Catalytic efficiency of immobilized glucose isomerase in isomerization of glucose to fructose

Glucose isomerase (GI) from Streptomycesrubiginosus was immobilized covalently onto Eupergit C 250 L made by copolymerization of N,N-methylene-bis-methacrylamide, glycidyl methacrylate, allyl glycidyl ether and methacrylamide. The catalytic efficiency of immobilized GI in isomerization of glucose to fructose was found as three fold higher than that of free GI. The residual activity of immobilized GI after 18 reuses in a batch type stirred reactor was about 85% of its initial activity. The thermal stability of immobilized GI was almost same with that of the free GI at 60 °C for 18 h preincubation time. The residual activities of immobilized GI when stored at 5 °C and 25 °C for four weeks were 72% and 69% of the initial activity, respectively. However, free GI retained 88% and 78% of its initial activity at 5 °C and 25 °C upon four weeks storage, respectively. Thus, the use of Eupergit C 250 L immobilized GI instead of free GI is suggested in enzymatic isomerization of glucose to fructose.     

糖浓度对酒精酵母GJ2008果糖与葡萄糖利用差异性的影响

在不同糖浓度条件下,以YPDF培养基模拟甘蔗汁进行酒精发酵,测定过程中各参数的变化,并对果糖与葡萄糖消耗过程进行曲线拟合,以拟合方程计算出果糖与葡萄糖代谢一半和代谢完全所需时间.结果表明,糖浓度为90gL～270g/L,酵母GJ2008始终会偏用葡萄糖,果糖利用一直受到葡萄糖的竞争性抑制.糖浓度为90g/L时,细胞生长受糖浓度抑制程度最小,但乙醇产率较低;糖浓度为230g/L,发酵液中葡萄糖含量较低时,果糖受葡萄糖的竞争性抑制得到了解除,果糖的利用急剧加快;糖浓度为250g/L和270g/L,发酵液中葡萄糖含量较低时,果糖受葡萄糖的竞争性抑制得到了解除,但果糖的利用并没有加快,表现为后期酵母数维持值较低.糖浓度对果糖代谢的影响要大于对葡萄糖代谢的影响,较低糖浓度有利于后期果糖与葡萄糖利用差异性的缩小.     

Comparative production of glucose and high fructose syrup from cassava and sweet potato roots by direct conversion techniques

High fructose syrup (HFS) is a highly valued liquid sweetener for beverage, confectionery and processed food industry, owing to its special attributes like high solubility and non-crystalline nature. Even though 85% HFS production is from corn, increased food demand has necessitated the search for alternative substrates and starchy root crops like cassava and sweet potato are potential raw materials. However, the economic production needs direct use of the roots and simplification of the cost-intensive steps. This study aims at the direct enzymatic conversion of roots for HFS production. Glucose yield was compared from six treatment systems viz., liquezyme–dextrozyme (T1), Stargen (T2), Stargen in two split doses (T3), Spezyme–Stargen (T4), Stargen (60 °C;T5) and Spezyme–Stargen (60 °C; T6). Glucose was higher (22–25%) from cassava than sweet potato (14.0–15.7%), owing to the high starch content in cassava. Conversion to glucose was higher in T1–T4 (95–98%) compared to 88–92% for T5 and T6. Although the fructose yield was more from cassava (8.36–9.78%) than sweet potato (5.2–6.0%), percentage conversion was similar (37–38%) for both the roots. The cost of production of HFS could be reduced by the direct hydrolysis of root slurry using Stargen.

Industrial relevance

The conventional process for HFS production involved three cost-intensive enzyme steps such as liquefaction, saccharification and isomerization and the major raw material is starch. Economic production using cheaper raw materials and simplification of the process are the decisive factors for the widespread use of HFS in the developing and less developed countries. The present study aimed at the direct conversion of cassava and sweet potato root slurry (without conversion to starch) through the use of improved enzymes like Spezyme and Stargen and mild operating conditions of pH and temperature. The cost of production of HFS could be reduced by using the wet root slurry and performing the Stargen aided saccharification at room temperature, followed by isomerization at 60 °C using Sweetzyme T.     

