the enzyme using agroindustrial residues as substrate. Optimization of inulinase activity as a function of pH, and concentrations of molasses, corn steep liquor (CSL) and yeast extract (YE) was carried out for the two strains. The maximum inulinase activity under optimized conditions was about 750 U mL1 . CONCLUSION: These values are approximately seven times greater than the values obtained using synthetic medium, showing the technical viability of the use agroindustrial residues for the inulinase production. c 2009 Society of Chemical Industry Keywords: inulinase; Kluyveromyces; agroindustrial residues; experimental design

INTRODUCTION
Inulinases are enzymes potentially useful with high fructose syrups (HFS) for enzymatic inulin hydrolysis, giving yields up to 95%.1 Conventional fructose production by starch hydrolysis includes three steps: -amylase, amyloglucosidase and glucose isomerase actions, yielding only 45% fructose in the nal product due to the thermodynamical equilibrium of the reaction.2 Inulinases are useful for the production of fructoligosaccharides production, which have functional and nutritional properties for use in lowcalorie diets, stimulation of Bidus and as a source of dietary bre in food preparations.3 The main raw material employed for inulinase production is inulin, which is expensive.3 The use of agroindustrial residues is a low cost alternative to for inulinase production, since the activity should be improved over, or at least remain the same as that obtained using a synthetic medium. The success of this substitute will depend on the choice of an appropriate microorganism that will grow on these substrates. Microorganisms used in food processes should meet the requirements for GRAS (Generally Recognized as Safe) as accepted by the FDA (Food and Drug Administration).2 Inulinase have been produced by several microorganisms, including Kluyveromyces marxianus,2 5 Aspergillus,6 8 Staphylococcus,5 9 Xantomonas,10 Pseudomonas.11 The Kluyveromyces strains have shown good potential, since they

meet the GRAS requirement and have been reported ato provide high inulinase production. This work is focused on the screening of Kluyveromyces strains with the potential for inulinase production using agroindustrial residues as substrates. Initially, 10 strains were tested in synthetic medium and, the best producers then tested in medium containing agroindustrial residues and yeast extract. For those strains with high potential for inulinase production, optimization of medium composition was carried out using an experimental design methodology.

EXPERIMENTAL
Screening The culture collection used in this work was composed of 10 Kluyveromyces strains: Kluyveromyces lodderi, Kluyveromyces

Correspondence to: Helen Treichel, Department of Food Engineering, URI Campus de Erechim, P.O. Box 743, CEP 99700-000, Erechim RS, Brazil. E-mail: helen@uricer.edu.br

a Department of Food Engineering, UNICAMP P.O Box 6121, CEP 13083-862, Campinas SP, Brazil b Department of Food Engineering, URI Campus de Erechim P.O. Box 743, CEP 99700-000, Erechim RS, Brazil

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Inulinase bio-production using agroindustrial residues marxianus NCYC 575, CBS 2103, NRRL Y-1550, NRRL Y-8280, NRRL Y-610, NCYC 587, NRRL Y-7571, NRRL Y-1196 and NRRL Y-8279. The microorganisms were grown on yeast malt agar medium (YMA) containing (g L1 ) yeast extract (Difco Laboratories, USA) 3.0, malt extract (Difco Laboratories) 3.0, peptone (Difco Laboratories) 5.0, glucose (Synth, Brazil) 10.0 and agar (Merck, Brazil) 20.0, and was sub-cultured every 3 weeks. To produce cells for the preinoculum, the cultures were grown in 500 mL asks with 100 mL of YM medium without agar, containing 20 g L1 of sucrose at pH 6.8, 30 C and 150 rpm for 24 h. Figure 1 presents a schematic diagram of the steps involved in the screening of microorganisms and process optimization. The preliminary screening was carried out employing synthetic medium previously optimized by Kalil et al.12 for Kluyveromyces marxianus ATCC 16 045 containing (g L1 ): sucrose 14.0, yeast extract 10.0, peptone 20.0, K2 HPO4 1.0. The pH was 3.5 or 6.5 at 30 C. Based on the results obtained, six strains were selected r further testing: Kluyveromyces marxianus NCYC 587, NRRL Y-610, NRRL Y-1196, NRRL Y-7571, NRRL Y-8279 and NRRL Y-8280. For these strains, runs employing agroindustrial residues as substrate were carried out. The media composition was that previously determined by Treichel13 (g L1 ): molasses 90.0, CSL 45.0, and yeast extract 4.0. The initials pH tested were 3.5 and 6.5 at 36 C for the strain NRRL Y-7571 and 30 C for the other strains. Based on the activity results, two strains were selected, Kluyveromyces marxianus NCYC 587 and Kluyveromyces marxianus NRRL Y-7571, for more tests. Process Optimization The fermentations were carried out in 500 mL conical asks containing 100 mL culture medium, with concentrations dened in the experimental design (Tables 1 to 3). Fermentations were started with inoculum 10% (v/v) at 150 rpm in an orbital shaker (PSYCROTHERM, New Brunswick Scientic, NJ). For the strain NRRL Y-7571 the temperature of fermentation was 36 C, as previously determined by Treichel et al.15 and 30 C was used for strain NCYC 587. The inulinase activity was assessed at 0, 24, 48 and 72 h of fermentation.

