Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field

A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field. With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activity, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism. The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion. To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditions has been found to be ca. 200,000 Da. These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended periods of time. As an example, in a penicillin G acylase reactor, >75% enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the same period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism.     

Application of cross-linked enzyme aggregates of Bacillus badius penicillin G acylase for the production of 6-aminopenicillanic acid

AIMS: Optimization of 6-aminopenicillanic acid (6-APA) production using cross-linked enzyme aggregates (CLEA) of Bacillus badius penicillin G acylase (PAC). METHODS AND RESULTS: CLEA-PAC was prepared using purified/partially purified PAC with phenylacetic acid as active-site blocking agent and glutaraldehyde as cross-linker. Conversion of penicillin G to 6-APA by CLEA-PAC was optimized using response surface methodology (RSM) (central composite rotatable design) consisting of a three-factor-two-level pattern with 20 experimental runs. CONCLUSION: Nearly, 80% of immobilization yield was obtained when partially purified enzyme was used for the preparation of CLEA-PAC. Quantitative conversion of penicillin G to 6-APA was observed within 60 min and the CLEA-PAC was reusable for 20 repeated cycles with 100% retention of enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The faster conversion of penicillin G to 6-APA by CLEA-PAC and efficient reusability holds a strong potential for the industrial application.     

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization

In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.     

Simple strategy of reactivation of a partially inactivated penicillin g acylase biocatalyst in organic solvent and its impact on the synthesis of β‐lactam antibiotics

Some reactions of organic synthesis require to be performed in rather aggressive media, like organic solvents, that frequently impair enzyme operational stability to a considerable extent. We have studied the option of developing a reactivation strategy to increase biocatalyst lifespan under such conditions, under the hypothesis that organic solvent enzyme inactivation is a reversible process. Glyoxyl agarose immobilized penicillin G acylase and cross‐linked enzyme aggregates of the enzyme were considered as biocatalysts performing in dioxane medium. Reactivation strategy consisted in re‐incubation in aqueous medium of the partly inactivated biocatalysts in organic medium, best conditions of reactivation being studied with respect to dioxane concentration and level of enzyme inactivation attained prior to reactivation. Best results were obtained with glyoxyl agarose immobilized penicillin G acylase at all levels of residual activity studied, with reactivations up to 50%; for the case of a biocatalyst inactivated down to 75% of its initial activity, full recovery of enzyme activity was obtained after reactivation. The potential of this strategy was evaluated in the thermodynamically controlled synthesis of deacetoxycephalosporin G in a sequential batch reactor operation, where a 20% increase in the cumulative productivity was obtained by including an intermediate stage of reactivation after 50% inactivation. Biotechnol. Bioeng. 2009;103: 472–479. © 2009 Wiley Periodicals, Inc.     

Attempts to relate enzyme inactivation to degradation in vivo

Inactivation of liver cytosol proteins has been measured in vitro in the presence of various membranes and disulphides. Inactivation rates correlate with the known degradation rate constants of the enzymes in the intact liver. More extensive studies were carried out with glucose-6-phosphate dehydrogenase (G6PD) and phosphoenolpyruvate carboxykinase (PEPCK) using either cytosol as a source of these enzymes or alternatively highly purified preparations of each enzyme. All membranes purified from liver had a considerable capacity to inactivate the enzymes with higher activity found in the hepatocyte plasma membrane. Various lipid preparations or plasma membranes from other tissues were virtually ineffective. Inactivation was dependent on disulphides in the membranes as shown by the inhibition of activity if membranes were pretreated with thiols. Preliminary experiments of the fate of inactivated G6PD or PEPCK show binding to membranes and subsequent proteolysis. A model is proposed for the degradation of labile enzymes.     

An integrated scale-down plant for optimal recombinant enzyme production by Pichia pastoris

An integrated bioprocess was created in a scale-down production plant by developing a two-stage enzyme production process with Pichia pastoris, containing a cell-breeding reactor and a production reactor in combination with a three-stage downstream process. To harvest the secreted enzymes, a disc separator and a cross-flow microfiltration clear the broth from the cells. Purification with hydrophobic interaction chromatography removes other proteins, concentrates the product, and prepares the enzyme solution for lyophilization. Fully automated and broad observable multi-stage parallel process courses have been developed using industrial process control systems and at-line measurements for enzyme concentration and enzyme activity. Optimal process conditions were found by application of Design of Experiments (DoE) for the production process.     

