Fructose uptake in Bifidobacterium longum NCC2705 is mediated by an ATP-binding cassette transporter

Recently, a putative ATP-binding cassette (ABC) transport system was identified in Bifidobacterium longum NCC2705 that is highly up-regulated during growth on fructose as the sole carbon source. Cloning and expression of the corresponding ORFs (bl0033-0036) result in efficient fructose uptake by bacteria. Sequence analysis reveals high similarity to typical ABC transport systems and suggests that these genes are organized as an operon. Expression of FruE is induced by fructose, ribose, or xylose and is able to bind these sugars with fructose as the preferred substrate. Our data suggest that BL0033-0036 constitute a high affinity fructose-specific ABC transporter of B. longum NCC2705. We thus suggest to rename the coding genes to fruEKFG and the corresponding proteins to FruE (sugar-binding protein), FruK (ATPase subunit), FruF, and FruG (membrane permeases). Furthermore, protein-protein interactions between the components of the transporter complex were determined by GST pulldown and Western blot analysis. This revealed interactions between the membrane subunits FruF and FruG with FruE, which in vivo is located on the external side of the membrane, and with the cytoplasmatic ATPase FruK. This is in line with the proposed model for bacterial ABC sugar transporters.     

长双歧杆菌NCC2705葡萄糖与果糖代谢的比较蛋白质组学

为揭示长双歧杆菌NCC2705 (Bifidobacterium longum NCC2705)果糖代谢途径, 建立其果糖发酵模型。以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础, 进行了果糖和葡萄糖生长的菌体比较蛋白质组学研究, 利用MALDI-TOF和ESI-MS/MS鉴定差异蛋白, 进一步通过半定量RT-PCR验证二者显著差异表达蛋白。果糖生长的菌体蛋白中鉴定到了所有葡萄糖降解途径中的酶和蛋白质, 另外鉴定到3倍以上差异蛋白点9个, 其对应的5个蛋白在果糖发酵中上调。半定量RT-PCR验证显著差异蛋白, 显示在果糖发酵中具有高水平表达是ABC 转运系统的果糖特异性-结合蛋白BL0033和ATP结合蛋白BL0034。果糖的发酵时间和浓度梯度试验显示诱导时间越长、浓度越高, BL0033的表达量越高。第一, 比较蛋白谱证明果糖和葡萄糖以相同途径降解。第二, BL0033的表达是受果糖诱导的, 果糖的吸收可能是通过一个特殊的转运系统, 即ABC转运系统将果糖从胞外转运到胞内, 其中BL0033和BL0034共同作为系统元件扮演了重要角色。     

A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.     

长双歧杆菌NCC2705株果糖结合蛋白BL0033基因的克隆及其在大肠杆菌中的表达

目的：克隆长双歧杆菌NCC2705株果糖结合蛋白BL0033的基因,利用大肠杆菌表达GST-BL0033融合蛋白并纯化。方法：以长双歧杆菌NCC2705株基因组为模板,PCR扩增BL0033基因,并将其插入pGEX-4T-1表达载体,转化至大肠杆菌DH5α;提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21,并对表达条件进行摸索;用谷胱甘肽-Sepharose4B树脂对可溶性GST-BL0033融合蛋白进行纯化。结果：PCR扩增的BL0033基因长度接近1000bp,与预期值一致;重组菌在IPTG浓度为0.05mmoL/L的条件下,于16℃诱导过夜后,SDS-PAGE分析可见可溶性表达条带,相对分子质量约60×103,与预期值一致;亲和纯化后,SDS-PAGE结果显示单一的表达条带。结论：克隆了BL0033蛋白的基因,并表达纯化了融合蛋白GST-BL0033,为进一步研究长双歧杆菌NCC2705株BL0033蛋白功能奠定了基础。     

