海因酶法耦合原位分离技术制备N-氨甲酰-D-苯丙氨酸

利用自行筛选的海因酶高产菌株Burkholderia cepecia njut01发酵后,经过硫酸铵分级沉淀、phenyl sepharose FF、DEAE sepharose FF等纯化步骤,得到初步纯化的D-海因酶;利用碳二亚胺法建立了一种基于EAH sepharose 4B的D-海因酶共价固定化方法,酶活回收率达到79.44%,固定化酶经100d 25个批次转化后仍可保持63.2％的初始酶活。将固定化酶填充为固定床反应器,考察了不同条件下固定床与固定床耦合离子交换原位分离（in situ product removal,ISPR）两种反应体系在转化过程中pH值变化及转化率的差异。结果表明,耦合离子交换原位分离技术可大幅度提高D,L-苄基海因转化率,经24h转化,N-氨甲酰-D-苯丙氨酸转化收率最高达62.725%,较采用单一固定床酶转化体系的转化率提高了89.3%。     

酶解反应与膜分离耦合连续制备酪蛋白磷酸肽

采用酶解反应与膜分离耦合新工艺连续水解全酪蛋白制备酪蛋白磷酸肽(CPPs)。考察了超滤膜对胰蛋白酶及底物溶液的截留效果;研究了初始底物质量浓度、初始酶质量浓度、反应体积、膜渗透通量等参数对反应器性能和反应转化率的影响规律;利用高效凝胶排阻色谱系统(HPSEC)对酶解产物进行检测分析;建立了酶膜反应器连续水解动力学模型,并对间歇与连续酶解过程进行比较分析,证明反应-分离耦合技术可使酶解效率及蛋白酶利用率大幅提高,并使产物得到调控与富集,为CPPs的酶法制备提供了一种更为有效的方法。     

Potential of Integrated Semi-Continuous Enzymatic Synthesis and Filtration Processes for Efficiency Enhancement

Galacto-oligosaccharides (GOS), increasingly popular prebiotics, are synthesized by enzymatic conversion of lactose. Among others, the total production costs are significantly influenced by the costly enzyme. Therefore, it was investigated if the reuse and full recovery of the enzyme is feasible, followed by the development of a semi-continuous process in order to maintain a consistent high GOS yield. As a preliminary step, the enzyme-catalyzed reaction was recorded within the permissible operating parameters. It was successfully shown that steady high GOS yields can be synthesized semi-continuously within a filtration plant functioning as an enzymatic membrane reactor.     

Enzymatic Hydrolysis of Cassava Starch into Maltose Syrup in a Continuous Membrane Reactor

This study deals with the use of a membrane reactor for the enzymatic conversion of cassava starch to maltose. The enzymes used were Maltogenase and Promozyme (Novo Nordisk). Maltogenase activity was unaffected after a 5 h incubation period at 65°C, but Promozyme was markedly heat-unstable even at 37°C. Batch hydrolysis of liquefied cassava starch (30% w/w) by Maltogenase and Promozyme resulted in a maximum degree of starch conversion to maltose of 72% (≈254 g dm⁻³ maltose). The conversion degree fell by 11% when no debranching enzyme was used. The residence time distribution of the ultrafiltration reactor (UFR) was that of an ideal continuously stirred tank reactor. Rejection of Maltogenase by Carbosep M4 membranes (MWCO: 50 kDa) was not total. The overall enzyme activity loss after a 5 h diafiltration period was 28%, however about half this loss appeared to be due to enzyme denaturation inside the reactor. During saccharification trials conducted in the UFR at a starch concentration of 30% (w/w), severe membrane fouling occurred. The average permeate fluxes obtained were 14 and 23 dm³ h⁻¹ m⁻² at constant transmembrane pressures of 100 and 200 kPa respectively. When the reactor was operated at a space-time of 4·2 h, the degree of starch conversion to maltose in the permeate rapidly stabilized around 55–56%. © 1997 SCI.     

