Bioresource Technology 99 (2008) 7–12

Characterization of a Saccharomyces cerevisiae mutant with enhancedproduction of �-D-fructofuranosidase

Ikram ul-Haq ¤, Sikander Ali, AaWa Aslam, M.A. Qadeer 1

Institute of Industrial Biotechnology, GC University Lahore, Pakistan

Received 22 July 2005; received in revised form 21 November 2006; accepted 22 November 2006Available online 26 February 2007

Abstract

The present study focused on the improvement of Saccharomyces cerevisiae through random mutagenesis for enhanced production of�-D-fructofuranosidase (FFase) using sucrose salt media. Sixty strains of S. cerevisiae were isolated from diVerent fruits and soil samplesand screened for FFase production. Enzyme productivity of diVerent yeast isolates ranged from 0.03 to 1.10 U/ml. The isolate with thehighest activity was subjected to ultraviolet (UV) radiation and mutagenesis using N-methyl N-nitro N-nitroso guanidine (MNNG). Onemutant produced FFase at a level of 17.8§ 0.9 U/ml. The MNNG-treated isolate was exposed to ethyl methane sulphonate (EMS), and amutant with an enzyme activity of 25.56§ 1.4 U/ml was obtained. Further exposure to UV radiation and chemicals yielded a mutantexhibiting an activity of 34.12§ 1.8 U/ml. After optimization of incubation time (48 h), sucrose concentration (5.0 g/L), initial pH (6.0) andinoculum size (2.0% v/v), enzyme production reached 45.65§ 4.6 U/ml with a noticeable greater than 40-fold increase compared to thewild-type culture. On the basis of kinetic variables, notably Qp (0.723§ 0.2 U/g/h), Yp/s (2.036§ 0.05 U/g) and qp (0.091§ 0.02 U/g yeastcells/h), the mutant S. cerevisiae UME-2 was found to be a hyperproducer of FFase (LSD 0.054, p 6 0.05).© 2007 Elsevier Ltd. All rights reserved.

Keywords: Saccharomyces cerevisiae; �-D-fructofuranosidase; Fermentation; Sucrose salt media; Kinetics; Random mutagenesis

1. Introduction

�-D-fructofuranosidase (FFase, EC 3.2.1.26) is a gly-coenzyme that hydrolyses �-D-fructofuranosides (raYnose,stachyose or sucrose) that is useful in the production ofconfectionery with liquid or soft centers and as an aid forfermentation of cane molasses into ethanol (Roitsch et al.,2003). Demand for this enzyme is increasing with growth ofthe confectionery industry. Microbial FFase is used in calffeed preparation and also for the manufacture of invertedsugars as nutrients for honeybees. Inverted solution ofsucrose is used in many industries (Sanchez et al., 2001).FFase is produced by a large number of organisms includ-ing Neurospora crassa, Candida utilis, Fusarium oxysporum,

* Corresponding author. Tel.: +92 42 9211634; fax: +92 42 7243198.E-mail address: ikrhaq@yahoo.com (I. ul-Haq).

1 Visiting Professor, CSO (Retd.), PCSIR Labs Complex, FerozepurRoad, Lahore, Pakistan.

0960-8524/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2006.11.047

Phytophthora meganosperma, Aspergillus niger, Saccharo-myces cerevisiae, Schizosaccharomyces pombe and Schwan-niomyces occidentalis (Lothe et al., 1999; Cuezzo et al.,2000). However, Saccharomyces cerevisiae is the organismof choice for FFase production because of its high sucrosefermentability (Rouwenhorst et al., 1991; Neto et al., 1996).A mutant with improved FFase production and the provi-sion of appropriate fermentation conditions are requiredfor better yield of enzyme (Gomez et al., 2000; ShaWq et al.,2004).