Production of mannitol and lactic acid by fermentation with Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius B-3693 was selected as a good producer of mannitol from fructose after screening 72 bacterial strains. The bacterium produced mannitol, lactic acid, and acetic acid from fructose in pH-controlled batch fermentation. Typical yields of mannitol, lactic acid, and acetic acid from 250 g/L fructose were 0.70, 0.16, and 0.12 g, respectively per g of fructose. The fermentation time was greatly dependent on fructose concentration but the product yields were not dependent on fructose level. Fed-batch fermentation decreased the time of maximum mannitol production from fructose (300 g/L) from 136 to 92 h. One-third of fructose could be replaced with glucose, maltose, galactose, mannose, raffinose, or starch with glucoamylase (simultaneous saccharification and fermentation), and two-thirds of fructose could be replaced with sucrose. L. intermedius B-3693 did not co-utilize lactose, cellobiose, glycerol, or xylose with fructose. It produced lactic acid and ethanol but no acetic acid from glucose. The bacterium produced 21.3 +/- 0.6 g lactic acid, 10.5 +/- 0.3 g acetic acid, and 4.7 +/- 0.0 g ethanol per L of fermentation broth from dilute acid (15% solids, 0.5% H(2)SO(4), 121 degrees C, 1 h) pretreated enzyme (cellulase, beta-glucosidase) saccharified corn fiber hydrolyzate.     

Synthesis and application of Fmoc-hydrazine for the quantitative determination of saccharides by reversed-phase high-performance liquid chromatography in the low and subpicomole range

A new derivatization reagent, Fmoc-hydrazine, has been synthesized from the reaction of Fmoc-chloroformate with hydrazine as a precolumn fluorometric labeling reagent for reducing sugars such as glucose, galactose, mannose, fructose, fucose, ribose, xylose, arabinose, lactose, and maltose. The optimization of derivatization conditions was examined in detail. Using a reversed-phase high-performance C-8 column and a mobile phase consisting of acetonitrile-aqueous acetic acid, seven sugar derivatives were separated under either isocratic or gradient conditions within 20 min. The Fmoc-hydrazine and sugar Fmoc-hydrazone derivatives exhibit excellent stability. The extent of the hydrazone formation was 77 and 82% for mannose and fucose as assessed by Dionex high-performance anion-exchange chromatography with pulsed amperometric detection. Linear calibration graphs were established in the range from 0.5 to 2 pmol and 12 to 110 pmol for individual sugar derivatives. The determination limits were 0.05-0.09 pmol for mannose, galactose, and ribose; 0.1 pmol for maltose, xylose, and glucose; 0.2 pmol for fucose and lactose; 0.3 pmol for arabinose; and 0.4 pmol for fructose. The component monosaccharides of ultramicroquantities of two glycoproteins (e.g., from 7 ng fetuin and ovalbumin) were determined in the subpicomole range.     

Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.     

Glass capillary or fused-silica gas chromatography—mass spectrometry of several monosaccharides and related sugars: Improved resolution

The gas chromatographic separation of several monosaccharides and related sugars derivatized by methoxylation and trimethylsilylation reactions was optimized with glass capillary (SP-2250) and fused silica (SP-2100) columns. Individual sugars included aldoses, ketoses, polyols, acidic forms and N-acetylated amino sugars. Peaks were detected by selected ion monitoring (SIM). The fused silica column gave complete resolution of all peaks (two per hexose and one per hexitol) arising from glucose, galactose, mannose, fructose, sorbitol, mannitol and dulcitol. The resolution of these sugars with the glass capillary column was not as good, but full differentiation was possible on the basis of SIM. Because the fused silica column gave a better resolution of 33 sugars tested and was more easily installed than the glass capillary column, it was utilized for quantitative analysis. A deuterated algal sugar mixture used for quantitation by isotope dilution was found to contain glucose, galactose, mannose, xylose, arabinose, ribose and rhamnose. Full recoveries were obtained of various amounts of glucose, galactose, mannose, fructose and xylose added to human serum.     

