Theoretical investigation of fructose 1,6-diphosphate production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow-fiber reactor

The performance of an ultrafiltration hollow-fiber reactor, in which enzymatic synthesis of fructose 1,6-diphosphate (FDP) from glucose and enzymatic ATP regeneration are performed simultaneously, was analyzed theoretically. The reaction system consists of three-step synthetic reactions catalyzed by glucokinase (GK), phosphoglucose isomerase and phosphofructokinase, and the ATP regeneration reaction catalyzed by acetate kinase. Based on a simple analytical model developed previously in which the liquid flowing in a tube was assumed to be plug flow and the radial concentration gradients in the tubes and shell side space were both neglected, a computer program was developed to calculate the concentration profiles of all the components along the flow direction in the tubes and shell side space of the reactor. From the FDP concentrations at the reactor outlet calculated under various operational conditions, reactor performances such as the FDP yield and the ATP recycle number were determined. The calculation showed the interesting phenomenon that under some conditions the FDP yield was higher when GK concentration was lower.     

Dissimilatory nitrate reduction metabolisms and their control in fungi

Most fungi grow under aerobic conditions by generating ATP through oxygen respiration. However, they alternatively express two pathways of dissimilatory nitrate reduction in response to environmental oxygen tension when the oxygen supply is insufficient. The fungus Fusarium oxysporum expressed the pathway of respiratory nitrate denitrification that is catalyzed by the sequential reactions of nitrate reductase and nitrite reductase. These enzymes are coupled with ATP generation through the respiratory chain and produce nitric oxide. Fungal nitric oxide reductase uses NADH as the direct electron donor in contrast to bacterial systems and thus might function in regeneration of NAD+ and detoxification of the toxic radical, nitric oxide. Another pathway of nitrate dissimilation by fungi reduces nitrate to ammonium and couples acetogenic reaction with substrate-level phosphorylation. This metabolic mechanism is also in feature of a variety of fungi and it is called ammonia fermentation. Thus, fungi adapt to various aerated conditions using these pathways of nitrate dissimilation in addition to conventional oxygen respiration.     

Enzymatic synthesis of fructose 1,6-diphosphate with ATP regeneration in a batch reactor and a semibatch reactor using purified enzymes of Bacillus stearothermophilus

The enzymatic synthesis of fructose 1,6-diphosphate (FDP), an important glycolytic intermediate whose applications in the field of medicine have generated a great deal of interest, was performed in a batch reactor and a semibatch reactor. Using the batch reactor, FDP was first synthesized from glucose by three enzymatic reactions and the ATP consumed was regenerated simultaneously using conjugated enzymes, all of which were purified from crude cell extract of thermophilic Bacillus stearothermophilus. The results of the experiments performed using several enzyme concentrations suggest the existence of an optimum concentration for each enzyme at which the maximum FDP yield can be attained. Since the thermal decomposition of acetyl phosphate reduced the yield of FDP in the batch reactor, the use of a semibatch reactor in which acetyl phosphate was fed continuously was examined. The yield of FDP was improved but the time required to complete the reaction was longer, resulting in a lower productivity of FDP. The yields observed in the two reactors using various enzyme and substrate concentrations were in good agreement with the theoretical predictions calculated based on differential equations derived for the system using the rate equations and the kinetic parameters determined previously. This means that these equations can be used for the analysis of the experimental results as well as for determining the optimum experimental conditions.     

Thermostable ATP regeneration system using polyphosphate kinase from Thermosynechococcus elongatus BP-1 for D-amino acid dipeptide synthesis

D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) was a useful biocatalyst for synthesizing D-amino acid dipeptides. TmDdl showed a broad substrate specificity at a high temperature; however, ATP was required for its reaction. One of the methods for an effective ATP supply was the coupling reaction with an ATP regeneration system. However, ATP regeneration systems consisted of enzymes from mesophiles and were difficult to operate at high temperatures. Therefore, an ATP regeneration system that could be used at high temperatures was desired to utilize TmDdl for the effective production of D-amino acid dipeptides. To establish a thermostable ATP regeneration system, polyphosphate kinase from a thermophile, Thermosynechococcus elongatus BP-1 (TePpk), was characterized. TePpk showed thermostability up to 70 degrees C; therefore, it was considered that a thermostable ATP regeneration system could be established using TePpk. In the coupling reaction with purified TmDdl and TePpk at 60 degrees C, the amount of ATP required for D-alanyl-D-alanine synthesis could be reduced to 1% of the theoretical amount required when there was no ATP regeneration. When the coupling reaction was applied to a resting cell reaction, ATP was regenerated from an adenosine scaffold in the cell, and D-alanyl-D-alanine was successfully synthesized in the maximum yield of 80% (mol/mol) without the addition of ATP. Thus, an effective synthesis of D-amino acid dipepitides was achieved using the thermostable ATP regeneration system.     

Strategies to remove water formed as by-product on the monoolein synthesis by enzymatic esterification performed on packed bed reactor

Different strategies to remove water generated on the monoolein synthesis by enzymatic esterification of oleic acid with glycerol were evaluated. Esterification reactions were catalyzed by Penicillium camembertii lipase immobilized on SiO₂-PVA and runs carried out on a packed bed reactor (PBR) under continuous mode. The use of molecular sieves was the selected technique to remove the water formed, and for this purpose, two experimental setups were performed: (1) reactor packed with alternate layers of molecular sieves and immobilized lipase and (2) three reactors in series having a water column extractor between two immobilized packed beds, making it possible to replace the saturated molecular sieves by a regenerated one at a given time. Continuous run without applying dehydration techniques to remove the formed water was used as a control. The best performance was obtained using a water column extractor as verified by an increase in the monoolein formation from 22 to 35%. This strategy also provided a good performance of the bed reactor as a result of the high operational stability estimated for the immobilized derivative (half-life = 19 days).     

A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.     

酶膜反应器制备燕麦蛋白质ACE抑制肽的研究

通过研究酶膜反应器系统对酶活的影响,可知酶膜反应器系统对碱性蛋白酶(Alcalase)具有很好的保留效果.蠕动泵运转过程对酶造成的破坏以及超滤膜对酶的吸附是导致碱性蛋白酶酶活损失的主要原因.通过利用Design Expert 6.0.5设计四因素三水平的响应面分析试验,求得酶膜反应器制备燕麦蛋白质ACE抑制肽的最佳工艺条件为底物质量分数3％、加酶量3.5％、料液流量45 L/h、操作压力0.07 MPa、温度61℃、pH 8,在该备件下产物的ACE抑制率可达67.86％.     

Orange peel degradation and enzyme recovery in the enzymatic peeling process

A detailed study was carried out on enzymatic peeling of oranges in a reactor using an enzyme preparation. This work was focused on determining the changes that happen in the peel albedo of Navelina oranges. Thus, the conditions of temperature and concentration of the enzymatic preparation were optimized in order to produce the maximum weight loss, which indicates good peeling efficiency. Experiments to study the efficiency of the reactor effluents by reusing them for successive enzymatic peelings were also carried out with the aim of diminishing the cost of the process. Finally, the recovery of the enzymes after use in multiple enzymatic peelings by means of ultrafiltration was tested and an increment in their enzymatic activity was observed.     

醇脱氢酶催化合成(S)-哌啶醇及产物抑制机制

(S)-N-Boc-3-羟基哌啶((S)-NBHP)是治疗套细胞淋巴瘤药物——依鲁替尼的关键手性中间体。利用醇脱氢酶催化N-Boc-3-哌啶酮(NBPO)的不对称还原是最具开发潜力的(S)-NBHP合成方法。对醇脱氢酶库进行筛选发现,来源于多孢克鲁维酵母的Kp ADH对NBPO具有最佳的催化效果,纯酶活力高达83.9 U/mg,产物对映体过量值为97.0%(S)。将其与葡萄糖脱氢酶偶联,并考察了单水相、两相、离子液体和不同底物浓度对转化反应的影响。结果表明:高浓度产物(S)-NBHP对Kp ADH存在抑制作用,抑制动力学分析表明属于非竞争性抑制。采用单水相体系可降低(S)-NBHP对Kp ADH催化反应的抑制,有利于转化效率的提高。初步探究了醇脱氢酶催化NBPO不对称还原反应的抑制现象,对酶法合成(S)-NBHP具有指导意义。     

Treatment of model soils contaminated with phenolic endocrine-disrupting chemicals with laccase from Trametes sp. in a rotating reactor

An enzymatic treatment system for the remediation of sand contaminated with endocrine-disrupting chemicals (EDCs) was studied. Laccase from Trametes sp. (Laccase Daiwa) decreased the amounts of nonylphenol, octylphenol, bisphenol A and ethynylestradiol (synthetic estrogen) adsorbed on sea sand (2 micromol g(-1)) in a test tube with shaking. The phenolic endocrine-disrupting chemicals might have polymerized via enzymatic conversion to their phenoxy radicals. The optimum pH for the enzymatic treatment was approximately 5. A rotating reactor was used for scaling up the enzymatic treatment. The reaction rate increased by rotating the reactor. The optimum speed of revolution was 10-15 rpm for the treatment of nonylphenol. The amounts of octylphenol, bisphenol A, and ethynylestradiol also decreased enzymatically in the reactor. Our enzymatic treatment system with a rotating reactor will be useful for the treatment of soil highly polluted with phenolic EDCs.     