www.soci.org For strain NCYC 587, a central composite rotatable design (CCRD) was carried out to optimize the concentrations of molasses, CSL and yeast extract for inulinase activity. To optimize inulinase production for the strain Kluyveromyces marxianus NRRL Y-7571 the sequential experimental design methodology was adopted.14 Initially, a 24 1 fractional design was carried out to evaluate the effect of molasses, CSL, yeast extract concentrations, and pH. Based on the effects analysis (P < 0.1) a CCRD was carried out to optimize the concentration of yeast extract and to optimize pH (Table 3). The results were analysed using Statistica 6.0 (Statsoft Inc, USA). Inulinase assay Activity was assayed as follows: 1 mL enzyme solution was mixed with 9 mL of 2% (w/w) sucrose in sodium acetate buffer 0.1 mol L1 , pH 4.8. The mixture was maintained at 50 C and the rate of appearance of total reducing sugars (TRS) was determined by the DNS method.16 One unit of inulinase activity is dened as the amount of enzyme necessary to hydrolyse 1 mol of sucrose per min under the above-dened conditions.3

RESULTS AND DISCUSSION

Screening Figure 2(a) and 2(b) presents the kinetics of the inulinase activity in synthetic medium at pH 3.5 and 6.5, respectively. It can be observed that the strains NCYC 575 and K. lodderi gave lower inulinase production at both pH levels. The strains CBS 2103, NRRL Y-1550, NRRL Y-8280 and NRRL Y610 produced enzyme activities around 20 U mL1 , and the strain NRRL Y-610 showed an intermediate production at pH 3.5. At pH 3.5, the strains NCYC 587, NRRL Y-7571, and NRRL Y-1196 were the highest producers (80130 U mL1 ). At pH 6.5, the strains NRRL Y-7571, NCYC 587, and NRRL Y-8279 were the highest producers (50100 U mL1 ). Based on these results six strains were selected for the enzyme production in medium containing agroindustrial residues: NCYC

SCREENING IN SYNTHETIC MEDIUM Medium (g.L-1) : sucrose 14.0, yeast extract 10.0, K2HPO4 1.0, and peptone 20.0 Microorganisms: Kluyveromyces marxianus NCYC 575, NCYC 587, CBS 2103, NRRL Y-7571, NRRL Y- 610, NRRL Y- 1196, NRRL Y- 1550, NRRL Y- 8279, NRRL Y- 8280, and Kluyveromyces lodery

SCREENING IN INDUSTRIAL MEDIUM Medium (g.L-1) : Molasses 90.0, corn steep liquor 45.0, and yeast extract 4.0 Microorganisms: Kluyveromyces marxianus NCYC 587, NRRL Y-7571, NRRL Y- 610, NRRL Y- 1196, NRRL Y- 8279, and NRRL Y- 8280

OPTIMIZATION NRRL Y-7571 2 + 3 Central points Factors: Molasses, CSL, YE, pH CCRD Factors: YE, pH
4-1

OPTIMIZATION NCYC 587 CCRD Factors: Molasses, CSL, YE

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Figure 1. Schematic diagram for screening and optimization.

