Production of structured lipids by lipase-catalysed interesterification in an ultrafiltration membrane reactor

The application of membranes to the enzymatic production of structured lipids has been investigated using an ultrafiltration membrane reactor for a reaction between medium chain triacylglycerols (MCT) and n-3 polyunsaturated fatty acids from fish oil. Lipozyme IM was used as the biocatalyst. The incorporation of polyunsaturated fatty acids into MCT was increased by about 15% over 80 h by simultaneous separation of the released medium chain fatty acids compared to control.     

Lipase-catalyzed selective synthesis of monolauroyl maltose using continuous stirred tank reactor

Monolauroyl maltose was synthesized by an immobilized lipase that catalyzed condensation of maltose and lauric acid in acetone using a batch reactor or a continuous stirred tank reactor. Mono- and di-lauroyl maltoses were identified by FT-IR, ¹H NMR, ¹³C NMR and MS. Monolauroyl maltose was selectively synthesized in a continuous stirred tank reactor and no diester was detected. The highest concentration of monolauroyl maltose at 28 mmol/l was obtained in 250 ml acetone when maltose was added at 4 g/d and the molar ratio of lauric acid to maltose was fixed at 4:1 at a flow rate of 0.15 ml/min for both influx and effluent without supplement of fresh molecular sieve.     

Continuous enzymatic production of lactobionic acid using glucose-fructose oxidoreductase in an ultrafiltration membrane reactor

Glucose-fructose oxidoreductase from Zymomonas mobilis catalyzed the oxidation of various aldose sugars to the corresponding aldonic acids. The enzyme was used for the selective and high-yield conversion of lactose to lactobionic acid in batch, fed-batch and continous reaction mode. A productivity of 110 g L d was obtained in an ultrafiltration membrane reactor, operated for 70 h.     

Enzymatic hydrolysis of sodium caseinate in a continuous ultrafiltration reactor using an inorganic membrane

Continuous hydrolysis of sodium caseinate by alcalase was investigated in a recycle bioreactor coupled to an inorganic M5 membrane module. The effects of various substrate concentrations and the role of an ultrafiltration membrane on conversion rate were reported. Although a high level of conversion was obtained in the retentate side at a steady state, only part of the products formed was transmitted through the inorganic membrane. Degree of hydrolysis and product concentration in the reactor seem to be the main factors limiting product output during the continuous hydrolysis.     

Enzyme inactivation and stabilization studies in an ultrafiltration reactor

Summary An ultrafiltration membrane enzymatic reactor is used in connection with different reacting systems.The experimental conditions are such that the enzyme, which operates at fairly high concentration levels because of the concentration polarization phenomena taking place in the reactor, is still in soluble form.The analysis of the system unsteady-state response enables the identification of the mechanism of enzyme deactivation and the extraction of the kinetic parameters of both the deactivation and the main reaction.The stabilizing effect observed in connection with enzyme entrapment within an inert gel deposited onto the U.F. membrane active surface is also discussed.List of Symbols A U.F. membrane cross sectional area cm² - C_E Enzyme concentration mg/ml - C_EI Enzyme concentration at the active membrane surface mg/ml - C_E Mean enzyme concentration mg/ml - c _s ^o Substrate concentration in the feed m moles/ml - c _s ^u Substrate concentration in the outlet m moles/ml - D_E Enzyme diffusivity cm²/s - Km michaelis constant mM - k₂ Kinetic constant of the enzymatic reaction m moles/mg s - k_d Kinetic constant of the enzyme deactivation reaction s^–1 - N^o Initial amount of active enzyme mg - N(t) Active enzyme amount at reaction time t mg - Q Flow rate ml/s - r Rate of the main reaction m moles/ml s - t Reaction time s - t^* Reaction time at which product concentration in the outlet is within ± 2% of the steady-state value s - v Fluid velocity cm/s - V Cell volume ml - V_B Volume within which 99% of the enzyme fed is contained at the steady-state ml - V_S Volume within which 99% of the total substrate concentration drop occurs at the steady-state ml - x Distance upstream the membrane measured from the membrane surface cm     

Enzymatic hydrolysis of sodium-hydroxide-pretreated sallow in an ultrafiltration membrane reactor

An ultrafiltration membrane reactor was used to investigate the recovery of biocatalysts during enzymatic hydrolysis of pretreated sallow. Product inhibition could be eliminated by continuous removal of products through the ultrafiltration membrane, thus retaining the macromolecular substrate and enzymes. In this way, the degree of conversion was improved from 40% in a batch hydrolysis to 95% (within 20 h), and the initial hydrolysis rate was increased up to seven times. The recovery studies were focused on mechanical deactivation and irreversible adsorption on to the nonconvertible fraction of the substrate. Cellulase deactivation during mechanical agitation was not significant, and the loss of activity was attributed mainly to strong adsorption of the enzymes onto undigested material. This process was studied in semicontinuous hydrolyses, where fresh substrate was added intermittently. The amount of reducing sugars produced in this experiment was 25.7 g/g enzyme, compared to 4.7 g/g enzyme in a batch hydrolysis.     