Kinetics of the digestion products and effect of temperature on the enzymatic peeling process of oranges

A detailed study was done on enzymatic peeling of oranges in a reactor using an enzymatic preparation. This work was focused on determining the changes that happen in the peel albedo of Navelina oranges by studying the variation in the effluent content from the reactor, not only to determine the loss of components from the albedo of the skin, but also as possible environmental information. The effect of temperature on the peeling process rate was also studied, determining the kinetic constants at different temperatures and also the activation energy of the degradation of the peel albedo.     

A new method of ATP regeneration rate measurement using a pH meter

A new method was developed for measuring the ATP regeneration rate by creatine kinase from creatine phosphate and ADP. The ATP regeneration rate was calculated to be 0.20 mM x s(-1) at room temperature from the pH change of the regeneration mixture.     

转谷氨酰胺酶途径的酶法糖基化修饰在蛋白质/多肽改性中的研究进展

为了拓展蛋白质的应用范围,常对蛋白质进行改性,其中转谷氨酰胺酶(TGase)催化的酶法糖基化反应已应用于多种蛋白质/多肽的改性中。为了对蛋白质/多肽的酶法糖基化技术的广泛应用提供参考,简述了TGase的来源、催化的反应类型和底物类型,重点阐述了糖基化蛋白/多肽糖基化程度的评价方法以及修饰产物功能性质的变化。目前,TGase主要来源于微生物,其可催化底物的交联、酰基转移(酶法糖基化)和脱酰基3种类型的反应。在TGase催化的酶法糖基化反应中,常用RP-HPLC、邻苯二甲醛(OPA)法和3,5-二硝基水杨酸(DNS)法评价糖基化程度。通过酶法糖基化反应,可以不同程度地改善底物的溶解性、乳化性、热稳定性等功能性质。TGase催化的酶法糖基化反应还存在反应体系复杂、糖基化效率低等问题,需要进一步研究解决。     

ATP再生系统及其应用

构建ＡＴＰ再生系统是提高生物合成酶反应经济性的一个有效措施。本文讨论了ＡＴＰ再生系统的两种形式自耦合反应系统与种间耦合反应系统在生产ＧＭＰ、ＡＴＰ、ＩＭＰ、ＧＳＨ和ＣＤＰ胆碱中的应用，提出了构建ＡＴＰ再生系统的必要条件并分析了该系统目前存在的问题，以期为国内同行开展类似研究提供参考。     

Enzyme Recovery and Effluents Generated in the Enzymatic Peeling Process of Lemons

This research focused on determining the variation in the effluent content from the reactor in enzymatic peeling of lemons, not only to determine the loss of components from the albedo of the skin but also to study the potential environmental impact. Experiments to study the efficiency of the reactor effluents by reusing them for successive enzymatic peelings were also carried out with the aim of reducing the cost of the process. Finally, the recovery of the enzymes after use in multiple enzymatic peelings by means of ultrafiltration was tested and an increase in their enzymatic activity was observed.     

Enzyme Recovery and Effluents Generated in the Enzymatic Peeling Process of Lemons

This research focused on determining the variation in the effluent content from the reactor in enzymatic peeling of lemons, not only to determine the loss of components from the albedo of the skin but also to study the potential environmental impact. Experiments to study the efficiency of the reactor effluents by reusing them for successive enzymatic peelings were also carried out with the aim of reducing the cost of the process. Finally, the recovery of the enzymes after use in multiple enzymatic peelings by means of ultrafiltration was tested and an increase in their enzymatic activity was observed.     

Enzymatic production of fructose 1,6-diphosphate using crude cell extract of Bacillus stearothermophilus

The enzymatic production of fructose 1,6-diphosphate (FDP) from glucose was performed in a batch reactor and a semibatch reactor using the crude cell extract of Bacillus stearothermophilus which contains all four enzymes required for the synthesis. The experimental results of the yield and the time courses of FDP production obtained using various enzyme concentrations were in good agreement with the theoretical predictions calculated based on the differential equations including the rate equations of the four enzymes, which were determined using the purified enzymes of B. stearothermophilus.     