The production level of FFase depends to a great extenton the microorganism, basal substrate and the microbialproduction process. Submerged fermentation has been pre-ferred over solid-state for FFase production as it is envi-ronmentally friendly, requires less manpower and giveshigher yields (Koo et al., 1998). Workers have optimizedthe cultural conditions and nutritional requirements for theenhanced production of FFase by S. cerevisiae in batch cul-ture (Herwig et al., 2001). The composition of basal

8 I. ul-Haq et al. / Bioresource Technology 99 (2008) 7–12

medium may be either yeast extract, peptone, carbohy-drates, salt and vitamin solution (ArW et al., 2003). Indus-trial molasses media with ethanol and NaCl have also beenemployed for FFase production (Zech and Goerisch, 1995)and improved FFase production has been reported inmedium containing corn-steep liquor (Barlikova et al.,1991). Yeast biosynthesis of FFase is controlled by speciWccarbon sources such as sucrose or raYnose in the culturemedium. An extracellular FFase was secreted by S. cerevi-siae when grown on a medium containing the �-fructofur-anosides sucrose or raYnose, indicating that synthesis wassubjected to induction by the substrate (Mormeno et al.,1989). The use of FFase is limited due to its high cost, thusoptimization of the production process is critical. The pres-ent study is concerned with the isolation, screening andimprovement of S. cerevisiae isolates for FFase productionby submerged fermentation in shake Xasks as well as thekinetic characterization of the FFase production by themutant culture.

2. Methods

2.1. Isolation and screening of microorganism

Sixty S. cerevisiae strains were isolated from diVerentsoil samples and fruits such as plum, peach, date, banana,mango and guava obtained from diVerent localities ofLahore District, Pakistan. Isolations were carried out byserial dilutions on yeast peptone sucrose agar (YPSA)medium containing (g/L): yeast extract 3.0, peptone 5.0,sucrose 20.0 and agar 20.0 (pH 6.0). The petri plates wereincubated at 30 °C for 2–3 days. IdentiWcation of the yeastisolates by culture and morphological characteristics wasdone as described by Onion et al. (1986). Sub-culturing ofthe isolates was carried out every two weeks. These strainswere screened for FFase productivity and stored at 4 °C.

2.2. Cell growth and FFase productivity

S. cerevisiae was grown in 50 ml of YPS medium in 250-ml Erlenmeyer Xasks. For production of FFase, themedium was inoculated with 1.2£ 106 cells. The Xasks wereincubated in a rotary shaking incubator (Gallenkamp, Lon-don, UK) at 30 °C for 48 h. The agitation rate was kept at200 revolutions per minute.

2.3. Mutagenesis

For UV irradiation 5.0 ml of an 8-h culture of S. cerevi-siae IS-14 was subjected to centrifugation in a centrifugerefrigerated (Model 1134, Matlab, London, UK) at 6000revolutions per minute for 15 min. The cells were suspendedin 5.0 ml of sterilized 0.5% weight/volume (w/v) sucrose ace-tate buVer, pH 4.5, washed twice and resuspended in 50 mlof the buVer. Five milliliter of this suspension was trans-ferred to individual sterile petriplates and exposed to UVlight for diVerent time intervals (5–120 min) at a Wxed dis-

tance of 5 cm (dose 1.2£ 102 J/m2/s). Approximately 0.1 mleach of the UV-irradiated cell suspensions were transferredto the petri plates containing YPSA media. Colonies thatappeared within 72 h of incubation at 30 °C were trans-ferred to YPSA slants. Chemical mutagenesis with MNNGand EMS was carried out as described by Ginka et al.(2004). Mutagenized cells were plated onto YPSA mediumcontaining 12% (w/v) sucrose. Colonies exhibiting the mostgrowth were replica plated, and one set of the colonies wasexposed to a glucose measuring kit solution (Sigma, St.Louis, USA). Colonies that were surrounded by the largestpinkish zones were selected for further study.

Potential mutant strains were cultured overnight in YPSmedium, harvested during the exponential phase of growth(1.2£ 106 cells/ml), washed with sterilized distilled waterand plated on 2 dg-YPRA medium containing (mg/ml):yeast extract 3.0, peptone 5.0, raYnose 20.0, agar 20.0 and2-deoxy-D-glucose 0.02–0.10. RaYnose was used instead ofsucrose because sucrose hydrolysis by yeast FFase canresult in glucose formation (Rincon et al., 2001). Coloniesappearing between three and Wve days were subcultured onthesame medium, and colonies exhibiting the most vigorousgrowth were tested for FFase production by shake Xask fer-mentation. Samples weredrawn periodically, washed andplated on YPRA medium to select for strains resistantto 2-deoxy-D-glucose. The master culture was preserved in ster-ile paraYn oil.

2.4. Analytical methods

Yeast dry cell mass was determined after drying har-vested cells at 105 °C for 1 h. Sugar was estimated by thedinitro salicylic acid (DNS) method (Miller, 1959). Theenzyme activity was determined according to Akgol et al.(2001). One FFase unit was deWned as the amount ofenzyme which released 1.0 mg of inverted sugar in 5 min at35 °C, pH 5.5. For FFase activity measurement, 2.5 ml ace-tate buVer (50 mM, pH 5.5) and 0.1 ml sucrose (300 mM)was added into individual test tubes. The tubes were pre-incubated at 35 °C for 5 min. After the addition of 0.1 ml ofappropriately diluted enzyme extract, incubation was con-tinued for another 5 min. The reaction mixture was placedin a boiling water bath for 5 min to stop the reaction andallowed to cool at room temperature. A blank with distilledwater instead of the enzyme solution was run parallel. To1.0 ml of each mixture 1.0 ml of DNS reagent was addedand was placed in boiling water for 5 min. After cooling toambient temperature (20 °C), the volume was raised to10.0 ml with distilled water. A UV/VIS double beam scan-ning spectrophotometer (Cecil CE 100-series, AquariusInc., London, UK) was used to determine color intensity.

Kinetic variables were studied according to the proce-dure of Pirt (1975). The values for speciWc growth rate i.e., �(h¡1) were calculated from the plots of ln(X) versus time offermentation. The growth yield coeYcient (Yx/s) was calcu-lated as the dry cell mass divided by the amount of saccha-ride utilized during fermentation. The product yield

I. ul-Haq et al. / Bioresource Technology 99 (2008) 7–12 9

coeYcients namely Yp/s and Yp/x were determined by usingthe relationships Yp/sDdP/dS and Yp/xDdP/dX, respec-tively. The volumetric rates for substrate utilization (Qs)and product formation (Qp) were determined from themaximum slopes in plots of substrate utilized and FFaseproduced versus the time of fermentation. The volumetricrate for biomass formation (Qx) was calculated from themaximum slope in a plot of cell mass formation versus theincubation time. The speciWc rate constants for product for-mation (qp) and substrate utilization (qs) were determinedby the equations qpD�£Yp/x and qsD�£Ys/x, respec-tively. The speciWc rate for cell mass formation (qx) was, cal-culated by multiplying the speciWc growth rate (�) with thegrowth yield coeYcient (Yx/s).

Treatment eVects were compared by the method ofSnedecor and Cochran (1980). Duncan’s multiple range test(SPSS-10, version 4.0) was applied under one-way analysisof variance (ANOVA). SigniWcance is given as probability(p < 0.05) values. Y-error bars and § indicate the standarderror of means among three parallel replicates.

3. Results and discussion

The present study deals with the isolation, screening andimprovement of S. cerevisiae for FFase production by sub-merged fermentation in shake Xasks. Sixty S. cerevisiaestrains were isolated from diVerent samples of fruits andsoil and screened for their FFase productivities. The activi-ties of these isolates ranged from 0.03 to 1.10 U/ml. The iso-late IS-14 with the highest productivity was selected forimprovement through mutagenesis. The culture wasexposed to UV radiation, but no mutants with improvedFFase activity were detected. Therefore, chemical mutagen-esis using MNNG and EMS was undertaken. Eighteen iso-lates were obtained after MNNG treatment that hadproduced at least a 90% death rate. One mutant, MNNG-5,gave an approximately 3-fold increase in FFase productionand was exposed to EMS. One mutant (EMS-7) gave25.56§ 1.4 U/ml FFase activity, and it was selected for fur-ther improvement by alternate treatment with UV radia-tion and chemicals (MNNG and EMS). The best FFaseproducing mutant, UME-2, (34.12§1.8 U/ml) was selectedfor culture and nutritional studies in shake Xasks. Theenzyme productivity was signiWcantly diVerent (p 6 0.05)from that of all other isolates. High yielding isolates wereobtained at 2-deoxy-D-glucose (2 dg) concentration of0.02 mg/ml; however, their enzyme production phenotypebecame unstable after approximately two weeks. The rea-son may be the development of resistance in yeast cells aftera few generations that permitted a few unstable mutants tothrive. To mitigate this problem, isolates were again grownon medium containing diVerent concentrations of 2 dg. Theconcentration of 0.04 mg/ml was found optimal, as at thislevel UME-2 gave a consistent FFase yield.

In batch-wise FFase fermentation, enzyme productionbegan after a lag phase of approximately 8–12 h andreached a maximum at the onset of stationery phase. After-

wards, enzyme productivity declined sharply possibly dueto the decrease in nutrient availability in the medium, orcarbon catabolite repression, as the expression of FFase inyeast is repressed by monosaccharides such as glucose orfructose (Herwig et al., 2001). Therefore the growth stage ofa culture is a critical factor for optimal enzyme production.The time course proWles for FFase production by wild-typeS. cerevisiae IS-14 and 2 dg-stabilized mutant UME-2 areshown in Fig. 1. Maximum FFase production by mutantUME-2 (34.72§ 2.6 U/ml with 17.05§ 1.2 g/L sugar con-sumption and 7.85§1.8 g/L dry cell mass) was observed48 h after the onset of incubation (p 6 0.05). Therefore therate of volumetric productivity was improved approxi-mately 31-fold over the parental strain. Longer incubationtimes did not increase FFase production possibly due to thedecrease in available nitrogen, the age of the cells, inhibitorsproduced by yeast itself and protease production. Otherworkers have reported maximum FFase production by

Fig. 1. FFase production in submerged culture by Saccharomyces cerevi-siae. IS-14 (top) and mutant UME-2 (bottom), sucrose concentration 30 g/L, temperature 30 °C, initial pH 6.0, agitation rate 200 revolutions perminute. Y-error bars indicate standard deviation among three parallelreplicates.

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8 16 24 32 40 48 56 64 72 80

Incubation period (h)

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/ml),

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Sugar consumption (g/l)

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Sugar consumption (g/l)

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10 I. ul-Haq et al. / Bioresource Technology 99 (2008) 7–12

S. cerevisiae incubated for 48–96 h (Barlikova et al., 1991;Gomez et al., 2000).

The eVect of sucrose concentrations (1.0–10.0 g/L) onFFase production by the mutant S. cerevisiae UME-2 wasstudied (Fig. 2). Maximum enzyme activity (35.56§3.1 U/ml) was obtained at a sucrose concentration of 5.0 g/L.Sucrose concentrations higher than 5.0 g/L caused anincrease in sugar consumption and cell biomass; however,there was no net increase in FFase productivity. The reasonmight be the generation of inverted sugar in the medium ata level that results in glucose-induced repression of FFase.At concentrations of sucrose less than 5.0 g/L, enzyme pro-duction was signiWcantly (p 6 0.05) less. As sucrose is thecarbon source in the medium, lower concentrations mightlimit growth of yeast, resulting in a lower yield of FFase(ArW et al., 2003). The production of FFase is largely depen-dent on the initial pH of medium. The eVect of initial pH onenzyme production by S. cerevisiae UME-2 is shown inFig. 3. Maximum production of FFase was obtained whenthe pH of the medium was 6.5. Similarly, dry cell mass andsugar consumption were optimal at pH 6.5 reading7.43§ 1.2 and 4.99§0.6 g/L, respectively. The Wnal pH ofthe medium was 6.7. Similar results were obtained by Silve-ira et al. (1996) who also observed maximum FFase pro-duction by yeast at pH 6.5.