膜反应器中辅酶再生体系的构建与研究进展

在膜反应器中基于膜介质支持构建辅酶再生的酶催化体系并实现酶促反应的连续进行是辅酶依赖型生物酶工业化应用的一个有效技术手段。本文总结了辅酶再生的意义与辅酶再生体系在膜反应器中的构建情况,并提出了在这过程中所需注意的问题及解决思路。     

Micro-kinetic model of fructo-oligosaccharide synthesis for prebiotic products

A new micro-kinetic model of the enzyme-catalyzed synthesis of fructo-oligosaccharides (FOS) was developed. A commercial enzyme mixture Pectinex® Ultra SP-L derived from Aspergillus aculeatus was used. A variety of initial enzyme concentrations (1–5 vol%) and sucrose concentrations (400–600 g/L) were experimentally investigated and included in kinetic modeling. Several variations of kinetic mechanisms and corresponding models have been examined. A hybrid genetic algorithm was used to predict the kinetic parameters simultaneously for all experimental data. The best fitting model has been adopted, and with an average error of 13.34%, it describes the experimental data very well. The influence of initial concentrations on the conversion of sucrose and production of FOS is being carefully investigated. It was shown that the initial sucrose concentration significantly affects the highest level of FOS concentration, but the enzyme concentration controls the time at which maximum is reached as well as the rate of FOS decomposition.     

Effect of enzymatic pretreatment on acid fermentation of food waste

Although food waste is a valuable carbon source for biological nutrient removal systems with low organic wastewater because of high C/N and C/P ratios, it must be pretreated to promote the hydrolysis of particulates, which is considered as a rate‐limiting step. This study investigated the effects of enzymatic pretreatment on hydrolytic solubilization of food waste with commercial enzyme. Both acidification efficiency and volatile fatty acid (VFA) production potential of enzymatically pretreated food waste were examined under controlled laboratory conditions. Experimental results indicated that protease exhibited the highest VSS reduction rate among three types of enzymes: carbohydrase, protease and lipase. A mixed enzyme treatment showed better reduction efficiency than a single enzyme treatment, and the highest volatile suspended solids (VSS) reduction was observed at an enzyme mixture ratio of 1:2:1 with carbohydrase:protease:lipase, respectively. It has been noted that pretreatment resulted in both maximum VFA production and the highest VFA content of soluble chemical oxygen demand at an enzyme mixture dosage of 0.1% (v/v). VFA production at this dosage revealed a 3.3 times higher rate than that of no‐enzyme added fermenter. The dominant VFAs were n‐butyrate followed by acetate. Copyright © 2006 Society of Chemical Industry     

Preparation and characterization of PSSA/PVA catalytic membrane for biodiesel production

Poly(styrene sulfonic acid) (PSSA)/Poly(vinyl alcohol) (PVA) blend membranes prepared by the solution casting were employed as heterogeneous acid catalysts for biodiesel production from acidic oil obtained from waste cooking oil (WCO). The membranes were annealed at different temperature in order to enhance their stability. The structure and properties of the membranes were investigated by means of Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TG), X-ray diffraction (XRD). It is found that the crosslinking structure among PVA and PSSA chains formed when the thermal treatment temperature was higher than 80 °C. The retention of PSSA in the blend membranes in the methanol/water solvent was markedly increased from 50% to 85% with the increase of the annealing temperature from room temperature (for the untreated membrane) to 150 °C due to the formation of the crosslinking structure. The results of esterification of acidic oil show that the conversion was slightly improve with the PVA content in the membrane at a fixed PSSA content. The thickness of the catalytic membrane had no significant effect on the conversion in the end. The membrane annealed at 120 °C exhibited the best catalytic performance among the membranes, with a stable conversion of 80% with the runs.     

