cellobiose fermentation by the yeast dekkera bruxellensis and implications for

7/27/2019 Cellobiose Fermentation by the Yeast Dekkera Bruxellensis and Implications For

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Biotechnology Letters

Cellobiose fermentation by the yeast Dekkera bruxellensis and implications forproduction of second generation ethanol.

--Manuscript Draft--

Manuscript Number:

Full Title: Cellobiose fermentation by the yeast Dekkera bruxellensis and implications for

production of second generation ethanol.Article Type: Manuscript

Section/Category: Microbial and Enzyme Technology

Keywords: BGL gene; -glucosidase; hydrolyzed bagasse; lignocellulose

Corresponding Author: Marcos Antonio de Morais Jr, Ph.D.Federal University of PernambucoRecife, Pernambuco BRAZIL

Corresponding Author SecondaryInformation:

Corresponding Author's Institution: Federal University of Pernambuco

Corresponding Author's SecondaryInstitution:

First Author: Alexandre LS Reis, MSc

First Author Secondary Information:

Order of Authors: Alexandre LS Reis, MSc

Rochane RNB Torres, MSc

Fernanda CB Leite, Ph.D.

Raquel FR de Souza, BSc

Thiago H Napoleo, Ph.D.

Patrcia Maria G Paiva, Ph.D.

Marcos Antonio de Morais Jr, Ph.D.

Order of Authors Secondary Information:

Abstract: In the present work we confirmed the potential of the yeast Dekkera bruxellensis toproduce ethanol from cellobiose both in synthetic medium, which has been recentlyreported, as well as in enzyme-treated steam-exploded sugarcane bagasse. It isshown the main features of the purified cellobiase (-glucosidase, E.C. 3.2.1.21).

Additional in silico analysis identified the corresponding BGL gene and revealed themain structural characteristics of the coded intracellular enzyme, which is similar toKluyveromyces marxianus counterpart. Physiological and enzyme data pointed thatlow assimilation capacity maybe the limiting step for the complete and fast conversionof cellobiose towards ethanol, besides to already known negative Custer effect for disaccharide fermentation. However, the overpowering capacity of this yeast to settleand stay in industrial environments such as the ethanol fermentation process makes ita promising yeast to ferment lignocellulosic substrates.

Suggested Reviewers: Ken Tokuyasu, Ph.D.Researcher, National Agriculture and Food Research [email protected]. Tokuyasu works on fermention of plant biomass and has been recently publishig inthe subject, such as:Guan et al (2012) Sequential incubation of Candida shehatae and ethanol-tolerantyeast cells for efficient ethanol production from a mixture of glucose, xylose andcellobiose. Bioresour Technol 132:419-422.

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Badal C Saha, Ph.D.Researcher, National Center for Agricultural Utilization [email protected]. Saha works on the fermentation of xylose and with production of hydrolyticenzymes and enzymes involved in the metabolism of alternative sugars for secondgeneration etahnol production.

Lisbeth Olsson, Ph.D.Professor, Technical University of [email protected]

Dr. Olsson works on the production and characterization of oligosaccharides-degradingenzymes for convertion of plant biomass to second generation ethanol:Krogh et al (2010) Characterization and kinetic analysis of a thermostable GH3 beta-glucosidase from Penicillium brasilianum. Appl Microbiol Biotechnol 86:143-154.

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Journal: Biotechnology L etters 1

Section: M icr obial and En zyme Technology 2

3

4

Cellobiose fermentation by the yeast Dekkera bruxell ensis and implications for5

production of second generation ethanol6

7

Alexandre Libanio Silva Reis 1, Rochane Regina Neves Baptista Torres 2, Fernanda Cristina Bezerra8

Leite2

, Raquel de Ftima Rodrigues de Souza1,2

, Thiago Henrique Napoleo3

, Patrcia Maria9

Guedes Paiva 3 and Marcos Antonio de Morais Jr 1,3,4* 10

11

12

1Laboratory of Bioprocessing, CETENE. 50740-540 Recife, PE, Brazil.13

2Interdepartmental Research Group on Metabolic Engineering, 3Department of Genetics and14

4Department of Biochemistry, Federal University of Pernambuco. 50670-901 Recife, PE, Brazil.15

