Xylulokinase activity in various yeasts includingSaccharomyces cerevisiae containing the cloned xylulokinase gene

d-Xylose is a major constituent of hemicellulose, which makes up 20–30% of renewable biomass in nature.d-Xylose can be fermented by most yeasts, includingSaccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase, and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source of xylose isomerase produced by recombinantE. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level of xylulokinase activity in S.cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene with an effective promoter. The resulting genetically-engineered yeasts can ferment xylose and xylulose more than twice as fast as the parent yeast.     

Cloning and expression of the gene for xylose isomerase fromThermus flavus AT62 inEscherichia coli

The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene. The XI gene was stably expressed inE. coli under the control oftac promoter. XI produced inE. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn²⁺, Co²⁺, and Mg²⁺ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

Enzymatic production and analysis of d-xylulose with a recirculating flow system and post-column liquid chromatographic detection using a co-immobilized enzyme reaction and a chemically modified electrode

A pure d-xylulose and standard was produced by isomerization of d-xylose in a recirculating flow system incorporating an enzyme reactor containing immobilized xylose isomerase. The d-xylulose formed was purified chromatographically. A selective chromatographic detection system was used in the post-column mode. It consisted of a co-immobilized enzyme reactor (CIMER) with xylose isomerase, mutarotase and glucose dehydrogenase on-line with a chemically modified electrode for selective elctrochemical oxidation of NADH. The pure standard was compared with commercially available d-xylulose, which was confirmed to contain impurities of d-glucose and d-xylose.     

A flow-injection system for the amperometric determination of xylose and xylulose with co-immobilized enzymes and a modified electrode

A flow-injection system is described for the detection of xylose and xylulose. The detection is based on three successive reactions taking place in an enzyme reactor containing xylose isomerase, mutarotase and glucose dehydrogenase co-immobilized on controlled-pore glass. The final product, NADH, is electrocatalytically oxidized at 0 mV vs. SCE at a chemically modified electrode. Straight calibration graphs for the two pentoses were obtained between 50 μM and 2 mM. The maximal sample throughput was 30 h^?1.     

Effect of Fermentation Conditions on the Flocculation of Recombinant Saccharomyces cerevisiae Capable of Co-fermenting Glucose and Xylose

Flocculation is a desirable property in industrial yeasts and is particularly important in the fuel ethanol industry because it provides a simple and cost-free way to separate yeast cells from fermentation products. In the present study, the effect of pH and lignocellulose-derived sugars on yeast flocculation was investigated using a flocculent Saccharomyces cerevisiae, MA-R4, which has been recombinantly engineered to simultaneously co-ferment glucose and xylose to ethanol with high productivity. The flocculation level of MA-R4 dramatically decreased at pH values below 3.0 during co-fermentation of glucose and xylose. Sedimentation and microscopic observation revealed that flocculation was induced in MA-R4 when it fermented glucose, a glucose/xylose mixture, or mannose, whereas attempts to ferment xylose, galactose, and arabinose led to the loss of flocculation. MA-R4 fermented xylose and galactose more slowly than glucose and mannose. Therefore, the various flocculation behaviors shown by MA-R4 should be useful in the control of ethanol fermentation processes.     

毛细管区带电泳法分离发酵液中的木糖和木糖醇

 建立了利用毛细管区带电泳分离发酵液中木糖和木糖醇的新方法。研究表明 :采用硼砂缓冲溶液时 ,木糖和木糖醇的分离度随硼砂浓度的增高而加大 ,在室温下硼砂最高浓度为 130mmol/L ;分离度还与溶液的 pH有关 ,在pH 9 5 5处分离度有最大值 ;缓冲液中十六烷基三甲基溴化铵的浓度为 4× 10 -6mmol/L～ 8× 10 -4 mmol/L时对分离度无显著影响 ;在优化的分离条件下 ,木糖和木糖醇可在 6min内基线分离。测定了发酵过程中样品各组分的含量和加标回收率 ,5次测定木糖的相对标准偏差 (RSD)为 1 42 %～ 3 11% ,回收率为 96 0 %～ 10 8 0 % 。     

介孔磷酸铌一锅法高效催化木糖制备糠醛

木糖转化到糠醛一般包括两步: 首先在酶、碱或路易斯(L)酸的催化作用下异构化木糖到木酮糖, 接下来木酮糖在酸的作用下脱水得到糠醛. 针对木糖水相脱水一步制备糠醛, 利用十六烷基三甲基溴化铵(CTAB)为模板剂, 借助软模板合作策略制备了一种抗水的新型固体酸催化剂, 介孔磷酸铌, 并利用X射线衍射(XRD)、N₂吸脱附、透射电镜(TEM)、氨气程序升温脱附(NH₃-TPD)和吡啶吸附傅里叶变换红外(Py-FTIR)光谱对材料的结构和酸性质进行了表征. 研究发现介孔磷酸铌不仅具有很高的比表面积(>200 m²·g^-1), 比较窄的孔径分布(3.5nm), 同时还具有很强的L酸性和布朗斯特(B)酸性. 通过L酸催化的木糖异构化为木酮糖/来苏糖和B酸催化的木酮糖/来苏糖进一步脱水得到糠醛, 实现了一步由木糖到糠醛的高效转化. 为了优化反应条件, 考察了水溶液中反应温度、投料质量比及反应时间对木糖转化率和糠醛收率的影响, 在最佳的反应条件下, 木糖的转化率为96.5%, 糠醛的收率达49.8%. 进一步地, 为了提高收率且易于分离, 利用4-甲基-2-戊酮(MIBK)/NaCl水溶液(体积比为7:3)作为反应混合溶剂, 使糠醛收率提高到68.4%.     

Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4 (pZB5)

Fermentation of glucose-xylose mixtures to ethanol was investigated in batch and continuous experiments using immobilized recombinant Zymomonas mobilis CP4(pZB5). This microorganism was immobilized by entrapment in κ-carrageenan beads having a diameter of 1.5–2.5 mm. Batch experiments showed that the immobilized cells cofermented glucose and xylose to ethanol and that the presence of glucose improved the xylose utilization rate. Batch fermentation of rice straw hydrolysate containing 76 g/L of glucose and 33.8 g/L of xylose gave an ethanol concentration of 44.3 g/L after 24 h, corresponding to a yield of 0.46 g of ethanol/g of sugars. Comparable results were achieved with a synthetic sugar control. Continuous fermentation experiments were performed in a laboratory-scale fluidized-bed bioreactor (FBR). Glucose-xylose feed mixtures were pumped through the FBR at residence times of 2–4 h. Glucose conversion to ethanol was maintained above 98% in all experiments. Xylose conversion to ethanol was highest at 91.5% for a feed containing 50 g/L of glucose and 13 g/L of xylose at a dilution rate of 0.24/h. The xylose conversion to ethanol decreased with increasing feed xylose concentration, dilution rate, and age of the immobilized cells. Volumetric ethanol productivities in the range of 6.5–15.3 g/L·h were obtained. The improved productivities achieved in the FBR compared to other bioreactor systems can help in reducing the production costs of fuel ethanol from lignocellulosic sugars. This article has been authored by a contractor of the US go vernment under contract DE-AC05-96OR22464. Accordingly, the US government retains a nonexclusive, royaltyfree license to publish or reproduce the published form of the contribution, or allow others to do so, for US government purposes.     

Optimization of seed production for a simultaneous saccharification cofermentation biomass-to-ethanol process using recombinantZymomonas

The five-carbon sugard-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain ofZymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective seed production that are compatible with the SSCF process design. Two-level and three-level full factorial experimental designs were employed to characterize efficiently the growth performance of recombinantZ. mobilis CP4:pZB5 as a function of nutrient level, pH, and acetic acid concentration using a synthetic hardwood hemicellulose hydrolyzate containing 4% (w/v) xylose and 0.8% (w/v) glucose. Fermentations were run batchwise and were pH-controlled at low levels of clarified corn steep liquor (cCSL, 1-2% v/v), which were used as the sole source of nutrients. For the purpose of assessing comparative fermentation performance, seed production was also carried out using a “benchmark” yeast extract-based laboratory medium. Analysis of variance (ANOVA) of experimental results was performed to determine the main effects and possible interactive effects of nutrient (cCSL) level, pH, and acetic acid concentration on the rate of xylose utilization and the extent of cell mass production. Results indicate that the concentration of acetic acid is the most significant limiting factor for the xylose utilization rate and the extent of cell mass production; nutrient level and pH exerted weaker, but statistically significant effects. At pH 6.0, in the absence of acetic acid, the final cell mass concentration was 1.4 g dry cell mass/L (g DCM/L), but decreased to 0.92 and 0.64 g DCM/L in the presence of 0.5 and 1.0% (w/v) acetic acid, respectively. At concentrations of acetic acid of 0.75 (w/v) or lower, fermentation was complete within 1.5 d. In contrast, in the presence of 1.0% (w/v) acetic acid, 25% of the xylose remained after 2 d. At a volumetric supplementation level of 1.5–2.0% (v/v), cCSL proved to be a cost-effective single-source nutritional adjunct that can support growth and fermentation performance at levels comparable to those achieved using the expensive yeast extract-based laboratory reference medium.     

Production of ethanol from cellulosic biomass hydrolysates using genetically engineered saccharomyces yeast capable of cofermenting glucose and xylose

Recent studies have proven ethanol to be the idael liquid fuel for transportation, and renewable ligno cellulosic materials to be the attractive feed stocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Sacharomyces yeast, 424A (LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.     

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation. Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that d-xylulokinase limits metabolic flux in recombinant S. cerevisiae.     

Evaluation of recombinant strains of Zymomonas mobilis for ethanol production from glucose/xylose media

The fermentation characteristics of two recombinant strains of Zymomonas mobilis, viz. CP4 (pZB5) and ZM4 (pZB5), capable of converting both glucose and xylose to ethanol, have been characterized in batch and continuous culture studies. The strain ZM4 (pZB5) was found to be capable of converting a mixture of 65 g/L glucose and 65 g/L xylose to 62 g/L ethanol in 48h with a yield of 0.46 g/g. Higher sugar concentrations resulted in incompletexylose utilization (80h) presumably owing to ethanol inhibition of xylose assimilation or metabolism. The fermentation results with ZM4 (pZB5) show a significant improvement over results published previously for recombinant yeasts and other bacteria capable of glucose and xylose utilization.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose,a Functional Sweetener,Utilizing Alginate Immobilized <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> CGMCC2921 Cells

d-tagatose is a ketohexose that can be used as a novel functional sweetener in foods, beverages, and dietary supplements. This study was aimed at developing a high-yielding d-tagatose production process using alginate immobilized Lactobacillus fermentum CGMCC2921 cells. For the isomerization from d-galactose into d-tagatose, the immobilized cells showed optimum temperature and pH at 65 °C and 6.5, respectively. The alginate beads exhibited a good stability after glutaraldehyde treatment and retained 90% of the enzyme activity after eight cycles (192 h at 65 °C) of batch conversion. The addition of borate with a molar ratio of 1.0 to d-galactose led to a significant enhancement in the d-tagatose yield. Using commercial β-galactosidase and immobilized L. fermentum cells, d-tagatose was successfully obtained from lactose after a two-step biotransformation. The relatively high conversion rate and productivity from d-galactose to d-tagatose of 60% and 11.1 g l⁻¹ h⁻¹ were achieved in a packed-bed bioreactor. Moreover, lactobacilli have been approved as generally recognized as safe organisms, which makes this L. fermentum strain an attracting substitute for recombinant Escherichia coli cells among d-tagatose production progresses.     

Xylose fermentation

The economic impact of conversion of xylose to ethanol for a wood-to-ethanol plant was examined, and the maximum potential reduction in the price of ethanol from utilization of xylose is estimated to be 0.42 per gallon from a base case price of0.42 per gallon from a base case price of 1.65. The sensitivity of the price of ethanol to the yield, ethanol concentration and rate of the xylose fermentation was also examined, and the price of ethanol is most affected by changes in yield and ethanol concentration, with rate of lesser importance. Current performances of various xylose conversion biocatalysts were analyzed, andC. shehatae andP. stipitis appear to be the best yeasts.     

Immobilization of Phenylalanine Dehydrogenase and Its Application in Flow-Injection Analysis System for Determination of Plasma Phenylalanine

Phenylalanine dehydrogenase (l-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity. The optimum pH value of immobilized l-PheDH was shifted from pH 10.4 to 11.0. The immobilized l-PheDH showed activity variations close to the maximum value in a wider temperature range of 45–55 °C, whereas it was 40 °C for the native enzyme. The pH and the thermal stability of the immobilized l-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K _m values of the native and the immobilized l-PheDH were determined as K _{m Phe} = 0.118, 0.063 mM and K _{m NAD}⁺ = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5–600 μM Phe at 9 mM NAD⁺ with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life period of the reactor was 15 days.     

Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445)

Using the simultaneoussaccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lacticacid using NRRL B445. SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97%. The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium. On prolonged incubation, depletion of xylose with lactic acid production was observed. An experimental procedure with a nonglucose medium was developed to eliminate the lag phase. From xylose fermentation, Lactobacillus delbrueckii yielded 88–92% lactic acid and 2–12% acetic acid.     

Preparation and properties of immobilized pig kidney aminoa cylase and optical resolution of N-acyl-dl-alanine

Aminoacylase (EC 3.5.1.14) was immobilized into DEAE-Sephadex A-25 by ion-exchange absorption for optical resolution of N-acyl-dl-alanine. The effects of pH, temperature, and Co²⁺ concentration on the activity of free and immobilized enzymes were in vestigated along with the operational and the thermal stability of the immobilized enzyme. The immobilized enzyme retained high catalytic activity. The optimum pH and temperature for the hydrolysis of N-acyl-l-alanine in the dl-isomer mixture were 8.0 and 65°C, respectively. Co²⁺ was an activator for the immobilized enzyme in a similarroleas for the free enzyme. Nosignificant loss of activity was observed for at least 300 h of continuous operation. The yield of l-alanine was about 70% of the theoretical yield. The immobilized aminoacylase column decayed over a very long period of operation, but could be completely reactivated by regeneration.     

Ethanol Production from Wet-Exploded Wheat Straw Hydrolysate by Thermophilic Anaerobic Bacterium Thermoanaerobacter BG1L1 in a Continuous Immobilized Reactor

Thermophilic ethanol fermentation of wet-exploded wheat straw hydrolysate was investigated in a continuous immobilized reactor system. The experiments were carried out in a lab-scale fluidized bed reactor (FBR) at 70°C. Undetoxified wheat straw hydrolysate was used (3–12% dry matter), corresponding to sugar mixtures of glucose and xylose ranging from 12 to 41 g/l. The organism, thermophilic anaerobic bacterium Thermoanaerobacter BG1L1, exhibited significant resistance to high levels of acetic acid (up to 10 g/l) and other metabolic inhibitors present in the hydrolysate. Although the hydrolysate was not detoxified, ethanol yield in a range of 0.39–0.42 g/g was obtained. Overall, sugar efficiency to ethanol was 68–76%. The reactor was operated continuously for approximately 143 days, and no contamination was seen without the use of any agent for preventing bacterial infections. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol. The work reported here also demonstrates that the use of FBR configuration might be a viable approach for thermophilic anaerobic ethanol fermentation.     

Immobilization of β-Galactosidase onto Magnetic Beads

A study of the cross-linking of β-galactosidase on magnetic beads is reported here. The magnetic beads were prepared from artemisia seed gum, chitosan, and magnetic fluid in the presence of a cross-linking regent (i.e., glutaraldehyde). The reactive aldehyde groups of the magnetic beads allowed the reaction of the amino groups of the enzymes. The animated magnetic beads were used for the covalent immobilization of β-galactosidase. The effect of various preparation conditions on the activity of the immobilized β-galactosidase, such as immobilizing time, amount of enzyme, and the concentration of glutaraldehyde, were investigated. The influence of pH and temperature on the activity and the stability of the enzyme, both free and immobilized, have been studied. And o-nitrophenyl-β-d-galactopyranoside (ONPG) was chosen as a substrate. The β-galactosidase immobilized on the magnetic beads resulted in an increase in enzyme stability. Optimum operational temperature for immobilized enzyme was 10 °C higher than that of free enzyme and was significantly broader.