Production of cyclodextrins in ultrafiltration membrane reactor containing cyclodextrin glycosyltransferase from Bacillus macerans

An enzyme reactor installed with ultrafiltration membrane was developed to produce alpha-, beta-, and gamma-cyclodextrins (CDs) from soluble starch by Bacillus macerans cyclodextrin glycosyltransferase (CGTase) tagged with 10 lysines at its C-terminus (CGTK10ase). Ultrafiltration membrane YM10 with 10,000 of molecular cutoff was chosen for membrane modification and CD production. A repeated-batch type of the enzyme reaction with free CGTK10ase resulted in a alpha-CD yield of 24.0 (+/-1.5)% and a productivity of 4.68 (+/-0.88) g/l-h, which were 7 times higher that those for CGTK10ase immobilized on modified YM10 membrane. Addition of 1- nonanol increased CD yields by 30% relative to the control, which might be due to prevention of the reversible hydrolysis of CDs.     

Continuous production of isomalto-oligosaccharides from maltose syrup by immobilized cells of permeabilizedAureobasidium pullulans

Continuous production of isomalto-oligosaccharides from maltose syrup by the permeabilized cells ofAureobasidium pullulans immobilized into calcium alginate gel was studied using a column reactor. The immobilized cell column maintained its full activity over 45 days when the reactor was operated at a velocity of 0.1 h^–1 at 50°C using 60%(w/v) maltose syrup as a substrate, and the maximum productivity achieved was around 60 g/1h.     

Production of biodiesel using a continuous gas-liquid reactor

A novel continuous reactor process has been developed for the production of biodiesel from fats and oils. The key feature of the process is its ability to operate continuously with a high reaction rate, potentially requiring less post reaction cleaning and product/reactant separation than currently established processes. This was achieved by atomising the heated oil/fat and then spraying it into a reaction chamber filled with methanol vapor in a counter current flow arrangement. This allows the continuous separation of product and the excess methanol stream in the reactor. The overall conversion based on a single cycle of this process has been between 50% and 96% of the feed stock materials. Conversions of 94-96% were achieved while operating with 5-7 g of sodium methoxide/L of methanol at methanol flow rate of 17.2 L/h and oil flow rate of 10 L/h. Additional variations in the reactant stoichiometry (i.e. reactant flow rates), catalyst type/concentration, and reaction temperature on the overall product conversion were investigated.     

Continuous hydrolysis of goat whey in an ultrafiltration reactor: generation of alpha-lactorphin

Bovine whey hydrolysate has been developed and applied to areas such as nutrition, culture media, and isolation of bioactive peptides. In order to produce such a type of hydrolysate, it is possible to use goat whey, which constitutes also a food processing by-product. Enzymatic hydrolysis of goat whey by pepsin was carried out in a continuous ultrafiltration reactor. The permeate contained peptide hydrolysate that was resolved by RPHPLC. Second order derivative spectroscopy, amino acid analysis, and mass spectrometry revealed the presence of a biologically active peptide called alpha-lactorphin. This constitutes preliminary information about goat whey enzymatic degradation for future applications.     

双酶偶联转化果糖制备含有稀少糖的混合糖液

酶转化法是功能性稀少糖生产的重要途径,但单一稀少糖转化酶的转化率普遍较低。文中提出构建双酶偶联转化系统提高转化效率的思路,即利用D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase,DPE)和L-鼠李糖异构酶(L-rhamnose isomerase,L-RhI)双酶偶联反应,催化D-果糖生成D-阿洛酮糖和D-阿洛糖等功能性稀少糖。DPE和L-RhI加酶量的比例为1∶10,其中DPE的浓度为0.05 mg/mL;转化反应的最佳温度为60℃,最适pH为9.0。当D-果糖浓度为2%时,反应10 h达到平衡,此时D-阿洛酮糖和D-阿洛糖的产量分别为5.12和2.04 g/L。利用文中提出的双酶偶联系统可以将果葡糖浆等富含果糖的低附加值原料转化为含有功能性稀少糖的高附加值混合糖液。     

Production of high fructo-oligosaccharide syrup with two enzyme system of fructosyltransferase and glucose oxidase

Summary A novel two enzyme system of fructosyltransferase and glucose oxidase to enhance the content of the net fructo—oligosaccharide (FOS) fractions in the industrial production of FOS syrup from sucrose was devised. The net FOS content in the commercial FOS syrup has been limited only to 55–60 % due to the accumulation of glucose which acts as a feedback inhibitor of the fructosyltransferase. By supplementing glucose oxidase to the conventional FOS reaction system, we could convert the glucose to gluconic acid readily separable from neutral sugars by simple ion exchange operation in the next step. The simultaneous removal of glucose was proved effective in proceeding the reaction by fructosyltransferase further by relieving the product inhibition caused by glucose. By this way, we could raise the net FOS content as high as 90 %.     