一种连续分离纯化酪蛋白磷酸肽的新工艺

为建立一条制备酪蛋白磷酸肽(CPPs)的新工艺路线。采用酶解与超滤耦联技术初步分离制备酪蛋白生物活性肽,然后通过树脂层析将酪蛋白生物活性肽中的酪蛋白磷酸肽和非磷酸肽分离。选用碱性蛋白酶Alcalase在切向流酶膜反应器中(膜截留分子质量10kD)连续酶解3h、酶解温度50℃、pH8.5。重点研究大孔弱碱性阴离子交换树脂D303对酪蛋白生物活性肽的分离纯化工艺,并采用单因素试验优化CPPs的分离纯化工艺参数,确定D303树脂的较佳动态吸附解吸条件：上样流速1BV/h、上样量100mL、洗脱温度45℃、洗脱酸浓度0.15mol/L HCl,获得磷酸肽N/P比为7.21,P洗脱得率为93.11%。     

Characterization study and five-cycle tests in a fixed-bed reactor of titania-supported nickel oxide as oxygen carriers for the chemical-looping combustion of methane

Recent investigations have shown that in the combustion of carbonaceous compounds CO2 and NOx emissions to the atmosphere can be substantially reduced by using a two stage chemical-looping process. In this process, the reduction stage is undertaken in a first reactor in which the framework oxygen of a reducible inorganic oxide is used, instead of the usual atmospheric oxygen, for the combustion of a carbonaceous compound, for instance, methane. The outlet gas from this reactor is mostly composed of CO2 and steam as reaction products and further separation of these two components can be carried out easily by simple condensation of steam. Then, the oxygen carrier found in a reduced state is transported to a second reactor in which carrier regeneration with air takes place at relatively low temperatures, consequently preventing the formation of thermal NOx. Afterward, the regenerated carrier is carried to the first reactor to reinitiate a new cycle and so on for a number of repetitive cycles, while the carrier is able to withstand the severe chemical and thermal stresses involved in every cycle. In this paper, the performance of titania-supported nickel oxides has been investigated in a fixed-bed reactor as oxygen carriers for chemical-looping combustion of methane. Samples with different nickel oxide contents were prepared by successive incipient wet impregnations, and their performance as oxygen carriers was investigated at 900 degrees C and atmospheric pressure in five-cycle fixed-bed reactor tests using pure methane and pure air for the respective reduction and regeneration stages. The evolution of the outlet gas composition in each stage was followed by gas chromatography, and the involved chemical, structural, and textural changes of the carrier in the reactor bed were studied by using different characterization techniques. From the study, it is deduced that the reactivity of these nickel-based oxygen carriers is in the two involved stages and almost independent of the nickel loading. However, in the reduction stage, carbon deposition, from the thermal decomposition of methane, and CO emissions, mainly derived from the partial reduction of titania as support acting as an additional oxygen source, may impose some constraints to the efficiency of the overall chemical-looping combustion process in CO2 capture.     

Gas-Solid Hydroxyethylation of Potato Starch in a Stirred Vibrating Fluidized Bed Reactor

A novel reactor for modifying cohesive C-powders such as in the gassolid hydroxyethylation of semidry potato starch is characterized, the so-called stirred vibrating fluidized bed reactor. Good fluidization chracteristics are obtained in this reactor for certain combinations of stirring and vibration parameters. Channeling can be suppressed by stirring in the bed with equidistant flat blades whereas agglomerated are destroyed by vibration of the gas distributor. Also the complex sorption, diffusion and reaction behaviour of ethylene oxide in semi-dry starch was investigated. Reaction rate equations were obtained for all four parallel reactions involved: the catalyzed and uncatalyzed hydroxyethylation of starch and the catalyzed and uncatalyzed hydrolysis of ethylene oxide. By combining all results, a pressure-controlled stirred vibrating fluidized bed reactor could be designed for the gas-solid hydroxyethylation of potato starch. This process was optimized and compared to the classical slurry process. In our novel reactor the reaction time can be reduced at least by a factor of 10 relative to the slurry process whereas the selectivity is also higher. Products with a high molar substitution can be obtained by this novel process without loss of the granular structure of the starch.