Among the factors that determine cell morphology andyeast fermentation patterns, size and age of inoculum are ofprime importance. In earlier attempts to standardize theinocula for FFase production in shaking cultures, Gancedo(1998) found a 10% inoculum to be optimal. In the presentinvestigation, a 16-h old vegetative inoculum was optimalfor maximum FFase production (45.65§ 1.6 U/ml) in shakeXasks when added at a level of 2.0% v/v (Fig. 4). An inocu-

Fig. 2. EVect of sucrose concentration on the FFase production in sub-merged culture by the mutant Saccharomyces cerevisiae UME-2. Incuba-tion period 48 h, temperature 30 °C, initial pH 6.0, agitation rate 200revolutions per minute. Y-error bars indicate standard deviation amongthree parallel replicates.

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-D-fructofuranosidase activity (U/ml) Dry cell mass (g/l) Sugar consumption (g/l)β

β

lum greater or smaller than 2.0% (v/v) resulted in a reduc-tion of FFase production. Possibly, at a lowerconcentration there are not enough yeast cells available inthe medium to produce more enzyme. Heredia and Heredia(1988) found 3.0% (v/v) vegetative inoculum for FFase pro-duction to be optimal. In contrast to our studies, Roitschet al. (2003) found that 48-h old cells were as good as thosefrom a 72–96 h old slant culture for FFase production,which suggested that the age of yeast cells may not have abearing on the enzyme production. The lag associated with

Fig. 3. EVect of initial pH on the FFase production in submerged cultureby the mutant Saccharomyces cerevisiae UME-2. Incubation period 48 h,sucrose concentration 5.0 g/L, temperature 30 °C, agitation rate 200 revo-lutions per minute. Y-error bars indicate standard deviation among threeparallel replicates.

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Fig. 4. EVect of age of inoculum on the FFase production in submergedculture by the mutant Saccharomyces cerevisiae UME-2. Incubationperiod 48 h, sucrose concentration 5.0 g/L, temperature 30 °C, agitationrate 200 revolutions per minute. Y-error bars indicate standard deviationamong three parallel replicates.

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I. ul-Haq et al. / Bioresource Technology 99 (2008) 7–12 11

inoculum from the stationary phase of a culture may beattributed to the reorganization necessary in the cell toreverse the changes caused by cessation of growth.

In the present study, the comparison of Qs (g cells/L/h)for FFase productivity demonstrated that the mutant strainUME-2 has a higher value for volumetric rate of substrateconsumption (QsD0.355§0.02 g/L/h) than the wild-cultureIS-14. A several fold improvement in terms of volumetricFFase productivity was noted with the mutant UME-2 forall the rates examined (Table 1). Although wild-type IS-14achieved a higher value (Yx/sD0.793§0.2 g yeast cells/g)than the mutant, mutant UME-2 demonstrated a signiW-cant improvement in volumetric rate of product formation.In addition, when both of the cultures were monitored forspeciWc rate constant, the mutant UME-2 gave higher val-ues for qp (greater than 18-fold improvement). Therefore,on the basis of kinetic variables, the mutant UME-2 exhib-ited 2–6 fold improvement in values for Qp, Yp/x, Yp/s and qpover the parental strain (LSD 0.054) and this was supportedby the Wndings reported by Pirt (1975). Neto et al. (1996)found that the aeration rate and substrate moisture contentinXuenced the substrate consumption rate, speciWc growthrate and subsequent enzyme productivity.