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Table 1. Matrix of the CCRD (real and coded values) with responses in terms of inulinase activity (after 72 h fermentation) using Kluyveromyces marxianus NCYC 587 Molasses (g L1 ) 70 (1) 110 (1) 70 (1) 110 (1) 70 (1) 110 (1) 70 (1) 110 (1) 56.4 (1.68) 123.6 (1.68) 90 (0) 90 (0) 90 (0) 90 (0) 90 (0) 90 (0) 90 (0) CSL (g L1 ) 30 (1) 30 (1) 60 (1) 60 (1) 30 (1) 30 (1) 60 (1) 60 (1) 45 (0) 45 (0) 19.8 (1.68) 70.2 (1.68) 45 (0) 45 (0) 45 (0) 45 (0) 45 (0) YE (g L1 ) 3 (1) 3 (1) 3 (1) 3 (1) 5 (1) 5 (1) 5 (1) 5 (1) 4 (0) 4 (0) 4 (0) 4 (0) 2.3 (1.68) 5.68 (1.68) 4 (0) 4 (0) 4 (0) Measured activity (U mL1 ) 334 128 394 90 386 213 397 371 295 302 394 657 60 631 765 723 717 Predicted activity (U mL1 ) 270 168 270 168 473 371 473 371 340 168 481 481 131 471 740 740 740 RED (%) Yexp Ymodel 100 Yexp 19.03 31.45 31.36 86.94 22.48 73.97 19.08 0.12 15.21 44.31 22.09 26.78 118.46 25.36 3.23 2.39 3.24

Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

RED =

Table 2. Matrix of the 2(4 1) fractional design (real and coded values) with responses in terms of inulinase activity (after 72 h fermentation) using Kluyveromyces marxianus NRRL Y-7571 Run 1 2 3 4 5 6 7 8 9 10 11 Molasses (g L1 ) 70 (1) 110 (1) 70 (1) 110 (1) 70 (1) 110 (1) 70 (1) 110 (1) 90 (0) 90 (0) 90 (0) CSL (g L1 ) 30 (1) 30 (1) 60 (1) 60 (1) 30 (1) 30 (1) 60 (1) 60 (1) 45 (0) 45 (0) 45 (0) YE (g L1 ) 3 (1) 3 (1) 3 (1) 3 (1) 5 (1) 5 (1) 5 (1) 5 (1) 4 (0) 4 (0) 4 (0) pH 3.5 (1) 5.5 (1) 5.5 (1) 3.5 (1) 5.5 (1) 3.5 (1) 3.5 (1) 5.5 (1) 4.5 (0) 4.5 (0) 4.5 (0) Activity (U mL1 ) 30 662 350 12 439 66 413 677 676 623 764

587, NRRL Y-610, NRRL Y-1196, NRRL Y-7571, NRRL Y-8279, and NRRL Y-8280. As suggested by the results presented in Fig. 2(a) and 2(b) the strains NCYC 587, NRRL Y-610 and NRRL Y-1196 showed better performance in pH 3.5 and the strains NRRL Y-7571, NRRL Y-8279 and NRRL Y-8280 showed better performance in pH 6.5. Fermentations were carried out at the ideal pH for each strain. Figure 2(c) shows that the strains NCYC 587 and NRRL Y-7571 presented the highest inulinase activities. The maximum activity was 296 U mL1 at 48 h for the strain NRRL Y-7571 and 740 U mL1 for the strain NCYC 587 after 72 h of fermentation. The strains NCYC 587 and NRRL Y-7571 were chosen for the optimization of medium composition using agroindustrial residues as substrate. The conditions used in the screening part of this study (90.0 g L1 molasses, 45.0 g L1 CSL and 4.0 g L1 yeast extract) was dened as the central point of the experimental designs presented in Tables 1 and 2.