16

17

*Author for correspondence:18

Marcos A de Morais Jr 19

Departamento de Gentica Universidade Federal de Pernambuco20

Av. Moraes Rego, 1235, Cidade Universitria 50670-901 Recife PE Brasil21

E-mail: [email protected] 22

Phone: 55-81-2126781723

Fax: 55-81-2126852224

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anuscriptck here to download Manuscript: Reis et al 2013 Cellobiose fermentation by Dekkera [Biotech Lett].doc
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Abstract1

In the present work we confirmed the potential of the yeast Dekkera bruxellensis to produce ethanol2

from cellobiose both in synthetic medium, which has been recently reported, as well as in enzyme-3

treated steam-exploded sugarcane bagasse. It is shown the main features of the purified cellobiase4

( -glucosidase, E.C. 3.2.1.21). Additional in silico analysis identified the corresponding BGL gene5

and revealed the main structural characteristics of the coded intracellular enzyme, which is similar 6

to Kluyveromyces marxianus counterpart. Physiological and enzyme data pointed that low7

assimilation capacity maybe the limiting step for the complete and fast conversion of cellobiose8

towards ethanol, besides to already known negative Custer effect for disaccharide fermentation.9

However, the overpowering capacity of this yeast to settle and stay in industrial environments such10

as the ethanol fermentation process makes it a promising yeast to ferment lignocellulosic substrates.11

12

13

Keywords: BGL gene, -glucosidase, hydrolyzed bagasse, lignocellulose.14

15

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Introduction1

2

The potential of the yeast Dekkera bruxellensis to ferment sucrose from sugarcane juice has been3

recently reported (Pereira et al. 2012; Leite et al. 2013) which, in connection to its high fitness in4

industrial production plants (De Souza Liberal et al. 2007), makes of this yeast a promising5

microorganism for first generation fuel ethanol production. Besides, another putative use of this6

yeast is for ethanol production from biomass hydrolysates, using resources that are inaccessible for 7

the fermenting yeasts (Blomqvist et al. 2011). Assimilation of cellobiose, a disaccharide generated8

by the incomplete hydrolysis of cellulose, is a welcome feature for yeasts that are intended to be9

used for this purpose. This trait have been reported long time ago for D. bruxellensis (Blondin et al.10

1982) despite the fact that this is not the characteristic described for the species from its type strain11

CBS 74 and many other strains (Blomqvist et al. 2010; Galafassi et al. 2011). Sequential12

fermentation approach was proposed for conversion the mixture of glucose, xylose and cellobiose13

after enzymatic hydrolysis of cellulosic biomass. This included the combination of Candida14

shehatae (for glucose and xylose fermentation) with D. bruxellensis (for cellobiose fermentation),15

with complete consumption of sugars and ethanol yield near to 0.4 g ethanol per gram of consumed16

sugar (Guan et al. 2013). In the present report we show the ability of D. bruxellensis strain GDB17

248 to ferment cellobiose for ethanol production. The advantages and constraints regarding to the18

biotechnological use of this yeast for production of second generation ethanol are discussed in the19

text.20

21

Materials and Methods22

23

Yeast strain and cultivation24

The strain Dekkera bruxellensis GDB 248 was maintained in YPD medium. Cells were pre-grown25

on liquid YPD at 30C and 150 rpm for 24 h. Aerobic growth experiments in different carbon26


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sources and analytical determination of extracellular metabolites by HPLC were performed as1

described (Leite et al. 2013). The experiments were performed in biological duplicate with two2

technical replicates for each point.3

4

Bagasse hydrolysis and fermentation assays5

Steam-exploded sugarcane bagasse was suspended in 100 mM Tris-Acetate pH 4.5 buffer to 20 g/l6

and treated with Fibrenzyme LWT commerc ial preparation (Dyadic International Inc., Jupiter,7

USA), with 1 ml of enzyme preparation for each 10 g of bagasse, at 50C for 72 h with gentle8

agitation. The hydrolysate was centrifuged at 1,200 x g for 5 minutes and the liquid part was used9

for fermentation assays. The initial sugar composition was evaluated by HPLC (Leite et al. 2013).10

Fermentation assays were performed as described by Pereira et al. (2012) with 10% (w/v) yeast11

biomass in synthetic fermentation medium (YNB at 1.7 g/l) containing sucrose or cellobiose at 2012

g/l or in the bagasse hydrolysate. The suspensions were incubated at 30C and 120 rpm in orbital13

shaker. Samples were collected every hour for cell density determination and metabolites analyses14

by HPLC. The experiments were performed in three biological replicates with two technical15

replicates for each point.16

17

Cellobiase ( -glucosidase) production18

Yeast cells were cultivated in synthetic cellobiose medium (1.7 g YNB/l and 1.0 g cellobiose/l)19

until 0.6 A600nm

and diluted 1/1000 to 250 ml fresh synthetic medium containing glucose, cellobiose20

or sucrose at 1.0 g/l. The flasks were incubated for 24 h at 33C and 130 rpm in orbital shaker.21

Afterwards, the cells were collected by centrifugation, re-suspended in 250 ml corresponding22

medium and cultivated as above. This recycling process was repeated by four times and after the23

last cycle the final cell density was determined (Leite et al. 2013). All cells were collected by24

centrifugation at 3,800 x g and 4C for 30 min. Cell pellet was re-suspended in two volumes of 1025

mM Acetate buffer pH 4 containing 1 mM -mercaptoethanol and lysed by maceration in liquid26


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nitrogen. The lysates were centrifuged at 21,000 x g for 15 mi nutes at 4C and the supernatant was1

recovered. Enzyme reaction were performed by mixing 100 g of protein from cell free extract and2

sugar solution diluted in 100 mM sodium citrate buffer pH 4.8 for final volume of one ml. The3

reaction was incubated 10 min at 30C and stopped by transferring the tubes to ice bath. Release4

glucose was measured by glucose oxidase kit (LabLabor, Brazil). Specific activity was recorded as5

the amount of glucose equivalent released per minute from the amount of protein in one gram of 6

yeast cells. For testing the presence of extracellular enzyme, supernatant of synthetic media7

containing sucrose or cellobiose was used for enzyme reactions.8

9

Cellobiase ( -glucosidase) purification 10

Yeast cells grown in synthetic-cellobiose medium were lysed as above and subjected to fractioning11

in ammonium sulfate from 0% to 60% saturation and the fraction were dialyzed in 100 mM sodium12

citrate buffer pH 5. Protein concentration was measured by Commasie Blue method. Fractions were13

tested for -glucosidase activity using the chromogenic substrate pNPG as below, and those14

containing enzyme activity were pooled (fraction EF1) and subjected to molecular exclusion15

chromatography in Sephadex G75 (26 mm diameter, 10 cm height columns equilibrated with 10016

mM citrate-phosphate buffer pH 5 at 6 ml/h). Fractions containing -glucosidase activity were17

pooled and subjected to ion exchange chromatography in CM-cellulose (15 mm diameter, 10 cm18

height columns equilibrated with 10 mM citrate-phosphate buffer pH 3.8 at 10 ml/h). Proteins19

linked to the matrix were eluted with 0.5 M NaCl solution at 10 ml/h flux and the fraction20

containing -glucosidase activity were pooled (fraction EF2). Protein concentration was measured21

and protein purity was checked by standard SDS-PAGE method in 12% acrilamide gel.22

Isoelectrofocusing was performed to determine the isoelectric point of the protein. Immobilized pH23

Gradient (IPG) strips with pH ranging from 3 to 10 were equilibrated for 30 min with solution24

containing 6.5 mM DTT and 134 mM IAA and the protein was submitted to electrophoretic run at25


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200 V (2 mA) for two hours followed 3,500 V (2mA) for 1.25 h. The strips were used for second1

dimension run in 12% acrylamide gel and revealed by Comassie Blue staining.2

3

Enzyme kinetics assays4

Substrate specificity for disaccharides was evaluated as described above. Kinetic profile of EF25

fraction was evaluated by using the chromogenic substrate p-nitrophenyl- -D-glucopyranoside6

(pNPG). Standard reactions used a volume of enzyme fraction containing 100 g protein, equal7

volume of 100 mM pNPG solution and 100 mM sodium citrate buffer pH 4.8 to one ml final8

volume. The reaction were incubated at 30C for 10 minutes and stopped by adding 100 L of 1 M9

sodium bicarbonate solution and the yellow color of pNP release was quantified at 410 nm. A10

standard curve was prepared with pNP to correlate the absorbance with the amount of product11

released and the specific activity was calculated as the amount of enzyme that release one mol12

pNP per minute per milligram of protein in the sample. Optimal pH was evaluated by using citrate-13

phosphate buffer adjusted for different pHs and the reactions were incubated at 30C for 10 minutes14

(see Figure 3A). For testing optimal temperature, pH was adjusted to 4.0 and the reactions were15

incubated in different temperatures for 10 minutes (see Figure 3B). Thermal stability of the enzyme16

was evaluated by incubating EF2 fraction for 10 minutes at temperatures ranging from 20C to17

60C. Afterwards, enzyme preparation was left at room temperature (c.a. 25C) for 10 min and then18

used for enzyme activity using pNPG at optimal pH and temperature. Maximum conversion rate19

(Vmax) and affinity constant (K M) were calculated from Lineweaver-Burk plot by varying pNPG20

concentration in the reactions, and were used to calculate catalytic constant (Kcat) of the purified21

enzyme. Inhibitory activity was measured by adding disaccharides or pNPGal at 10 mM in22

reactions containing pNPG and expressed as the percentage of pNPG cleavage. All those23

measurements were performed under optimal pH and temperature.24

25

Gene identification and in silico analysis26


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Searches by the keywords hydrolase, amylase, glucosidase and amyloglucosidase were performed1

in the Dekkera bruxellensis Genomic Database (http://www.lge.ibi.unicamp.br/dekkera/ ) and the2

nucleotide sequences of the retrieved contigs were used for BLASTx analysis at GenBank. The D.3

bruxellensis contig with higher similarity to -glucosidase encoding genes was recovered and the4

ORF determined by the ORF Finder tool at NCBI. The partial sequence of the -glucosidase protein5

was used for BLASTp analysis in the D. bruxellensis genome database of the Joint Genome6

Initiative-JGI (http://genome.jgi.doe.gov/Dekbr1/Dekbr1.home.html ) to recover the complete7

protein sequence. Phylogenetic analysis of the amino acids sequences coded by -glucosidase genes8

of D. bruxellensis and other fungi was performed as previously reported (De Souza Liberal et al.9

2012). Sequence of the bacterial Thermotoga neapolitana -glucosidase was used as outgroup.10

Functional domains of the putative -glucosidase of D. bruxellensis were identified by using the11

structural analysis tools available online at European Bioinformatic institute (http://www.ebi.ac.uk/ ) 12

and SIB Bioinformatic Resource Portal ( http://www.expasy.org/ ).13

14

Results and Discussion15

16

Aerobic growth and fermentation17

Aerobic growth curves (Fig. 1A) showed that GDB 248 strain can grow on cellobiose as carbon18

source at 0.13 h -1 that is lower than the growth rate of 0.19 h -1 calculated for sucrose. No glycerol19

was detected in the course of cell growth on both disaccharides (data not shown), indicating that the20

cultures were aerobic (Pereira et al. 2012; Leite et al. 2013). In this yeast most of the sucrose21

hydrolysis occurs inside the yeast cells since the invertase activity is mainly intracellular (Leite et22

al. 2013). Similarly, no cellobiase ( -glucosidase) activity was observed in the culture supernatant23

(see below), indicating that this enzyme is intracellular too. Therefore, differences for growth rates24

might be related to differences observed in the uptake of sucrose or cellobiose (Fig. 1B). Ethanol25

production was higher in cellobiose than in sucrose, although ethanol consumption was observed26
http://www.lge.ibi.unicamp.br/dekkera/http://www.lge.ibi.unicamp.br/dekkera/http://www.lge.ibi.unicamp.br/dekkera/http://genome.jgi.doe.gov/Dekbr1/Dekbr1.home.htmlhttp://genome.jgi.doe.gov/Dekbr1/Dekbr1.home.htmlhttp://genome.jgi.doe.gov/Dekbr1/Dekbr1.home.htmlhttp://www.ebi.ac.uk/http://www.ebi.ac.uk/http://www.ebi.ac.uk/http://www.expasy.org/http://www.expasy.org/http://www.expasy.org/http://www.ebi.ac.uk/http://genome.jgi.doe.gov/Dekbr1/Dekbr1.home.htmlhttp://www.lge.ibi.unicamp.br/dekkera/


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even in the presence of this sugar (Fig. 1C). This feature remains to be nvestigated. Acetate1

production during aerobic growth was similar in both sugars (Fig. 1D) and reached almost one third2

in mass (one forth in molar ratio) of the ethanol production in aerobic cultures as previously3

reported (Aguilar-Uscanga et al. 2003; Leite et al. 2013).4

Fermentation of cellobiose was assayed under high cell density condition in order to simulate the5

industrial processes, a situation that differed from the previous reports. However, the experiments6

were performed under gently agitation to ensure minimal oxygenation of the cultures due to the7

Custer effect, in which D. bruxellensis cells stopped ethanol production when oxygen is depleted8

from the medium ( Leite et al. 2013 ). This situation of oxygen supply was attested by the production9

of acetate (Table 1). No variation in cell mass was observed (data not shown). Only 44% of the10

cellobiose was consumed after six hour of fermentation, while no sucrose was left in the medium11

(Table 1), once again pointing out for differences in sugar uptake as probably the limiting step in12

cellobiose assimilation. Sugar consumption rate ( qS) and ethanol production rate ( qE) were higher 13

in sucrose medium, while acetate production rate ( qA) was higher in cellobiose medium (Table 1).14

Mass balance was closed for sucrose medium, with almost all carbon being recovered from the15

fermentation products (Table 1). This was not observed when cellobiose was the carbon source.16

Since no other fermentation metabolites were detected, we supposed that CO 2 production was17

underestimated in cellobiose medium. As the fermentation continues to 24 h, all cellobiose was18

consumed and ethanol production reached 3.7 g/l, even though ethanol yield still in the range of 19

0.18 g/g. Cellobiose fermentation by D. bruxellensis CBS 11269 was reported to be performed for 20

long period of incubation (Blomqvist et al. 2010), although ethanol yield with that strain of 0.29 g/g21

was higher than the strain in the present study (Table 1). High ethanol yield on cellobiose by the22

strain CBS 5512 of Brettanomyces custersii (the former epithet of D. bruxellensis ) in the range of 23

0.40 g/g was reported (Spindler et al. 1992).24

Steam-exploded enzyme-treated sugarcane bagasse hydrolysates used in the present work contained25

5 times more glucose than cellobiose. Under fermentation with agitation, glucose was rapidly26

Figure 1

Table 1


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consumed by D. bruxellensis cells while cellobiose was uptake slowly (Fig. 2). Acetate but not1

glycerol was detected, attesting the presence of oxygen in the fermentation. Ethanol and acetate2

production and yield (0.25 g/g) were similar. The stoichiometric CO 2 yield was calculated as 0.483

g/g, closing the mass balance at 98%. Similar production of ethanol and acetate corroborates the4

tendency of D. bruxellensis for respiration (Leite at al. 2013).5

6

Production and purification of D. bruxell ensis cellobiase ( -glucosidase, E.C. 3.2.1.21)7

Yeast cells cultivated in synthetic media containing different sugars were tested for the cross8

production of the main gluco-hydrolases: invertase, cellobiase and maltase. None of the tested9

enzymes were detected in the supernatant of the growth of fermentation cultures (data not shown),10

confirming their intracellular localization. Cellobiase as well as invertase were highly produced11

when yeast cells were cultivated on glucose, while they were more produced when the cells were12

cultivated on the respective sugars (Table 2). In Kluyveromyces marxianus the production of 13

cellobiase was highly induced by cellobiose (Rajoka et al. 2004). In order to decrease the14

background of invertase, yeast cells were cultivated in cellobiose medium for preparation of cell-15

free extract to purify cellobiase (Table 3). Enzyme purification was confirmed by the presence of a16

band of 93 KDa in SDS-PAGE and the isoelectric point of 6.13 was calculated from17

isoelectrofocusing method (data not shown), similar to the enzyme from K. marxianus (96 KDa)18

(Yoshida et al. 2009).19

20

Kinetics and optimal activity of the D. bruxel lensis cellobiose21

Purified cellobiase (EF2) from D. bruxellensis displayed a narrow range of pH with its optimal22

activity pH 4.2 (Fig. 3A). On the other hand, it showed a wider range of optimal temperature (Fig.23

3B). At pH 5 the enzyme preparation EF2 maintained 100% of the activity after 60 minutes of 24

incubation at 40C (data not shown). Thus, the optimal conditions for enzyme activity were25

established as 30C and pH 4.2 in phosphate-citrate buffer and all experiments hereafter were26

Figure 2

Table 2

Table 3


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performed in this condition. The specificity assays showed that D. bruxellensis cellobiase has broad1

substrate specificity (Table 4), while no activity was detected for pNP- -D-galactopyranoside2

(Table 4) neither for carboxymethyl cellulose (data not shown). By using the chromogenic substrate3

pNPG it was observed that disaccharides competitively inhibited cellobiase activity, while the4

presence of pNPGal did not prevent the interaction of pNPG with the active site of the enzyme5

(Table 4). The following kinetics parameters were determined using pNPG: K M = 0.58 mM; V max =6

154 mol/min.mgProtein; K cat = 12.84 min -1. Although the kinetics parameters were not high than7

reported for other yeasts, a clear advantage concerning to D. bruxellensis enzyme is its stability at8

high temperatures allied to the capacity of the cells to settle the industrial process to produce9

ethanol (De Souza Liberal et al. 2007).10

11

Gene identification and protein structure12

Computational analysis identified a nucleotide sequence in the genome of D. bruxellensis whose13

theoretical protein showed similarity to -glucosidase of the yeasts K. marxianus and14

Schwanniomyces etchellsii (Fig. 4). The theoretical protein contained 840 amino acids with15

predicted molecular weight of 93 KDa, compatible with the experimental data above of the purified16

enzyme. Its predicted structure presents three major domains. The N-terminus presented a glucosyl-17

hydrolase family 3 motif followed by the PA14 -barrel, this last one being thought to be involved18

in carbohydrate binding in the K. marxianus enzyme (Yoshida et al. 2010). At C-terminus there was19

a fibronectin type III-like domain that is also present in the structure of K. marxianus cellobiase20

(Yoshida et al. 2010), whose function still unknown. It may be possible that such domain is21

involved in protein-protein interaction to form a homodimer structure of the enzyme. Neither 22

transmembrane nor signal peptide domain or glycosylation sites were identified, which corroborates23

with the results above showing the intracellular localization of the enzyme. Theoretical pI was24

calculated as 6.1, which corresponded to the experimentally determined pI value using IEF25

procedure. These results confirmed the identification of the BGL gene that codes for cellobiose ( -26

Figure 3

Table 4


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glucosidase) of D. bruxellensis (for complete nucleotide and amino acids sequences consult1

http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Dekbr2&id=34222 ).2

3

Conclusion and Perspectives4

It has been recently reported the increasing number of yeasts capable of hydrolyzing cellobiose for 5

the production of ethanol, for example Candida queiroziae, Clavispora sp., Spathaspora6

passalidarum (Long et al. 2012; Liu et al. 2012; Santos et al. 2011), and D. bruxellensis (Blomqvist7

et al. 2010; Galafassi et al. 2011; Leite 2013). However, only the last species has been reported as8

able to settle and survive in industrial environments (Passoth et al. 207; De Souza Liberal et al.9

2007; Pereira et al., 2012). Thus, the great challenging for its use as fermenting yeast is regarded to10

the low conversion rates when facing simulated industrial conditions. Studies are being undertaken11

in our laboratory in order to identify the main metabolic bottlenecks of this feature and to evaluate12

its capacity towards the conversion hydrolysates of sugarcane and sweet sorghum bagasse into13

ethanol at high industrial yields.14

15

16

Acknowledgements17

This work was sponsored by the Bioethanol Research Network of the State of Pernambuco (CNPq-18

FACEPE/PRONEM program, grant n APQ-1452-2.01/10), by CNPq-Universal program (grant n19

472106/2012-0) and by the Ministry of Science and Technology of Brazil (SIGTEC number 20

PRJ03.33). 21

22

23

References24

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Dekkera strain. Biotechnol Bioeng 24:2031-2037. doi: 10.1002/bit.260240910.26

Figure 4
http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Dekbr2&id=34222http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Dekbr2&id=34222http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Dekbr2&id=34222


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Kumagai H (2009) Purification, crystallization and preliminary X-ray analysis of beta-16

glucosidase from Kluyveromyces marxianus NBRC1777. Acta Crystallogr Sect F Struct Biol17

Cryst Commun 65:1190-1192. doi: 10.1107/S1744309109042948.18

Yoshida E, Hidaka M, Fushinobu S, Koyanagi T, Minami H, Tamaki H, Kitaoka M, Katayama T,19