4. Conclusions

A mutant strain of S. cerevisiae, UME-2, with a 40-foldimproved FFase yield (45.65§ 4.6 U/ml) compared to wild-

Table 1Comparison of kinetic variables for FFase productivity by Saccharomycescerevisiae in shake Xask at 48 h

Kinetic variables: � (h¡1)D speciWc growth rate, Qp D �-D-fructofurano-sidase units/g/h, Yp/s D �-D-fructofuranosidase units/g substrate consumed,Yp/x D �-D-fructofuranosidase units/g yeast cells formed, qp D �-D-fructofu-ranosidase units/g yeast cells/h, Yx/s D g yeast cells/g substrate utilized,Qs D g substrate consumed/l/h, qs D g substrate consumed/g yeast cells/h,Qx D g yeast cells formed/L/h. HS is for the ‘highly signiWcant’ while S for‘signiWcant’ values. LSD denotes least signiWcant diVerence, p is for proba-bility. § indicates standard deviation among three parallel replicates. Thevalues in each row diVer signiWcantly at p 6 0.05.

Kinetic variables FFase productivity (U/ml)

IS-14 (wild-type) UME-2 (mutant)

SpeciWc growth rate� (h¡1) 0.092 § 0.02 0.166 § 0.01

FFase formation variablesQp (U/g/h) 0.023 § 0.02 0.723 § 0.2Yp/s (U/g) 0.190 § 0.01 2.036 § 0.05Yp/x (U/g) 0.239 § 0.02 4.356 § 0.8qp (U/g yeast cells/h) 0.005 § 0.001 0.091 § 0.02

Substrate consumption variablesYx/s (g yeast cells/g) 0.793 § 0.2 0.467 § 0.1Qs (g/L/h) 0.121 § 0.02 0.355 § 0.02qs (g/g yeast cells/h) 0.026 § 0.01 0.045 § 0.02Qx (g yeast cells/L/h) 0.096 § 0.02 0.174 § 0.02

Least signiWcant diVerence (LSD) 0.013 0.054

SigniWcance level �p� S HS

culture was isolated. The values of kinetic variables, nota-bly Qp (0.723§0.2 U/g/h), Yp/s (2.036§0.05 U/g) and qp(0.091§ 0.02 U/g yeast cells/h) demonstrated that themutant has a faster growth rate and subsequently a higherenzyme production capability (LSD 0.054, p 60.05).

References

Akgol, S., Kacarb, Y., Denizlia, A., ArÂcab, M.Y., 2001. Hydrolysis ofsucrose by invertase and novel magnetic polyvinylalcohol micro-spheres. Food Chem. 74, 281–288.

ArW, K., Tache, R., Spinnler, H.E., Bonnarme, P., 2003. Dual inXuence of thecarbon source and L-methionine on the synthesis of sulphur compoundsin the cheese-ripening yeast. Appl. Microbiol. Biotechnol. 61, 359–365.

Barlikova, A., Svorc, J., Miertus, S., 1991. �-D-fructofuranosidase forinverted syrup production and sugar determination. Anal. Chem. Acta247, 83–87.

Cuezzo, S., Maldonado, M.C., Valdez, G., 2000. PuriWcation and charac-terization of �-D-fructofuranosidase from Lactobacillus reuteri CRL1100. Curr. Microbiol. 40, 181–184.

Gancedo, J.M., 1998. Yeast carbon catabolite repression. Microbiol. Mol.Biol. Rev. 62, 334–361.

Ginka, I., Frengova, P., Emilina, D.S., Beshkova, M., 2004. Improvementof carotenoid-synthesizing yeast Rhodotorula rubra by chemical muta-genesis. Naturforsch. 59, 99–103.