Mead production: fermentative performance of yeasts entrapped in different concentrations of alginate

Mead is an alcoholic drink known since ancient times, produced by yeast fermenting diluted honey. However, the production of mead has suffered in recent years, partially owing to the lack of scientific progress in this field. In this study, two strains of Saccharomyces cerevisiae, QA23 and ICVD47, were immobilized in 2 or 4% (w/v) alginate beads to assess the most effective alginate concentration for yeast immobilization to produce mead. Neither of the alginate concentrations was able to prevent cell leakage from the beads. The fermentation length was 120 h for both yeast strains. In all cases, at the end of the fermentation, the number of cells entrapped in the beads was higher than the number of free cells, and the total 4% alginate bead wet weight was significantly higher than the 2% alginate bead wet weight. In addition, the evaluation of mead quality showed that the yeast strain had significantly more influence on the physicochemical characteristics than the alginate concentration. Although the yeasts immobilized in the two alginate concentrations were able to perform the fermentation, further research is needed in order to understand the evolution of the yeast population inside the beads throughout the fermentative process. Copyright © 2014 The Institute of Brewing & Distilling     

Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol.     

Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol

Growth efficiency and regulation of key enzyme activities were studied in carbon- and energy-limited chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol at a fixed dilution rate. Biomass yields on substrate carbon and oxygen could be adequately described as the net result of growth on the single substrates. Activities of isocitrate lyase and malate synthase were not detected in cell-free extracts of glucose-limited cultures. However, both enzymes were present when the ethanol fraction in the reservoir medium exceeded the theoretical minimum above which the glyoxylate cycle is required for anabolic reactions. Fructose-1,6-bisphosphatase activity was only detectable at high ethanol fractions in the feed, when activity of this enzyme was required for synthesis of hexose phosphates. Phospho-enol-pyruvate-carboxykinase activity was not detectable in extracts from glucose-grown cultures and increased with the ethanol fraction in the feed. It is concluded that, during carbon-limited growth of S. cerevisiae on mixtures of glucose and ethanol, biosynthetic intermediates with three or more carbon atoms are preferentially synthesized from glucose. Synthesis of the key enzymes of gluconeogenesis and the glyoxylate cycle is adapted to the cells′ requirement for these intermediates. The gluconeogenic enzymes and their physiological antagonists (pyruvate kinase, pyruvate carboxylase and phosphofructokinase) were expressed simultaneously at high ethanol fractions in the feed. If futile cycling is prevented under these conditions, this is not primarily achieved by tight control of enzyme synthesis.     

双菌共固定化耦合催化发酵生产红色染色剂的研究

根据微生物的协同作用理论。采用酿酒酵母与红曲双菌共固定化耦合发酵生产红曲色素。实验结果表明：红曲霉菌与酿酒酵母菌体量配比为4：1时,固定化细胞双菌耦合发酵产红曲色素是红曲霉菌单菌固定化发酵产色素量的约1．6倍。     

酿酒酵母GY-1的固定化及其对魔芋寡糖中单糖的利用

以酿酒酵母（Saccharomyces cerevisiae）GY-1作为实验菌株,利用魔芋粉和海藻酸钠搭配作为固化交联剂进行固定化酵母制备,并通过固定化酵母去除魔芋水解产物中的还原单糖。结果表明,2%的魔芋粉和1%的海藻酸钠组合作为固定化剂,并混合5%的粉状酵母GY-1添加到40 ℃热水中,搅拌混合形成多糖胶体,通过微量泵加入到4%的CaCl2溶液中,在低温条件下交联反应10 h,形成固定化酵母颗粒均匀,平均直径为4～5 mm,包埋的酵母死亡率低于10%;采用薄层层析（TLC）法检测,发现固定化酵母GY-1能够利用魔芋胶中还原单糖（葡萄糖、甘露糖）,从而达到纯化魔芋寡糖的目的。     

Near-infrared spectroscopic monitoring of biomass, glucose, ethanol and protein content in a high cell density baker's yeast fed-batch bioprocess

The use of at-line NIRS to monitor a high cell density fed-batch baker's yeast bioprocess was investigated. Quantification of the key analytes (biomass, ethanol and glucose) and the product quality indicator (percentage protein content) was studied. Biomass was quantitatively modelled using whole matrix samples (as was percentage protein content). The dominance of the whole matrix spectrum by biomass, and its associated light scattering effects, were overcome by use of filtrate samples and adapted (semi-synthetic) filtrate samples, which allowed successful ethanol and glucose modelling, respectively. Calibrations were rigorously challenged via external validation with large sample sets relative to the calibration sample size, ensuring model robustness and potential practical utility. The standard errors of calibration for biomass, glucose, ethanol and total intracellular protein were (g/l) 1.79, 0.19, 0.79 and 0.91, respectively, comparable to those of the primary assays. The calibration strategies necessary to generate quantitative models for this range of analytes in such a complex high cell density bioprocess fluid are discussed.     