长双歧杆菌NCC2705菌株应激蛋白的研究

研究长双歧杆菌NCC2705菌株发酵至稳定期时应激蛋白的表达情况。根据乳酸乳球菌IL1403菌株蛋白质参考图谱及长双歧杆菌NCC2705基因组注释中应激蛋白的分子量与等电点,确定应激蛋白在双向电泳凝胶上的相应蛋白点,并利用MALDI-TOF和/或ESI-MS/MS对相应蛋白质点进行鉴定。每个蛋白质点的肽指纹图谱均在长双歧杆菌NCC2705的蛋白质数据库用Mascot进行检索,共鉴定到44个蛋白点对应8个应激蛋白。这些蛋白为亲水性酸性蛋白,大多具有翻译后修饰现象,它们基因的CAI值除DnaJ外,其余均在0.5以上,在全细胞表达谱中为高丰度蛋白;此外,菌体具有较强的抗脂质过氧化和清除DPPH自由基的能力,而对羟自由基和超氧负离子的清除力较弱,推测鉴定到的具有逆转氧化损害作用的碱性过氧化氢还原酶(ahpC)可能是体内表达的降低氧损伤的主要酶。     

Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum

A lacto-N-biose phosphorylase (LNBP) was purified from the cell extract of Bifidobacterium bifidum. Its N-terminal and internal amino acid sequences were homologous with those of the hypothetical protein of Bifidobacterium longum NCC2705 encoded by the BL1641 gene. The homologous gene of the type strain B. longum JCM1217, lnpA, was expressed in Escherichia coli to confirm that it encoded LNBP. No significant identity was found with any proteins with known function, indicating that LNBP should be classified in a new family. The lnpA gene is located in a novel putative operon for galactose metabolism that does not contain a galactokinase gene. The operon seems to be involved in intestinal colonization by bifidobacteria mediated by metabolism of mucin sugars. In addition, it may also resolve the question of the nature of the bifidus factor in human milk as the lacto-N-biose structure found in milk oligosaccharides.     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Fermentation of lignocellulosic sugars to acetic acid by <Emphasis Type="Italic">Moorella thermoacetica</Emphasis>

A systematic study of bioconversion of lignocellulosic sugars to acetic acid by Moorella thermoacetica (strain ATCC 39073) was conducted. Four different water-soluble fractions (hydrolysates) obtained after steam pretreatment of lignocellulosic biomass were selected and fermented to acetic acid in batch fermentations. M. thermoacetica can effectively ferment xylose and glucose in hydrolysates from wheat straw, forest residues, switchgrass, and sugarcane straw to acetic acid. Xylose and glucose were completely utilized, with xylose being consumed first. M. thermoacetica consumed up to 62 % of arabinose, 49 % galactose and 66 % of mannose within 72 h of fermentation in the mixture of lignocellulosic sugars. The highest acetic acid yield was obtained from sugarcane straw hydrolysate, with 71 % of theoretical yield based on total sugars (17 g/L acetic acid from 24 g/L total sugars). The lowest acetic acid yield was observed in forest residues hydrolysate, with 39 % of theoretical yield based on total sugars (18 g/L acetic acid from 49 g/L total sugars). Process derived compounds from steam explosion pretreatment, including 5-hydroxymethylfurfural (0.4 g/L), furfural (0.1 g/L) and total phenolics (3 g/L), did not inhibit microbial growth and acetic acid production yield. This research identified two major factors that adversely affected acetic acid yield in all hydrolysates, especially in forest residues: (i) glucose to xylose ratio and (ii) incomplete consumption of arabinose, galactose and mannose. For efficient bioconversion of lignocellulosic sugars to acetic acid, it is imperative to have an appropriate balance of sugars in a hydrolysate. Hence, the choice of lignocellulosic biomass and steam pretreatment design are fundamental steps for the industrial application of this process.     

Sugar transport systems of Bifidobacterium longum NCC2705

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.     