Gomez, S.J., Augur, C., Gozalez, G., 2000. �-D-fructofuranosidase produc-tion by Aspergillus niger in submerged and solid-state fermentation.Biotechnol. Lett. 22, 1255–1258.

Heredia, M.F., Heredia, C.F., 1988. Saccharomyces cerevisiae acquiresresistance to 2-deoxyglucose at a very high frequency. J. Bacteriol. 170,2870–2872.

Herwig, C., Doerries, C., Marison, I., Stockar, U., 2001. Quantitative anal-ysis of the regulation scheme of �-D-fructofuranosidase expression inSaccharomyces cerevisiae. Biotechnol. Bioeng. 76, 247–258.

Koo, J., Hyup, K., Young, K.S., Choel, R.Y., Nansoo, H., Jin, S., 1998.Invertase production by fed-batch fermentation of recombinant Sac-charomyces cerevisiae. J. Microbiol. Biotechnol. 8, 203–207.

Lothe, R.R., Purohit, S.S., Shaikh, V.C., Pandit, A.B., 1999. PuriWcation of�-D-fructofuranosidase from bakers’ yeast on modiWed polymeric sup-ports. Bioseparation 8, 293–306.

Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for the determina-tion of reducing sugar. Anal. Chem. 31, 426–428.

Mormeno, S., Zueco, J., Iranzo, M., Sentandreu, R., 1989. O-Linked man-nose composition of secreted �-D-fructofuranosidase of Saccharomycescerevisiae. FEMS Microbiol. Lett. 48, 271–274.

Neto, J., Infanti, P., Vitolo, M., 1996. Hexokinase production from Saccha-romyces cerevisiae: culture conditions. Appl. Biochem. Biotechnol. 58,407–412.

Onion, A.H., Allosopp, D., Eggins, H.O., 1986. Smith’s Introduction toIndustrial Mycology, second ed. Edward Arnold Pub., London. pp.217–226.

Pirt, S.J., 1975. Principles of Microbes and Cell Cultivation, second ed.Blackwell ScientiWc Corporation, London. p. 116.

Rincon, A.M., Codon, A.C., Castrejon, F., Benitez, T., 2001. Improvedproperties of baker’s yeast mutants resistant to 2-deoxy-D-glucose.Appl. Environ. Microbiol. 67, 4279–4285.

Roitsch, T., Balibrea, M.E., Hofmann, M., Proels, R., Sinha, A.K., 2003.Extracellular �-D-fructofuranosidase: key metabolic enzyme and pro-tein. J. Exp. Biol. 54, 513–524.

Rouwenhorst, R.J., Van-Der-Baan, A.A., ScheVers, W.A., Van-Dijken, J.P.,1991. Production and localization of �-fructosidase in asynchronousand synchronous chemostat cultures of yeasts. Appl. Environ. Micro-biol. 57, 557–562.

Sanchez, M.P., Huidobro, J.F., Mato, I., Munigategui, S., Sancho, M.T.,2001. Evolution of �-D-fructofuranosidase activity in honey. J. Agr.Food Chem. 49, 416–422.

12 I. ul-Haq et al. / Bioresource Technology 99 (2008) 7–12

ShaWq, K., Ali, S., Haq, I., 2004. Analysis of nutritional strategies forinvertase production by Saccharomyces cerevisiae KR18. Pak. J. Bio-technol. 1, 25–30.

Silveira, M.C., Carvajal, E., Ban, E.S., 1996. Assay for in vitro yeast �-D-fructofuranosidase activity using NaF. Anal. Biochem. 238, 26–28.

Snedecor, G., Cochran, W.G., 1980. Statistical Methods, seventh ed. IowaState Univ.. pp. 80–86.

Zech, M., Goerisch, H., 1995. �-D-fructofuranosidase from Saccharomycescerevisiae: reversible inactivation by components of industrial molassesmedia. Enzyme Microbiol. Technol. 17, 41–46.