Performance of Perstractive Enzyme Reactor for Synthesis of Aspartame Precursor

A perstractive enzyme reactor was used for the synthesis of N-(benzyloxycarbonyl)-L -aspartyl-L -phenylalanine methyl ester (ZAPM), the precursor of the artificial sweetener, aspartame. The synthesis of ZAPM in the reactor proceeded by an enzymatic reaction between N-(benzyloxycarbonyl)-L -aspartic acid (ZA) and L -phenylalanine methyl ester (PM) in the aqueous phase. The synthesized ZAPM in the aqueous phase was mainly extracted into the organic phase, therefore, the concentration of ZAPM in the aqueous phase could be kept low. As a result, high conversion of ZAPM was obtained with this system. The partition coefficients of substances in the aqueous/butyl acetate biphasic system, the mass transfer coefficients of substances through the membrane and the enzymatic kinetics of ZAPM synthesis were determined experimentally. The reaction model which was based on the material mass balance equations was discussed to estimate the performance of the perstractive enzyme reactor system. The calculation values using the model and the experimental data showed good agreement with the concentration changes of the substances in the system. © 1997 SCI     

Continuous phenol hydroxylation over ultrafine TS-1 in a side-stream ceramic membrane reactor

A side-stream ceramic membrane reactor system was developed that can facilitate the in situ separation of ultrafine catalysts from the reaction mixture and make the production process continuous. Continuous hydroxylation of phenol to dihydroxybenzene over ultrafine titanium silicalites-1 (TS-1) was taken as a model reaction to evaluate the feasibility and performance of the membrane reactor system. The effects of membrane pore size and operation conditions (residence time, temperature, catalyst concentration, phenol/H₂O₂ molar ratio) on the performance of the reactor system were examined via single factor experiments. We demonstrated that the membrane pore size and operation conditions greatly affect the conversion, selectivity and filtration resistance. The phenol conversion and dihydroxybenzene selectivity remain stable at about 11% and 95% in a 20-h continuous run, respectively.     

Effects of lyophilization on catalytic properties of immobilized fructosyltransferase from Rhodotorula sp. LEB-V10

The extracellular fructosyltransferase (FTase) from Rhodotorula sp. LEB-V10 was immobilized on particles of niobium–graphite alloy and freeze dried (lyophilized), with and without additives. Twelve additives commonly applied as cryoprotectants were selected and evaluated both individually and in formulation; the biocatalyst was then studied according to its catalytic properties. Lyophilization with or without additives did not significantly affect the immobilized enzyme. After a period of 6 months, reductions in the initial enzymatic activity of about 7 and 4% were observed for the lyophilized enzyme when using 50 and 200 mM sodium acetate buffers, respectively. CMC, sorbitol, inositol and trehalose as single additives (all at 2.5%, w/v) in 100 mM sodium acetate buffer were capable to preserve the enzymatic activity after 6 months. However, formulations with more than one additive resulted in 36–14% less enzymatic activity after 6 months. After lyophilization, FOS synthesis features changed positively, by increasing the FOS yield from a non-lyophilizated yield of 58–68% with lyophilization. FOS composition changed as well, with 1^F-fructofuranosyl-nystose (GF₄) content increasing to 61% with lyophilization, which is 76 times higher than with the non-lyophilized enzyme.     

High efficiency bioethanol production from OPEFB using pilot pretreatment reactor

BACKGROUND: Current ethanol production processes using crops such as corn and sugar cane are well established. However, the utilization of cheaper biomasses such as lignocellulose could make bioethanol more competitive with fossil fuels while avoiding the ethical concerns associated with using potential food resources. RESULTS: Oil palm empty fruit bunches (OPEFB), a lignocellulosic biomass, was pretreated using NaOH to produce bioethanol. The pretreatment and enzymatic hydrolysis conditions were evaluated by response surface methodology (RSM). The optimal conditions were found to be 127.64 °C, 22.08 min, and 2.89 mol L^?1 for temperature, reaction time, and NaOH concentration, respectively. Regarding enzymatic digestibility, 50 FPU g^?1 cellulose of cellulase was selected as the test concentration, resulting in a total glucose conversion rate (TGCR) of 86.37% using the Changhae Ethanol Multi Explosion (CHEMEX) facility. Fermentation of pretreated OPEFB using Saccharomyces cerevisiae resulted in an ethanol concentration of 48.54 g L^?1 at 20% (w/v) pretreated biomass loading, along with simultaneous saccharification and fermentation (SSF) processes. Overall, 410.48 g of ethanol were produced from 3 kg of raw OPEFB in a single run, using the CHEMEX_50 L reactor. CONCLUSION: The results presented here constitute a significant contribution to the production of bioethanol from OPEFB. Copyright © 2011 Society of Chemical Industry     