不同碳源对大肠杆菌lpdA突变菌累积丙酮酸的影响

为了利用大肠杆菌构建模式"细胞工厂",必须了解在构建过程中各种因素的影响。本研究选用敲除了lpdA基因的大肠杆菌作为模型细胞,考察了该突变菌在合成培养基中利用葡萄糖、果糖、木糖和甘露糖累积丙酮酸的能力。结果显示,在初始糖浓度为10g/L的情况下,lpdA突变菌可以很好地利用葡萄糖、果糖、木糖和甘露糖转化丙酮酸,其得率分别达到了0.884g/g、0.802g/g、0.817g/g和0.808g/g,且在以葡萄糖、果糖和木糖发酵时,丙酮酸的积累过程与细胞生长偶联。甘露糖发酵的情况则不同:菌浓度很快达到平台期,随后丙酮酸积累和甘露糖消耗都表现为线性变化。当在考察了不同的接种量对lpdA突变菌发酵葡萄糖的影响时发现,大接种量能加快葡萄糖消耗速率、丙酮酸的积累速率和细胞生长速率,但丙酮酸得率却明显下降。这些结果对构建以大肠杆菌为母体的模式"细胞工厂"有参考价值。     

长双歧杆菌NCC2705葡萄糖与乳糖代谢的比较蛋白质组学

[目的]以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础,研究长双歧杆菌发酵乳糖和葡萄糖的比较蛋白质组学.[方法]采用ImageMaster 2D Elite Platnum Version 5.0比较分析3倍以上蛋白差异点;利用MALDI-TOF进行差异蛋白鉴定,每个蛋白质点的肽指纹图谱在长双歧杆菌NCC2705菌株的蛋白质数据库用Mascot进行检索;采用Pro-Q磷酸化试剂进行磷酸化蛋白的染色.[结果]鉴定到31个蛋白表达发生显著变化,在乳糖发酵中14个蛋白上调17个蛋白下调.这些蛋白为亲水性酸性蛋白,它们基因的CAI值均在0.5以上,主要包括糖代谢相关蛋白、应激蛋白、转录和翻译相关蛋白,还有一些未知功能的蛋白.此外,有两个蛋白:转醛缩酶(BL0715,transaldolase,tal)L3蛋白点和丙酮酸激酶(BL0988,pyruvate kinase,pyk)G9蛋白点发生了磷酸化作用.[结论]长双歧杆菌NCC2705在乳糖中生长快于葡萄糖,它们的降解途径是相同的;转醛缩酶和丙酮酸激酶发生了翻译后修饰作用,推测转醛缩酶在43T和47S发生了磷酸化,而丙酮酸激酶在65S发生了磷酸化.     

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

Golgi Apparatus Mediated Polysaccharide Secretion by Outer Root Cap Cells of Zea mays. III. Control by Exogenous Sugars

Sugars supplied to germinating seedlings of maize (Zea mays L.) regulate the secretion of polysaccharides by the outer cells of the root cap. The polysaccharide secreted by these cells adheres to the root tip as a droplet and the size of the droplet was used to quantitate polysaccharide secretion. The polysaccharide contains glucose, galacrose, and galacturonic acid residues with smaller quantities of mannose, arabinose, xylose, fucose and rhamnose. These sugars supplied to maize seedlings had marked effects on the rate of polysaccharide secretion by root tips. The effects on secretion were independent of the growth rates of the roots. Glucose, fucose and xylose increased droplet size 1.5–2 fold (as did sucrose, maltose, lacrose, fructose and ribose) whereas galactose, arabinose and galacturonic acid were inhibitory. Mannose increased dropler size 5–7 fold. The marked effect of mannose on polysaccharide secretion was due to an increased rate of secretion combined with a longer phase of extrusion of polysaccharide into the forming droplet. The effect of mannose was partially reversed by inorganic phosphate and other sugars (except for fucose which had no effect or promoted secretion in the presence of mannose). In contrast to sucrose, mannose stimulated secretion in a maize variety having a high sugar endosperm (high endogenous sugar). The results suggest that regulation of secretion by mannose is due to an alteration of normal sugar metabolism; whereas stimulation of secretion by sucrose and other sugars may be due to an increased availability of sugars for metabolism.     

Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant.

From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein).     

Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant.

From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein).