105 8 Bioethanol from Starchy Biomass Part II Hydrolysis and Fermentation Sriappareddy Tamalampudi, Hideki Fukuda, and Akihiko Kondo ABSTRACT Bioethanol, which is derived from starchy and cellulosic biomass, is becoming impor- tant as an alternative fuel due to diminishing petroleum resources and environmental impacts. Acid and enzymatic methods have been developed for the hydrolysis of starchy biomass in order to release fermentable sugars. Acid hydrolysis results in the production of unnatural compounds that have adverse effects on yeast fermentation. In enzymatic hydrolysis of starch, the biomass has to be cooked at high tempera- tures and large amounts of amylolytic enzymes have to be added to hydrolyze the starchy biomass prior to fermentation. Recent advances in yeast cell surface engi- neering developed the strategies to genetically immobilize amylolytic enzymes like CONTENTS Abstract 105 8.1 Introduction 106 8.2 Yeast Cell Surface Engineering: A Tool for Direct Ethanol Production from Starch 106 8.3 Ethanol Production from Soluble Starch 108 8.3.1 Displayed Glucoamylase 108 8.3.2 Co-Displayed Glucoamylase and Amylase 109 8.4 Ethanol Production from Low-Temperature Cooked Corn Starch 111 8.5 Ethanol Production from Raw Corn Starch 113 8.6 Evaluation of Surface Engineered Yeast Strains 117 8.7 Conclusions 117 References 118 © 2009 by Taylor & Francis Group, LLC 106 Handbook of Plant-Based Biofuels α-amylase and glucoamylase on the yeast cell surface. As a means of reducing the cost of ethanol production, occulent and nonocculent yeast strains co-displaying amylolytic enzymes have been developed and used successfully for direct ethanol production from raw starch. Hence, the cell surface engineered yeast appears to have great potential in industrial application. 8.1 INTRODUCTION The utilization of biomass as the starting material for various chemicals and for the production of biofuels has received considerable interest in recent years. Starchy and cellulosic materials of plant origin are the most abundant utilizable biomass resources. Starchy biomass has to be hydrolyzed either by enzymatic or acid hydro- lysis to release fermentable sugars. However, acid hydrolysis results in the formation of by-products such as levulinic acid and formic acid which have adverse effects on yeast growth during the fermentation process (Kerr 1944). The enzymatic hydrolysis of starchy material for ethanol production via fermentation consists of two or three steps and requires improvement if it is to realize efcient production at low cost. There are two main reasons for the present high cost: one is that starchy materials need to be cooked at a high temperature (140 to 180°C) to obtain high ethanol yield and the other is that large amounts of amylolytic enzymes, namely glucoamylase (EC 3.2.1.3) and α-amylase, need to be added. To reduce the energy cost of cook- ing starchy materials, previously reported noncooking and low-temperature cooking fermentation systems have succeeded in reducing energy consumption by approxi- mately 50% (Matsumoto et al. 1982), but it is still necessary to add large amounts of amylolytic enzymes to hydrolyze the starchy materials to glucose. Many researchers have reported attempts to resolve this problem by using recombinant glucoamylase-expressing yeasts with the ability to ferment starch to ethanol directly (Ashikari et al. 1989; Inlow, McRae, and Ben-Bassat 1988). Recom- binant yeast that co-produces glucoamylase and α-amylase has been developed to further improve the efciency of starch fermentation (Birol et al. 1998; De Moreas, Astol-Filho, and Oliver 1995; Eksteen et al. 2003). Recent advances in yeast cell surface engineering provided the tools for the display of amylolytic enzymes which allows the utilization of yeast whole-cell biocatalyst for direct ethanol production from starch. Moreover, integration of hydrolysis and fermentation steps by arming yeast cells can reduce the unit operations compared to that of hydrolysis by acids and isolated enzymes (Figure 8.1). This review summarizes the work on cell sur- face engineering systems that demonstrated direct ethanol production from soluble starch, low-temperature cooked starch, and raw starch. 