Optimization of inulinase production for the strain NCYC 587 The analysis of the data presented in Fig. 2(a) and 2(b) for the strain NCYC 587 showed that high activities were obtained at pH 3.5. This result is similar to that obtained by Kalil et al.12 for the yeast Kluyveromyces marxianus ATCC 16 045, where the analysis of the effects indicated that the most adequate pH for this strain was 3.5. A decreasing pH value affects the isoelectric point of the enzyme: pH was maintained at 3.5 for all the experiments. The effects of the molasses, CSL, and yeast extract (YE) concentrations were assessed in a CCRD. Table 1 presents the experimental results in terms of inulinase activity obtained after 72 h fermentation. Maximum inulinase activity was 735 26 U mL1 at the central point conditions of the CCRD (90 g L1 molasses, 45 g L1 CSL, and 4 g L1 yeast extract). This production level is six times higher than that obtained by Kalil et al. using synthetic medium and three times higher than that obtained in the screening tests for this strain.12 Data in Table 1 were used to generate a coded empirical model to predict the inulinase production as a function of the molasses (M), CSL and YE concentrations. This model was validated by analysis of variance (ANOVA), since the calculated F-test (F5;11 = 9.16) was about 2.9 times greater than an earlier F-test result in the literature (3.20).17 The goodness of t of the model was checked using the correlation coefcient: R = 0.898 indicating good correlation between the independent variables. The predicted inulinase activity and relative error deviation (RED) are also illustrated in Table 1, where one can see that for the higher values of inulinase activity, lower values of RED were obtained, so the model is satisfactory in the range of interest. From the model equation it can be seen that the non-signicant terms (P < 0.05) were the linear term in CSL and the interaction terms: ActivityNCYC587 = 740.3 51.1 M 172.3 M2 91.9 CSL2 + 101.2 YE 155.6 YE2 (1)

The response surface methodologies (RSM) and contour plots presented in Fig. 3 are graphical representations of the regression

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Table 3. Matrix of the CCRD (real and coded values) with responses in terms of inulinase activity (after 72 h fermentation) by Kluyveromyces marxianus NRRL Y - 7571 YE (g L1 ) 3(1) 5(1) 3(1) 5(1) 2.6 (1.41) 5.4 (1.41) 4(0) 4(0) 4(0) 4(0) 4(0) Measured activity (U mL1 ) 558 557 359 147 477 473 554 321 761 745 661 Predicted activity (U mL1 ) 547 547 312 312 450 450 579 247 723 723 723 RED (%) Yexp Ybmod el 100 Yexp 1.91 1.73 13.02 112.42 5.60 4.80 4.43 23.03 5.06 3.02 9.31

Run 1 2 3 4 5 6 7 8 9 10 11

pH 4.8 (1) 4.8 (1) 6.2 (1) 6.2 (1) 5.5 (0) 5.5 (0) 4.5 (1.41) 6.5 (1.41) 5.5 (0) 5.5 (0) 5.5 (0)

RED =

(a) 100 80 Activity (U/mL) 60 40 20 0 0 24 48 72 Time (h) (c) 800 700 Activity (U/mL) 600 500 400 300 200 100 0 0

Activity (U/mL)

CBS 2103 NCYC 587 NCYC 575 K. loderi NRRL Y-610 NRRL Y-1196 NRRL Y-1550 NRRL Y-7571 NRRL Y-8279 NRRL Y-8280

(b) 100 80 60 40 20 0 0 24 48 Time (h)

NCYC 587 (pH 3.5) NRRL Y-1196 (pH 3.5) NRRL Y-610 (pH 3.5) NRRL Y-7571 (pH 6.5) NRRL Y-8279 (pH 6.5) NRRL Y-8280 (pH 6.5)

CBS 2103 NCYC 587 NCYC 575

K. Loderi NRRL Y-610 NRRL Y-1196 NRRL Y-1550 NRRL Y-7571 NRRL Y-8279 NRRL Y-8280

72

24 Time (h)

48

72

Figure 2. Kinetic behavior of inulinase production during the screening stage: (a) synthetic medium at pH 3.5; (b) synthetic medium at pH 6.5; and (c) agroindustrial residues.

equation for the inulinase activity obtained by the strain NCYC 587. It is clear that the process variables investigated in this study are at optimum, since the maximum inulinase activity was predicted at around the central point of the CCRD. For all the residues employed in the medium formulation, the maximum inulinase activity was obtained for an ample range of concentration. These results are important when agroindustrial residues are employed in fermentation, since the variability existing in the raw material will not affect the process yield. Optimization of inulinase production by the strain NRRL Y7571 Figure 2(a) and 2(b) presents the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using synthetic medium, with similar behavior at both pH levels. Figure 2(c) (using agroindustrial residues) shows that the activity decreased abruptly after

48 h fermentation. A possible explanation for this is that the pH of the medium affected the enzyme stability, suggesting the possibility of an optimal pH value to improve enzyme production. The inuence of the initial molasses, CSL and yeast extract concentrations and pH was evaluated using a 2(4 1) fractional design. The results obtained are listed in Table 2, where the enzyme activity ranged from 12 to 688 71 U mL1 . These data were used to compute the main effect of the independent variables (P < 0.1) and the results are shown in Fig. 4. The molasses and CSL concentration were not signicant in the range investigated and their values were maintained at 90 g L1 and 45 g L1 , respectively, in the next experimental design. pH was the most important variable in this part of the study, such that an increment in its value can improve production. YE concentration was not signicant, but it was considered in the next experimental design, because of its importance to the process and the P-value obtained for