Mathematical modeling of maltose hydrolysis in different types of reactor

A commercial enzyme Dextrozyme was tested as catalyst for maltose hydrolysis at two different temperatures: 40 and 65 °C at pH 5.5. Its operational stability was studied in different reactor types: batch, repetitive batch, fed-batch and continuously operated enzyme membrane reactor. Dextrozyme was more active at 65 °C, but operational stability decay was observed during the prolonged use in the reactor at this temperature. The reactor efficiencies were compared according to the volumetric productivity, biocatalyst productivity and enzyme consumption. The best reactor type according to the volumetric productivity for maltose hydrolysis is batch and the best reactor type according to the biocatalyst productivity and enzyme consumption is continuously operated enzyme membrane reactor. The mathematical model developed for the maltose hydrolysis in the different reactors was validated by the experiments at both temperatures. The Michaelis–Menten kinetics describing maltose hydrolysis was used.     

Production of vitamin B12 in an upflow anaerobic filter continuous reactor using Acetobacterium sp.

The accumulation of biofilm by Acetobacterium sp. during continuous culture in an upflow anaerobic filter (UAF) growing on methanol-formate was the result of space velocity and inlet concentrations of substrate and Co⁺². To achieve good development of biofilm, a space velocity of 0.38 h^–1, inlet substrate concentrations of 125 mM of both methanol and formate, and Co⁺² at 0.16 mM were required. Cell productivities in the effluent of the UAF-reactor were about 6-fold higher than in chemostat cultures (0.20 g l^–1 h^–1 for UAF and 0.035 g l^–1 h^–1 for chemostat) (previous studies), and the maximum vitamin B₁₂ specific concentration was 5.1 mg g cell^–1.     

Production of high ethanol concentrations from glucose using a vertical rotating immobilized cell reactor of the bacterium zymomonas mobilis

Long-term continuous ethanol production of up to 80 g.l¹ with a volumetric ethanol productivity of 63 g. l^?1. h^?1 was maintained for more than 72 days using a Vertical Rotating Immobilized Cell Reactor of the bacterium Z. mobilis. Continuous production of higher ethanol concentration was unsuccessful due to an inhibition of cell growth by long exposure to high ethanol concentrations. However, ethanol concentration as high as 120g. l^?1 and volumetric ethanol productivity of 13g. l^?1. h^?1 were achieved in a repeated-batch fermentation system using the same bioreactor. By a simple washing operation at the end of each run, immobilized biomass could be effectively regenerated and used to carry out more than 10 successive fermentation cycles.     

Production of penicillins and cephalosporins in an immobilized enzyme reactor

Summary Four enzymes required for the biosynthesis of pencillins and cephalosporins by Streptomyces clavuligerus have been immobilized on an anion exchange resin. The capabilities of the system have been studied by circulation of reaction mixtures through the immobilized enzyme reactor. Within 30 min, all of the substrate -(l--aminoadipyl)-l-cysteinyl-d-valine is consumed and converted to a mixture of penicillins and cephalosporins. After 60 min the major antibiotic products are (iso)penicillin N and desacetylcephalosporin C. The activity of the immobilized enzyme reactor activity is stable to storage at temperatures below 4°C but activity is lost on repeated use.     

Production of bacterial cellulose by Acetobacter xylinumwith an air-lift reactor

Bacterial cellulose was produced by Acetobacter xylinum subsp. surcrofermentans BPR2001 in a 50 liter air-lift reactor using fructose as the main carbon source. When air was supplied, the production of the cellulose was only 2.3 g/l in 80 h but when O -fortified air was supplied, the cellulose concentration increased to 5.63 g/l in 28 h and the productivity of the cellulose in an air-lift reactor with O -fortified air supply was comparable to that in a mechanically agitated jar fermenter.     

β-d-Glucosidase stabilization in a polarized ultrafiltration membrane reactor

Cellobiose hydrolysis by β-d-glucosidase (β-d-glucoside glucohydrolase EC 3.2.1.21) can become the rate-limiting step in the hydrolysis of cellulosic wastes because of inhibition phenomena involving other enzymes of the cellulase complex. Enhancement of the overall rate can therefore be obtained by increasing the amount of β-d-glucosidase present in the reactor. Unfortunately, the thermal stability of β-d-glucosidase is rather poor compared to $\text{endo}\text{-(1 → 4)-β-}\text{d}\text{-}\text{glucanase}$ and cellobiohydrolase. A novel stabilization method is proposed that exploits the polarization phenomena that take place in an unstirred ultrafiltration membrane enzymatic reactor. As much as a 20-fold increase in half-life compared to the native enzyme is obtained by injecting small amounts of hydroxyethyl cellulose into the system. No reduction in enzyme activity levels is observed.