ENZYMATIC CATALYSIS USING ASYMMETRIC HOLLOW FIBER MEMBRANES

An enzyme immobilization technique employing enzyme sequestration within the porous support regions of asymmetric hollow fiber membranes is described and experimentally evaluated. Reactor conversion data over a wide range of operating conditions agrees well with predictions obtained from a mathematical model developed previously. β-galactosidase immobilized by this technique was found to retain 100% activity for 60 hours of continuous reactor operation, and for 140 days when stored at 3°C. The effects on reactor performance of (1) enzyme adsorption by the membrane, and (2) axial redistribution of enzyme accompanying radial flow of fluid through the fiber wall, have been evaluated; neither process significantly alters conversion kinetics or efficiency for the substrate/enzyme system investigated.     

Modelling of combined mass transfer-kinetic effects in an enzyme membrane reactor system with a direct approach to the identification of intrinsic rate parameters

A methodology for identifying non-linear parameters in a generalised Briggs-Haldane rate expression for immobilised enzyme membrane flow reactors has been developed for the intermediate case where mutually rate-controlling processes of mass transfer and enzymatic reaction prevail. The technological significance of the estimation procedure lies in the ability of the algorithm to handle parameter identification in non-linear, two-point boundary value problems which are prevalent in both immobilised enzyme and non-enzymic heterogeneous catalysed reactor systems. The methodology was successfully implemented on an experimental level using quasi-steady-state conversion data for the model system of urea/collagen-urease in a spiral-wound, biocatalytic reactor module. By using a novel procedure of pepsin pretreatment of the collagen host membrane before enzyme impregnation, followed by glutaraldehyde crosslinking, it was possible to enhance the activity and stability of the immobilised enzyme complex to relatively high levels where interphase and intraphase diffusional limitations mutually controlled the rate of reaction. Information on the coefficient of mass transfer at the interface between solid and fluid phases was also obtained as part of the estimation procedure.     

The use of hybrid anaerobic solid–liquid (HASL) system for the treatment of lipid‐containing food waste

The hybrid anaerobic solid–liquid (HASL) system was a modified two‐phase anaerobic digester developed for bioconversion of food waste. The aim of this study was to estimate the feasibility of the HASL system for the treatment of food waste with a high content of lipids. The presence of lipids in food waste increased the energy value of nutrients but could inhibit growth of methanogens. The positive effect of lipids on the performance of anaerobic digestion dominated when the contents of lipids were in the range from 20 to 30% of total solids of food waste. Lipid contents of 40% diminished the production of volatile fatty acids in the acidogenic reactor as well as biogas production and the concentration of total bacteria and methanogens in the methanogenic reactor. Therefore, the HASL system can be used for the treatment of lipid‐containing food wastes if the lipid content is below 40% of total solids. Copyright © 2005 Society of Chemical Industry     

反应器操作方式对酶动力学拆分立体选择性的影响

描述了在批式反应器和连续流搅拌反应器（ＣＳＴＲ）中酶动力学拆分对映异构体的不同之处，从宏观反应器平衡角度，推导出了在ＣＳＴＲ反应器中不同于在批式反应器中的一定酶立体选择性（Ｅ）下，底物或产物的对映体过量值与反应的转化率之间关系的定量关系式。并通过商品脂肪酶及芽胞杆菌Ｅ－５３脂肪酶催化的萘普生甲酯的不对称水解反应得到了证实。分别在批式反应器和ＣＳＴＲ反应器中进行萘普生的酶法拆分，在一定转化率下，批式     

Comparisons of existing pretreatment,saccharification, and fermentation processes for butanol production from agricultural residues