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Figure 3. Response surfaces and contour curves for inulinase production by the strain NCYC 587: (a) and (b) effect of the concentrations of molasses and corn steep liquor (CSL); (c) and (d) effect of the concentrations of molasses and yeast extract; (e) and (f) effect of the concentrations of yeast extract and CSL.

(4) pH

6.674431

(3) YE

2.246961

(2) CSL

1.059104

(1) Molasses

.7683694

p=.1 Effect Estimate (Absolute Value)

Figure 4. Pareto chart for 24 1 fractional design for inulinase activity by Kluyveromyces marxianus NRRL Y 7571.

this variable. According to Haaland, the P-values are useful for screening experiments, and it is probably better to accept higher P-values rather than take the chance of missing an important factor.18 Based on the results obtained in the fractional design, a CCRD was carried out to evaluate the effect of YE concentration and pH on inulinase production. The results obtained are presented in Table 3. The inulinase activity ranged from 147 to 722 54 U mL1 . Maximum production was obtained in the central point of the CCRD: 90 g L1 molasses, 45 g L1 CSL, 4 g L1 YE and pH 5.5. The activity obtained in the optimized conditions (central point) was improved about 2.5 times compared with that obtained in the screening tests (294 U mL1 ). Thus, pH 5.5 is better than pH 3.5 and 6.5. Analogous to the strain NCYC 587, the data in Table 3 were used to t a coded empirical model for the inulinase production by the strain NRRL Y-7571 as a function of YE concentration and

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Figure 5. Response surfaces (a) and contour curves (b) for inulinase production by the strain NRRL Y-7571.

pH. The coded model was validated using analysis of variance (ANOVA), since the calculated F-test (F3;7 = 16.23) was about 3.7 times greater than F-test listed one (F3;7 = 4.35), allowing for validation of the second order codied model (Equation 2). The goodness of t of the model was checked using the correlation coefcient (R = 0.935). From the model equation (Equation (2)), it can be seen that the most signicant variables (P < 0.05) were the quadratic term in YE concentration and both linear and quadratic terms in pH. This implies satisfactory representation of the process by the model. This is illustrated by the predicted inulinase activity and relative error deviation (RED) shown in Table 3, where one can see that at the central point (optimized condition), the relative error is less than 10%. It is worth noting that the higher values of RED were obtained at low values of inulinase activities, so this fact will not compromise the model reliability. ActivityNRRLY 7571 = 722.5 136.9 YE 117.5 pH 155.8 pH
2 2

60 Cells, TRS (g/L), S/I (-) 50 40 30 20 10 0 0 12 24

Cells TRS S/I Inulinase

800 700 Inulinase (U/mL) 600 500 400 300 200 100 0

36 Time (h)

48

60

72

(2)

Figure 6. Validation kinetic for the strain NRRL Y-7571.

The RSM and contour plots of model 2 for the inulinase activity obtained by the strain NRRL Y-7571 are presented in Fig. 5. It is clear that the process is robust, since optimized conditions are available over wide ranges of pH and YE concentration (4.7 to 5.7 for pH and 3.5 g L1 to 4.5 g L1 for YE). Validation of the model predictions for the strain NRRL Y-7571 The validity and condence of the coded model predictions of Equation. (2) were checked. For this, an additional experiment using different values for the independent variables from those used in the CCRD was carried out. The kinetic behaviour of the process was monitored (Fig. 6) over 72 h using a medium composition of molasses 90 g L1 , CSL 45 g L1 , YE 4 g L1 , and pH 5.0. After 72 h fermentation, the experimental inulinase activity was about 700 U mL1 , which is in agreement with the model predictions (728 U mL1 ). Figure 6 presents data on the microbial cell concentration, TRS consumption and sucrose/inulin ratio (S/I). TRS concentration decreased by approximately 90% in the rst 12 h, and the cell concentration reach the maximum value at 24 h. Inulinase production is partially growth-associated, since enzyme production was veried in the dead cell phase. The criterion used to conrm that the enzyme produced was an inulinase was based on the S/I value, which should be lower than 50.19 In this work, the S/I value was lower than 20 during all fermentations. After process optimization for the two strains selected (NRRL Y-7571 and NCYC 587) the maximum inulinase activity obtained was approximately 710 U mL1 . This activity is greater than