High-affinity glucose uptake in Saccharomyces cerevisiae is not dependent on the presence of glucose-phosphorylating enzymes

Glucose uptake in Saccharomyces cerevisiae is believed to consist of two kinetically distinguishable components, the affinity of which is modulated during growth on glucose. It has been reported that triple hexose-kinase deletion mutants do not exhibit high-affinity glucose uptake. This raises the question of whether and how high-affinity glucose uptake is related to the presence of glucose-phosphorylating enzymes. In this study the kinetics of glucose uptake in both wild-type cells and cells of hexose-kinase deletion mutants, grown on either glycerol or galactose, were determined using a rapid-uptake method. In wild-type cells glucose uptake measured over either 5 s or 200 ms exhibited high affinity. In contrast, in cells of hexose-kinase deletion mutants the apparent affinity of glucose uptake was dependent on the time scale during which uptake was measured. Measurements on the 5-s scale showed apparent low-affinity uptake whereas measurements on the 200-ms scale showed high-affinity uptake. The affinity and maximal rate of the latter were comparable to those in wild-type cells. Using a simple model for a symmetrical facilitator, it was possible to simulate the experimentally determined relation between apparent affinity and the time scale used. The results suggest that high-affinity glucose transport is not necessarily dependent on the presence of glucose-phosphorylating enzymes. Apparent low-affinity uptake kinetics can arise as a consequence of an insufficient rate of removal of intracellular free glucose by phosphorylation. This study underlines the need to differentiate between influences of the translocator and of metabolism on the apparent kinetics of sugar uptake in yeast.     

Genetic control of chromosome stability in the yeast Saccharomyces cerevisiae

We have identified four new genetic loci: CHL2 (on chromosome XII), CHL3 (on chromosome XII); CHL4 (on chromosome IV), and CHL5 (on chromosome IX), controlling mitotic transmission of yeast chromosomes. The frequency of loss of chromosomes is 10-100-fold higher in chl5, chl2, chl3 and chl4 mutants than observed in wild-type strains. The mutants also show unstable maintenance of artificial circular minichromosomes with various chromosomal replicators (ARS) and one of the centromeric loci (CEN3, CEN4, CEN5 or CEN6). The instability of minichromosomes in the chl5, chl2, and chl4 mutants is due to the loss of minichromosomes in mitosis (1:0 segregation). In the chl3 mutant the instability of artificial minichromosomes is due to nondisjunction (2:0 segregation). The CHL3 gene therefore appears to affect the segregation of chromosomes during cell division.     

Enzymatic production of atranorin: a component of the oak moss absolute by immobilized lichen cells

Cells of the lichen, Evernia prunastri, immobilized in calcium alginate were able to produce the depside atranorin from acetate. The synthesis of the depside was enhanced by molecular oxygen and NADH. This enhancement suggested the participation of an oxidase and an alcohol dehydrogenase to produce an aldehyde-substituted phenolic acid, hematommic acid, as the most probable precursor of atranorin. The participation of both enzymes was confirmed by loading immobilized cells with sodium azide, an inhibitor of several metallo-oxidases, and pyrazole, an inhibitor of alcohol dehydrogenase, which impeded atranorin production and accumulated beta-methyl orsellinate (after azide loading) or its alcohol derivative (after pirazole treatment).     

Alterations of the glucose metabolism in a triose phosphate isomerase-negative Saccharomyces cerevisiae mutant

The absence of triose phosphate isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996). Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures. The Deltatpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Deltatpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Deltatpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Deltatpi1 mutant may occur in relation to the fraction of ethanol present in the media.     