Kumagai H (2010) Role of a PA14 domain in determining substrate specificity of a20

glycoside hydrolase family 3 -glucosidase from Kluyveromyces marxianus . Biochem J21

431:39-49. doi: 10.1042/BJ20100351.22

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Legend to figures1

2

Figure 1. Shake flask growth curve of Dekkera bruxellensis GDB 248 in synthetic medium3

containing sucrose ( ) or cellobiose ( ). Variation of cell density (A), consumption of sugar (B)4

and production of ethanol (C) and acetate (D) were plotted.5

6

Figure 2. Fermentation of steam-exploded enzyme-treated sugarcane bagasse by Dekkera7

bruxellensis GDB 248. The following parameters were measured: sugar consumption (total: ,8

straight line; glucose: , dotted line; cellobiose: , dotted line) and ethanol ( ) and acetate ( )9

production.10

11

Figure 3. Optimal pH (A) and temperature (B) of the -glucosidase (cellobiase) purified from12

Dekkera bruxellensis GDB 248 using pNPG as substrate.13

14

Figure 4. Phylogenetic analysis of the Dekkera bruxellensis -glucosidase (cellobiase) from yeasts15

and filamentous fungi. Amino acids sequences were used to prepare the p-distance matrix and16

clustered by maximum likelihood method.17

18

19


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ure 1ck here to download high resolution image
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13

Reis et al. Cellobiose fermentation by the yeast Dekkera br ux ell ensis and implications for1production of second generation ethanol 2

3

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Table 1. Global physiological and kinetics data for the eight-hour fermentation of cellobiose or 5

sucrose in agitated flasks by Dekkera bruxellensis GDB 248.6

Parameter Cellobiose Sucrose

Residual sugar (g/l) a 11.5 0.00

qSugar (mmol/gCel.h) 0.31 (0.08) 1.22 (0.01)

qEthanol (mmol/gCel.h) 0.66 (0.09) 2.62 (0.08)

qAcetate (mmol/gCel.h) 0.53 (0.03) 0.38 (0.03)

Y Ethanol (g/g) 0.18 (0.03) 0.34 (0.02)

Y Acetate (g/g) 0.25 (0.04) 0.12 (0.02)

Y CO 2 (g/g) 0.41 (0.01) 0.45 (0.02)

Y Biomass (g/g) 0.0 0.06 (0.02)

Mass balance (%) 84 97

aInitial sucrose 25 g L -1 and initial cellobiose 20 g L -1.7

bCalculated on the basis of stoichiometry of ethanol and acetate production.8

9

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e 1

k here to download table: Reis et al - Table 1.doc
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Table 2. Enzyme activity in the cell-free extracts from Dekkera bruxellensis GDB 248 cultivated in6

different sugars.7

Sugar in the

medium

Substrate of

enzyme reaction Enzyme assayed

Specific activity

(U/gCel)

Glucose Sucrose Invertase 0.998

Cellobiose -glucosidase 0.835

Maltose -glucosidase 0.000

Sucrose Sucrose Invertase 0.598



Cellobiose Sucrose Invertase 0.295



8

e 2

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Table 3. Purification of cellobiase from Dekkera bruxellensis GDB 248 grown on cellobiose6

medium.7

Enzyme fraction Protein (mg/ml)

Specific activity

( mol EqGlucose/min.mgProtein) Purification factor

Cell-free extract 0.95 0.104 1.00

EF1 1.19 0.113 1.09

EF2 0.11 0.848 8.16

8

e 3

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Table 4. Effect of disaccharides on the activity of the purified cellobiase from Dekkera bruxellensis6

GDB 248.7

Substrate Glucosyl link Relative activity (%) Inhibitory activity (%) a

Cellobiose Glucose- 1 4) -Glucose 100.0 100.0

Maltose Glucose- (1 4) -Glucose 27.7 94.9

Sucrose Glucose- (1 2) -Fructose 90.0 95.2

pNPG Glucose- 1 4) -phenyl 100.0 n.a.

pNPGal Galactose- 1 4) -phenyl 0.0 0.0

aDissacharides were added to reactions with the chromogenic substrate pNPG.8

bn.a.: not applicable9

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e 4

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April 2013

cellobiose fermentation by the yeast dekkera bruxellensis and implications for

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