8.2 YEAST CELL SURFACE ENGINEERING: A TOOL FOR DIRECT ETHANOL PRODUCTION FROM STARCH The cell surface is a functional interface between the inside and outside of the cell. Some surface proteins extend across the plasma membrane and others are bound by noncovalent interactions to the cell surface components. Cells have systems for anchoring surface-specic proteins and for conning surface proteins to particular © 2009 by Taylor & Francis Group, LLC Hydrolysis and Fermentation of Starchy Biomass 107 domains on the cell surface. In biotechnology, the cell surface can be exploited by making use of known mechanisms for the transport of proteins to the cell surface. In particular, Saccharomyces cerevisiae is useful as a host for genetic engineering, because it allows the folding and glycosylation of expressed heterologous eukaryotic proteins and can be subjected to many genetic manipulations. Moreover, the yeast can be cultivated to a high density in an inexpensive medium, so that the display of enzymes on yeast cell surface has several applications in bioconversion processes. Many glucoamylase-extractable proteins on the yeast cell surface, for example, agglutinin (Agα1 and Aga1) and Flocculin Flo1, Sed1, Cwp1, Cwp2, Tip 1, and Tir 1/Srp 1 have glycosylphosphotidylinositol (GPI) anchors which play an important role in the expression of cell surface proteins (Roy et al. 1991; Watari et al. 1994). GPI anchored proteins contain hydrophobic peptides at their C-termini. After the comple- tion of protein synthesis, the precursor protein remains anchored in the endoplasmic reticulum (ER) membrane by the hydrophobic carboxyl-terminal sequence, with the rest of the protein in the ER lumen. Within less than a minute, the hydrophobic car- boxyl-terminal sequence is cleaved at the site and concomitantly replaced with a GPI anchor, presumably by the action of a transamidase (Ueda and Tanaka 2000). Among the GPI anchor proteins α-agglutinin and Flocculin anchors are dem- onstrated to be suitable for the expression of hydrolytic enzymes. The molecular- level information on α-agglutinin is utilized to target the heterologous proteins of Starch Cooking Gelatinization α-Amylase Liquefaction Saccharification Arming yeast displaying amylolytic enzymes (C) Fermentation Yeast Ethanol (B) Acid hydrolysis (A) Glucoamylase FIGURE 8.1 Schematic diagram of starch hydrolysis and ethanol fermentation using differ- ent methods. (a) Acid hydrolysis, (b) enzymatic hydrolysis, and (c) arming yeast displaying amylolytic enzymes. © 2009 by Taylor & Francis Group, LLC 108 Handbook of Plant-Based Biofuels biotechnological importance to the outermost glycoprotein layer of the cell wall. In the α-agglutinin system, the C-terminal half of the α-agglutinin containing the GPI anchor attachment signal was used to anchor the heterologous proteins on the yeast cell surface (Capellaro et al. 1991). In the case of the occulin system two types of cell surface display methods were developed. In one system, the C-terminal region of Flo1p, contains a GPI-attachment signal; the second system, by contrast, attempts to utilize the ability of the occulation functional domain of Flo1p to create a novel surface display apparatus (Kondo and Ueda 2004). 8.3 ETHANOL PRODUCTION FROM SOLUBLE STARCH 8.3.1 d i S P l a y e d Gl u c o a m y l a S e Surface expression of the amylolytic enzymes was initiated by the pioneering work of Murai et al. (1997). They reported the strategy of developing recombinant S. cer- evisiae displaying amylolytic enzymes. The multi-copy plasmid pGA11 (Figure 8.2) was used for the expression of glucoamylase/α agglutinin fusion gene containing the secretion signal sequence of the glucoamylase under the control of the GAPDH pro- moter and was introduced into the S. cerevisiae MT8-1 as host strain. The displayed glucoamylase is from Rhizopus oryzae, an exo-type amylolytic enzyme, cleaving α-1,4-linked and α-1,6-linked glucose effectively from starch. The anchoring of the fusion gene on the cell wall of recombinant yeast harboring the plasmid pGA11 was demonstrated by immunouorescence labeling of the cells with anti-glucoamylase IgG (Murai et al. 1997; Ueda et al. 1998). Kondo et al (2002) used occulating yeast strain YF207 for the surface expres- sion of glucoamylase. The yeast strain YF207 is a tryptophan auxotroph with a strong occulation ability which was obtained from Saccharomyces diastaticus ATCC60712 and S. cerevisiae W303-1B by tetrad analysis and was transformed with pGA11 GAPDH terminator 3'-Half of α-agglutinin gene Glucoamylase gene GAPDH promoter pGA11 2µm TRP1 Col E1 ori Amp r Secretion signal sequence of R. oryzae glucoamylase gene FIGURE 8.2 Schematic representation of the expression plasmid for glucoamylase/α- agglutinin fusion gene. © 2009 by Taylor & Francis Group, LLC Hydrolysis and Fermentation of Starchy Biomass 109 constructed in the previous study (Murai et al. 1997). The cell surface glucoamylase does not show any effect on occulation ability during growth and ethanol fermenta- tion phases. Moreover, the glucoamylase activity displayed on the surface of occu- lent yeast strain was similar to that displayed on nonocculent yeast cells. Therefore, the occulent yeast cells displaying glucoamylase possess both strong occulation ability and glucoamylase activity; and hence they are considered more advantageous in industrial processes for ethanol production from starchy materials. The results shown in Figures 8.3a and 8.3b demonstrate that the cell-surface glu- coamylase is effective for direct ethanol fermentation from soluble starch, because high ethanol fermentation from soluble starch was obtained. In previous studies using recombinant S. cerevisiae secreting glucoamylase (Nakamura et al. 1997; Briol et al. 1998) both cell growth and fermentation were performed under anaerobic or minimal aerobic conditions; and hence over 150 h was necessary to attain ethanol concentra- tions of 20 to 30 g/l. Ideally, a large cell mass should be obtained by high-density cell culture under aerobic conditions and cells harvested by sedimentation were used for the ethanol fermentation. However, in secretory expression of amylolytic enzymes, this approach is not suitable because inoculated cells should produce a sufcient amount of amylases before ethanol fermentation. In the study by Kondo et al. (2002), recombinant yeast strain YF207/pGA11 displaying glucoamylase gene maintained a high ethanol production rate (approximately 0.6 to 0.7 g l -1 h -1 ) during repeated utilization for fermentation over 300 h. This is attributable to high plasmid stability during growth and fermentation phases, even though pGA11 is a multi-copy-type plasmid, based on pYE22m. The plasmid stability in cells cultivated in YPS medium was found to be higher than in cells cultivated with YPD medium. Since host cells could not metabolize soluble starch, the utilization of soluble starch as the carbon source would be a selection pressure for the yeast cells bearing plasmids. In the case of glucoamylase-displaying yeast cells, glucose was maintained at a very low concentration and, at the same time, a high ethanol production rate was achieved. This might be because the recombinant yeast cells metabolize the glucose as soon as glucose is released from soluble starch by the glucoamylase displayed yeast cells. However, a high ethanol production rate was obtained because local glucose concen- tration near the yeast cell surface was probably higher than that in the fermentation medium. This low concentration of glucose in the fermentation medium is advanta- geous in minimizing the risk of contamination. 8.3.2 co-di S P l a y e d Gl u c o a m y l a S e a n d am y l a S e Studies show the display of only glucoamylase leads to the accumulation of insolu- ble starch during fed-batch fermentation, because of the lack of liquefying enzyme α-amylase. In order to overcome this problem, Shigechi et al. (2002), developed two recombinant yeast strains co-expressing glucoamylase and α-amylase. Plasmids for the surface expression (pAA12) and secretory expression (pSAA11) of Bacillus stearothermophilus α-amylase were constructed and co-transformed into the occu- lent yeast strain YF207 along with the plasmid pGA11 for cell surface display of R. oryzae glucoamylase. The ethanol productivity by these two strains was examined by fed-batch fermentations using soluble potato starch as substrate. The amylolytic © 2009 by Taylor & Francis Group, LLC 110 Handbook of Plant-Based Biofuels 80 (a) Growth Ethanol fermentation 30 25 20 15 Ethanol concentration (g/l) 10 5 0 60 40 Starch concentration (g/l) Glucose concentration (g/l) 20 0 02040 Time (h) 60 80 80 (b) Growth Ethanol fermentation 80 60 Ethanol concentration (g/l) 40 20 0 60 40 Starch concentration (g/l) Glucose concentration (g/l) 20 0 04080 Time (h) 120 160 FIGURE 8.3 (a) Batch fermentation of starch to ethanol by YF207/pGA11. YF207/pGA11 cells were grown under aerobic conditions (2.0 ppm), harvested, and used for batch fermen- tation. The left side of the solid line in the gure is the growth phase and the right side is the ethanol-fermentation phase. (b) Fed-batch fermentation by YF207/pGA11. YF207/pGA11 cells were grown under aerobic conditions (2.0 ppm), harvested, and used for fed-batch fer- mentation under anaerobic conditions. The left side of the solid line in the gure is the growth phase and the right side is the ethanol-fermentation phase. © 2009 by Taylor & Francis Group, LLC Hydrolysis and Fermentation of Starchy Biomass 111 activity was detected by both the strains and ow cytometric analysis conrmed the successful co-expression of glucoamylase and α-amylase in strains YF207/ [pGA11, pAA12] and YF207/ [pGA11, pSAA11]. As shown in Figures 8.4a and 8.4b, both recombinant strains YF207/ [pGA11, pAA12] and YF207/ [pGA11, pSAA11] grew faster in the growth phase than the glucoamylase displaying yeast YF207/pGA11. The activities of glucoamylase and α-amylase displayed on the cell surface were maintained with YF207/ [pGA11, pAA12] during the ethanol fermentation phase, whereas in the case of YF207/ [pGA11, pSAA11] strain the secreted α-amylase was accumulated. The ethanol con- centration produced reached 60 g l -1 after 100 h of fermentation by both strains. But glucose concentration is slightly higher in the culture medium of YF207/ [pGA11, pSAA11] strain. This is probably due to the secretion of α-amylase, which decom- poses starch in the culture medium. In addition, the occulation ability of the yeast strain co-expressing glucoamy- lase and α-amylase did not change during the fed-batch fermentation and was almost the same as that of the yeast strains YF207 and YF207/pGA11. This nding sug- gested the co-display of two amylolytic enzymes on the cell surface does not inu- ence the occulation ability of yeast cells. 8.4 ETHANOL PRODUCTION FROM LOW-TEMPERATURE COOKED CORN STARCH In direct ethanol production, noncooking and low-temperature cooking fermenta- tion systems have several advantages over conventional high-temperature cooking (140 to 180°C) process (Matsumoto et al. 1982) because high-temperature cook- ing requires high energy and the addition of large amounts of amylolytic enzymes. Shigechi et al. (2000) performed direct ethanol production in a single step using corn starch cooked at low temperature (80°C) as the sole carbon source instead of soluble starch using yeast strains displaying amylolytic enzymes. The productivity of ethanol from corn starch cooked at low temperature was investigated by using the recombinant yeast strains that were developed in their previous study, that is, yeast strains displaying only glucoamylase on the cell surface (YF207/pGA11) or yeast strains displaying glucoamylase and either co-displaying (YF207/pGA11/pAA12) or secreting (YF207/pGA11/pSAA11) α-amylase. The ethanol production rate increased markedly under co-expression of glu- coamylase and α-amylase compared with the yeast strain displaying only glucoamy- lase (Figure 8.5). Specically, by co-displaying two amylolytic enzymes on the cell surface, strain YF207/pGA11/pAA12 was able to produce ethanol more rapidly than strain YF207/pGA11/pSAA11 and without time lag. These results indicated that α-amylase, which hydrolyzes α-1,4 linkages of starch in a random fashion, plays a very important role in efcient hydrolyzation of corn starch. It is probable that the cooperative and sequential reaction of two enzymes is crucial for efcient uti- lization of corn starch. Because the yeast strain YF207/pGA11/pAA12 possesses enough glucoamylase and α-amylase activity in the initial stages of cultivation and fermentation, it grows fast, produces ethanol without time lag, and achieves maxi- © 2009 by Taylor & Francis Group, LLC 112 Handbook of Plant-Based Biofuels 80 (a) 80 Ethanol concentration (g/l) 60 40 20 0 60 40 20 0 04080 Time (h) 120 160 80 (b) 80 60 Ethanol concentration (g/l) 40 20 0 60 40 20 0 04080 Time (h) 120 160 Starch concentration (g/l) Glucose concentration (g/l) Starch concentration (g/l) Glucose concentration (g/l) FIGURE 8.4 (a) Fed-batch fermentation of starch to ethanol by YF207/[pGA11, pAA12]. YF207/[pGA11, pAA12] cells were grown under aerobic conditions (2.0 ppm), harvested, and used for fed-batch fermentation under anaerobic conditions. To the left of the solid line in the gure is the growth phase and to the right the ethanol fermentation phase. (b) Fed-batch fer- mentation of starch to ethanol by YF207/[pGA11, pSAA11]. YF207/[pGA11, pSAA11] cells were grown under aerobic conditions (2.0 ppm), harvested, and used for fed-batch fermenta- tion under anaerobic conditions. © 2009 by Taylor & Francis Group, LLC Hydrolysis and Fermentation of Starchy Biomass 113 mum ethanol concentration (18 g/l) within the short time (36 h) of the recombinant yeast strains. A comparison of conventional high-temperature and low-temperature cooking fermentation systems using the yeast strain YF207/pGA11/pAA12 co-displaying glucoamylase and α-amylase (Figure 8.6) shows maximum ethanol concentration, ethanol-production rate, and substrate consumption rate were almost the same in the two fermentation systems. In high- and low-temperature cooking systems, the yield of ethanol produced was 0.50 g per gram of carbohydrate consumed. This corresponds to 97.2% of theoretical yield (0.51 g of ethanol per gram glucose). This indicates that the low-temperature cooking fermentation system based on YF207/ pGA11/pAA12 is cost effective in direct fermentation of corn starch. 8.5 ETHANOL PRODUCTION FROM RAW CORN STARCH The isolation of amylase enzyme from lactic acid bacteria Streptococcus bovis opened new horizons for the efcient hydrolysis of raw starch (Satoh et al. 1993). Shigechi et al. developed a novel noncooking fermentation system for direct ethanol production from raw corn with yeast strain YF207 that co-displayed R. oryzae glu- coamylase and S. bovis 148 α-amylase by using the C-terminal half of α-agglutinin (pBAA1) and the occulation domain of Flo1p (pUFLA) as anchor proteins (Fig- ures 8.7a and 8.7b). 60 50 40 30 20 10 0 010203040 Time (h) 50 60 70 0 5 10 Ethanol (g/l) Starch (g/l) 15 20 25 30 FIGURE 8.5 Time course of anaerobic ethanol fermentation from 50 g/l corn starch cooked at low temperature. Each group of cells was aerobically cultivated for 48 h on SDC medium, harvested, and used in ethanol fermentation with YPS medium cooked at 80°C for 5 min. (,). YF207; (, ) YF207/pGA11; (,). YF207/pGA11/pAA12; (,) YF207/pGA11/ pSAA11. Open and closed symbols show the starch and ethanol concentrations, respectively. © 2009 by Taylor & Francis Group, LLC 114 Handbook of Plant-Based Biofuels The α-amylase and glucoamylase activities conrmed the display of both amylolytic enzymes (Table 8.1). In glucoamylase-displaying yeast strains there is not much difference in the activities; whereas in the case of α-amylase-displaying yeast strains, the activity is dependent on the anchor protein. The yeast strains YF207/pBAA1 and YF207/pGA11/pUFLA which uses Flo1 anchor showed 40 times higher α-amylase activity than the yeast strains using a-agglutin anchor. It has been reported that several α-amylases have raw starch binding abilities and that the starch digesting domain is located in the C-terminal region (Lo et al. 2002). The two recombinant yeast strains YF207/pGA11/pBAA1 and YF207/pGA11/pUFLA were used in direct ethanol production from raw corn starch. The raw corn starch, which corresponds to 200 g of total sugar per liter, was used as the sole carbon source. As shown in Figure 8.8, strain YF207/pGA11, displaying only glucoamylase, and strains YF207/pBAA1 and YF207/pUFLA, displaying only α-amylase, produced almost no ethanol, while soluble sugar accumulated in the fermentation medium of strain YF207/pUFLA due to degradation of corn starch to oligosaccharides by the surface-displayed α-amylase. Although strain YF207/pGA11/pBAA1, co-displaying glucoamylase and α-amylase via α-agglutinin, did produce ethanol from the raw corn starch, the ethanol yield was low (23.5 g l -1 ) after 72 h of fermentation. On the other hand, the yeast strain co-displaying glucoamylase and α-amylase using α-agglutinin and Flo1P (YF207/pGA11/pUFLA) was able to produce ethanol directly from the raw corn starch without the addition of commercial enzymes. The concentration of raw corn starch decreased drastically during the fermentation, as the ethanol concentration increased to 61.8 g l -1 after 72 h of fermentation. A 60 50 40 30 20 10 0 010203040 Time (h) 50 60 70 0 5 10 Ethanol (g/l) Starch (g/l) 15 20 25 30 FIGURE 8.6 Comparison of ethanol production between high- and low-temperature cook- ing fermentation systems using yeast strain YF207/ pGA11/pAA12. YPS medium was cooked at 120°C for 20 min or at 80°C for 5 min. Ethanol fermentation started with initial starch concentration of 50 g/l. (,) .High-temperature cooking fermentation system; (,)low- temperature cooking fermentation system. Open and closed symbols show starch and ethanol concentrations, respectively. © 2009 by Taylor & Francis Group, LLC [...]... 2009 by Taylor & Francis Group, LLC 1 18 Handbook of Plant- Based Biofuels cells displaying α-amylase show low performance in batch culture, their efficiency is almost comparable to the α-amylase-secreting yeast strains during repeated batch fermentations In conclusion, the choice of α-amylase secretion/display depends on the nature of the substrate and on the type of process operation References Ashikari,... displaying α-amylase also acted efficiently on the raw starch with a specific ethanol production rate of approximately 0.06 and 0.04 g g-dry cell-1 h-1, respectively The comparatively high ethanol production rate in α-amylase-secreting nonflocculent yeast is because the diameter of the displayed α-amylase is 10 -6 m which is three orders of magnitude lower than the secreted α-amylase with a diameter of 10 -9 ... Hydrolysis and Fermentation of Starchy Biomass GAPDH promoter URA3 s.s Ampr pBAA1 2µm α-Amylase gene 3'-Half of α-agglutinin gene (a) Ampr 2µm ColE1ori GAPDH promoter URA3 pUFLA FL-anchor gene GAPDH terminator α-Amylase gene (b) Figure 8. 7 Expression plasmids for cell surface display of S bovis α-amylase (a) Plasmid pBAA1 for C-terminal immobilization using the α-agglutinin -based surface display system;... weight) of cells; values are averages of three independent experiments b ND, not detected © 2009 by Taylor & Francis Group, LLC 116 Handbook of Plant- Based Biofuels 70 60 50 150 40 100 30 Ethanol (g/liter) Total Sugar (g/liter) 200 20 50 10 0 0 10 20 30 40 Time (h) 50 60 70 0 Figure 8. 8 Time course of direct ethanol production via fermentation from raw corn starch, which corresponds to 200 g of total... strains secreting α-amylase (0. 18 g g-dry cell-1 h-1) showed threefold higher specific ethanol production rate than the α-amylase-displaying nonflocculent yeast strain (0.06 g g-dry cell-1 h-1) But the specific starch consumption rate in the third batch of fermentation was decreased significantly compared with the first two batches The decrease in the activity was due to the removal of α-amylase from the... 58: 291–296 Kondo, A and M Ueda 2004 Yeast cell-surface display: Applications of molecular display Appl Microbiol Biotechnol 64: 28 40 Lo, H F., L L Lin, W Y Chiang, M C Chie, W H Hsu, and C T Chang 2002 Deletion analysis of the C-terminal region of the α-amylase of Bacillus sp strain TS-23 Arch Microbiol 1 78: 115–123 Matsumoto, N., O Fukunishi, M Miyanaga, K Kakihara, E Nakajima, and H Yoshizumi 1 982 ... Hauser, V Mrsa, M Watzele, G Watzele, C Gruber, and W Tanner 1991 Saccharomyces cerevisiae a- and α-agglutinin: Characterization of their molecular interaction EMBO J 10:4 081 –4 088 De Moreas, L., S Astolfi-Filho, and S G Oliver 1995 Development of yeast strains for the efficient utilization of starch: Evaluation of constructs that express alpha amylase and glucoamylase separately or as bifunctional fusion... m (typical diameter of globular proteins) and the rate of association of the raw starch granule is (10 -5 ) with displayed α-amylase is expected to be much lower than secreted α-amylase 8. 7 Conclusions The yeast cells displaying amylolytic enzymes have been proved as potential biocatalysts for direct conversion of starchy materials to ethanol The genes encoding glucoamylase and α-amylase were fused... starch through development of novel flocculent yeast strains displaying glucoamylase and co-displaying or secreting α-amylase J Mol Cat B 17:179– 187 Ueda, M., T Murai, Y Shibasaki, N Kamasawa, M Osumi, and A Tanaka 19 98 Molecular breeding of polysaccharide-utilizing yeast cells by surface engineering Ann NY Acad Sci 13 (86 4):5 28 537 Ueda, M and A Tanaka 2000 Genetic immobilization of proteins on the yeast... plasmid pUFLA for N-terminal immobilization using the Flo1p -based surface display system s.s., secretion signal sequence of R oryzae glucoamylase gene Table 8. 1 Glucoamylase and α-Amylase Activities of Yeast Strains Carrying Different Plasmids Glucoamylase Activitya α-Amylase Activitya YF207 b ND NDb YF207/pGA11 42.5 NDb YF207/pBAA1 b ND 2.52 YF207/pUFLA NDb 90.1 YF207/pGA11/pBAA1 45.9 2. 38 YF207/pGA11/pUFLA . Group, LLC 1 08 Handbook of Plant- Based Biofuels biotechnological importance to the outermost glycoprotein layer of the cell wall. In the α-agglutinin system, the C-terminal half of the α-agglutinin. achieves maxi- © 2009 by Taylor & Francis Group, LLC 112 Handbook of Plant- Based Biofuels 80 (a) 80 Ethanol concentration (g/l) 60 40 20 0 60 40 20 0 04 080 Time (h) 120 160 80 (b) 80 60 Ethanol. Francis Group, LLC 114 Handbook of Plant- Based Biofuels The α-amylase and glucoamylase activities conrmed the display of both amylolytic enzymes (Table 8. 1). In glucoamylase-displaying yeast strains