航天诱变混菌固态发酵玉米芯生产单细胞蛋白的研究

黑曲霉ZM-8和啤酒酵母YB-6分别是经航天诱变而筛选出的优良纤维素降解菌和高生物积累量的单细胞蛋白生产菌.以玉米芯粉和麸皮为主要原料,采用混菌固态发酵方式,研究了啤酒酵母YB-6的接种量、接种时间、黑曲霉ZM-8和啤酒酵母YB-6共生发酵时间等因素对发酵产物中CMC酶活和单细胞蛋白含量的影响.结果表明,黑曲霉ZM-8固体曲发酵36h后接入啤酒酵母YB-6、接种量为15％ (v/w)、共生发酵为60h,有利于发酵体系中CMC酶的分泌和SCP的积累.在上述条件下,CMC酶活和SCP含量分别为32.36U/g和16.83％.     

固定化后期添加啤酒酵母菌降低啤酒中双乙酰含量的探讨

为探索用后期添加固定化酵母菌降低啤酒中双乙酰的工艺,通过单因素和正交试验,发现后期接入固定化啤酒酵母菌降低双乙酰含量的最佳工艺为:发酵温度为12℃,接入时间为发酵第16d,发酵再次接入固定化酵母菌的接种量为1.0%,此时双乙酰的量为0.083mg/L,啤酒口感达到9.2分。固定化的酵母菌可以重复利用3次,啤酒的双乙酰值和口感维持稳定。该研究表明利用固定化细胞的后期添加可以降低啤酒中的双乙酰含量。     

混菌固态发酵玉米秸秆生产单细胞蛋白的研究

利用经航天诱变筛选的高产纤维素酶活菌株黑曲霉ZM-8和高生物积累量的啤酒酵母YB-6,采用混菌固态发酵方式,研究了啤酒酵母YB-6的接种量、接种时间、黑曲霉ZM-8和啤酒酵母YB-6共生发酵时间等因素在玉米秸秆为主要原料的培养基中对黑曲霉ZM-8产纤维素酶活和发酵产物中单细胞蛋白(SCP)含量的影响.结果表明,当啤酒酵母YB-6的接种时间为黑曲霉ZM-8固体曲发酵24h、接种量为15%、黑曲霉ZM-8和啤酒酵母YB-6共生发酵48h时,较有利十产CMC酶、FPU酶和发酵原料中SCP的合成.     

Expression of recombinant platelet-derived endothelial cell growth factor in the yeast Saccharomyces cerevisiae.

A platelet-derived endothelial cell growth factor cDNA has been cloned, sequenced and expressed using the Saccharomyces cerevisiae PRB1 promoter. Soluble recombinant platelet-derived endothelial cell growth factor constituted 0.5-1.0% of total soluble protein. Yeast soluble protein extracts containing recombinant platelet-derived endothelial cell growth factor stimulate the growth of calf pulmonary artery endothelial cells in vitro.     

Spectrophotometric determination of glucose in foods by flow injection analysis with an immobilized glucose oxidase reactor

 A stopped-flow injection analysis system for the determination of glucose is described, based on the iodometric measurement of hydrogen peroxide generated in a glucose oxidase reactor. The detection of the iodine–starch complex was carried out spectrophotometrically. The calibration curve was linear up to 2 mmol l^-1 glucose, with a correlation coefficient, r, of 0.9996. The relative standard deviation was 1.6% (n=5) for 1.0 mmol l^-1 glucose. A sample frequency of 25 samples h^-1 was achieved. This method was applied to the determination of glucose in fruit products and the results obtained using this method were in good agreement with those of a routine enzymatic method. A high dilution of the sample avoided interference from ascorbic acid. The enzyme reactor was stable for 1 month after 200 measurements with no loss of activity. Received: 22 March 1996     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Serine metabolism contributes to cell survival by regulating extracellular pH and providing an energy source in Saccharomyces cerevisiae

Changes in extracellular pH affect the homeostasis and survival of unicellular organisms. Supplementation of culture media with amino acids can extend the lifespan of budding yeast, Saccharomyces cerevisiae, by alleviating the decrease in pH. However, the optimal amino acids to use to achieve this end, and the underlying mechanisms involved, remain unclear. Here, we describe the specific role of serine metabolism in the regulation of pH in a medium. The addition of serine to synthetic minimal medium suppressed acidification, and at higher doses increased the pH. CHA1, which encodes a catabolic serine hydratase that degrades serine into ammonium and pyruvate, is essential for serine-mediated alleviation of acidification. Moreover, serine metabolism supports extra growth after glucose depletion. Therefore, medium supplementation with serine can play a prominent role in the batch culture of budding yeast, controlling extracellular pH through catabolism into ammonium and acting as an energy source after glucose exhaustion.