Some of the most recent, relevant, industrial and academic contributions made in the field of butanol production are reviewed here. The focus on butanol is due to the growing demand for non‐fossil biofuels. In addition, butanol can be mixed with fossil fuels or can be used alone, allowing an alternative to gasoline. Butanol can be synthesised biologically using sugars extracted from biomass such as agricultural waste. This agricultural waste must be pretreated before it is suitable for sugar extraction. Following this stage, enzymatic hydrolysis is employed, before performing fermentation using microorganisms. This article summarises some of the economical methods such as simultaneous saccharification and fermentation (SSF). Different pretreatment and saccharification processes were compared. Acid pretreatment and saccharification achieved the highest sugar concentrations from wheat straw. Monoethanolamine pretreatment achieved highest sugars from hardwood. Comparisons and analysis of different types of fermentation processes illustrated that immobilised reactor provided the best butanol rate of production. Integration of fermentation with product removal process improved butanol production in immobilised reactor. Gas stripping method was illustrated to be the product removal process. © 2011 Canadian Society for Chemical Engineering     

操作参数对余热回收甲醇水蒸气重整制氢过程的影响

以模拟汽车尾气供热的甲醇水蒸气重整（MSR）制氢反应为研究对象,设计了集余热加热与MSR制氢反应于一体的肋式微反应器,考察了反应器进口热风速度、温度,反应物进口速度、温度、水醇比及顺逆流情况对MSR制氢过程的影响。计算结果表明,逆流、水醇比1.3、热风进口速度1.1 m/s、温度773 K、反应物进口速度0.1 m/s、温度493 K为该反应过程的最佳工况参数,此时甲醇转化率为99.4%,模拟汽车尾气余热的热效率为28%,反应器出口氢气的体积分数为69.6%。研究结果对开展余热综合利用及发动机尾气重整制氢掺氢燃烧的研究有借鉴意义。     

Use of membrane separation in enzymatic hydrolysis of waste paper

A three-stage process containing phosphoric acid pretreatment, enzymatic hydrolysis, and membrane filtration was performed on waste paper as a lignocellulosic material. In the first two stages, the effect of phosphoric acid concentration, enzyme loading, hydrolysis time, and substrate concentration on the amount of products was investigated. At the third stage using a proper membrane, the effect of substrate concentration and transmembrane pressure (TMP) on yield of the reducing sugars was studied. The novelty of the present study was to demonstrate the application of ultrafiltration membrane on the enzymatic hydrolysis process of waste paper. The reducing sugars concentration was determined by using the 3,5-dinitrosalicylic acid (DNS) reagent method. According to the results, a value of 0.5% was determined as the optimum concentration for phosphoric acid in the pretreatment stage. The reducing sugars yield was obtained as 67.4% in this concentration. Moreover, for the enzymatic hydrolysis of waste paper, the suitable amounts of cellulase enzyme loading and hydrolysis time were determined as 50 mg/g substrate and 48 h, respectively. In the filtration stage, increase of substrate concentration and decrease of TMP resulted in higher rejection of the reducing sugars. The experimental results revealed that the highest rejection was 19.2% at TMP of 3 bar and substrate concentration of 100 g/L.     

Galacto-oligosaccharide production with immobilized β-galactosidase in a packed-bed reactor vs. free β-galactosidase in a batch reactor

We report here that the usage of immobilized enzyme in a continuous packed bed reactor (PBR) can be a good alternative for GOS production instead of the traditional use of free enzyme in a batch reactor. The carbohydrate composition of the product of the PBR with immobilized enzyme was comparable to that of the batch reactor with free enzyme. The stability of the immobilized enzyme at a lactose concentration of 38% (w/v) and at 50 °C was very high: the half-life time of the immobilized enzyme was approximately 90 days. The enzymatic productivity of GOS production using immobilized enzyme in a PBR can be more than six times higher than that of GOS production with free enzyme in a batch reactor. Besides, when aiming for an equal volumetric productivity to the batch process in designing a PBR, the volume of the PBR can be much smaller than that of the batch reactor, depending on the enzyme dosage and the run time of a single batch.