other researchers had obtained using synthetic medium with the Kluyveromyces marxianus ATCC 16 045. Kalil et al.12 obtained 127 U mL1 and Silva-Santisteban and Maugeri obtained 176 U mL1 .3 Using inulin as carbon source, Singh et al., obtained 55.4 U mL1 using Kluyveromyces marxianus YS-1.20 Using agroindustrial residues in solid-state fermentation, Bender et al.21 and Mazutti et al.2 obtained 445 and 395 U g1 , respectively with Kluyveromyces marxianus NRRL Y-7571. Recently, an interesting study investigated inulinase production from Kluyveromyces marxianus YS-1 using root tubers of Aspargus ofcinalis as the source of raw inulin, and the maximum inulinase activity obtained was 40.2 U mL1 .22

CONCLUSIONS
This work has presented a sequential methodology of screening and optimization of Kluyveromyces strains for inulinase production using agroindustrial residues as substrate. Initially, 10 strains were tested in synthetic medium and six were selected to use in medium containing agroindustrial residues. Screening tests enabled selection of two strains with good potential for inulinase production in agroindustrial residues: Kluyveromyces marxianus NCYC 587 and Kluyveromyces marxianus NRRL Y-7571. At optimized conditions, the maximum inulinase activity obtained was 735 26 and 722 54 U mL1 for the strain NCYC 587 and NRRL Y-7571, respectively. This value is approximately seven times greater than that obtained using synthetic medium,

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ACKNOWLEDGEMENTS
The authors are grateful to CNPq, CAPES and FAPESP for their nancial support of this research and for the scholarships awarded.

REFERENCES
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10 Park JP, Bae JT, You DJ and Kim BW, Production of inulooligosaccharydes from inulin by a novel endo inulinase from Xanthomonas sp. Biotechnol Lett 21:10431046 (1999). 11 Jong WY, Yong JC, Chii HS and Seung KS, Microbial production on inulo-oligosaccharydes by an endoinulinase from Pseudomonas sp. expressed in Escherichia coli. J Biosci Bioeng 87:291295 (1999). 12 Kalil SJ, Suzan R, Maugeri F and Rodrigues MI, Optimization of inulinase production by Kluyveromyces marxianus using factorial design. Appl Biochem Biotechnol 94:257264 (2001). 13 Treichel H, Study of industrial medium for inulinase production by Kluyveromyces marxianus var. bulgaricus ATCC 16045. Master thesis, Campinas, BrazilUNICAMP (2001). 14 Rodrigues MI and Iemma AF, Planejamento de Experimentos o de Processos : Uma estrat e Otimizac a egia sequencial de planejamentos, Casa do P ao Editora, Campinas, Brazil (2005). 15 Treichel H, Mazutti MA, Maugeri Filho F and Rodrigues MI, Technical viability of the production, partial purication and characterisation of inulinase using pretreated agroindustrial residues. Bioprocess Biosyst Eng DOI:10.1007/s00449-008-0262-0, in press. 16 Miller GL, Use of dinitrosalisylic acid reagent for determination of reducing sugar. Anal Chem 31:426428 (1959). 17 Khuri AI and Cornell JA, Response Surface Design and Analysis. ASQC Quality Press, Marcel Dekker, New York (1987). 18 Haaland PD, Experimental Design in Biotechnology. Marcel Dekker, New York (1989). 19 Vandamme EJ and Derycked G, Microbial inulinases : fermentations process, properties and applications. Adv Appl Microbiol 139:7679 (1983). 20 Singh RS, Sooch BS and Puri M, Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1. Bioresources Technol 98:25182525 (2007). 21 Bender JP, Mazutti MA, Oliveira D, Di Luccio M and Treichel H, Inulinase production by Kluyveromyces marxianus NRRL Y7571 using solid state fermentation. Appl Biochem Biotechnol 129132:951958 (2006). 22 Singh RS and Bhermi HK, Production of extracelular exoinulinase from Kluyveromyces marxianus YS-1 using root tubers of Asparagus ofcinalis. Bioresources Technol 99:74187423 (2008).

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