Continuous production of maltotetraose using a dual immobilized enzyme system of maltotetraose-forming amylase and pullulanase

In order to produce a product with a high content of maltotetraose, dual-enzyme systems composed of immobilized maltotetraose-forming amylase (G(4)-forming amylase) and pullulanase were studied. The thermostability of individually immobilized enzymes was examined in continuous operation; studies revealed that the enzyme immobilized on "Chitopearl" was much more stable than that immobilized on Diaion HP-50. The effects of operating conditions on the stability of G(4) forming amylase immobilized on "Chitopearl" were examined to confirm that the apparent half-life data could be arranged using the immobilized enzyme stability factor, f(s). As for the dual immobilized enzyme system, six methods of usage were considered, with five yielding a 7-10% (w/w) higher content of maltotetraose product than the single-enzyme system. The effects of operating conditions on the maltotetraose production reaction were examined to confirm that the maltotetraose content of the products could be analyzed using the specific space velocity,SSV. In dual immobilized enzyme systems, pullulanase immobilized on the same carrier as the G(4)-forming amylase was found to be more stable than pullulanase immobilized on separate carriers. The effectiveness of using immobilized pullulanase along with the G(4)-forming amylase was confirmed from constant-conversion operations in which the maltotetraose content in the product was kept at 50% (w/w) in laboratory-scale experimentation.     

Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products

Inactivation of immobilized penicillin acylase has been studied in the presence of substrate (penicillin G) and products (phenylacetic acid and 6-aminopenicillanic acid), under the hypothesis that substances which interact with the enzyme molecule during catalysis will have an effect on enzyme stability. The kinetics of immobilized penicillin acylase inactivation was a multistage process, decay constants being evaluated for the free-enzyme and enzyme complexes, from whose values modulation factors were determined for the effectors in each enzyme complex at each stage. 6-Aminopenicillanic acid and penicillin G stabilized the enzyme in the first stage of decay. Modulation factors in that stage were 0.96 for penicillin G and 0.98 for 6-aminopenicillanic acid. Phenylacetic acid increased the rate of inactivation in both stages, modulating factors being -2.31 and -2.23, respectively. Modulation factors influence enzyme performance in a reactor and are useful parameters for a proper evaluation. (c) 1996 John Wiley & Sons, Inc.     

Production of galacto-oligosaccharides from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth.

The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae beta-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased approximately 25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40 degrees C and 48 days at 50 degrees C. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40 degrees C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported.     

Conversion of benzylpenicillin to 6-aminopenicillanic acid in a batch reactor and continuous feed stirred tank reactor using immobilized penicillin amidase

A rate equation has been derived to describe the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid by penicillin amidase. The integrated from of the rate equation has been shown to predict satisfactorily the progress of the reaction in a batch reactor using either soluble or immobilized penicillin amidase. The rate equation was also used to predict the performance of a continuous feed stirred tank reactor containing immobilized enzyme. There was good agreement with experimental measurements.     

Kinetic properties of gel-immobilized acid phosphatase in a tubular membrane reactor

Summary Acid phosphatase has been immobilized onto the internal surface of tubular ultrafiltration membranes by two different methods, namely copolymerization/gelation and co-gelation. Rate parameters for p-nitrophenyl phosphate hydrolysis by the enzyme in both gel-immobilization conditions have been determined and compared to the corresponding values obtained in previous work using a flat ultrafiltration membrane. Results indicate that the kinetic properties of the enzyme seems not substantially modified by the membrane geometry; however, for industrial purposes an enzyme reactor equipped with tubular membranes should be preferred.     

Enzyme recovery during gas/liquid two-phase flow microfiltration of enzyme/yeast mixtures

The effect of a gas/liquid two-phase flow on the recovery of an enzyme was evaluated and compared with standard crossflow operation when confronted with the microfiltration of a high-fouling yeast suspension. Ceramic tubular and flat sheet membranes were used. At constant feed concentration (permeate recycling) and transmembrane pressure, the results obtained with the tubular membrane were dependent on the two-phase flow pattern. In comparison with single-phase flow performances at the same liquid velocity, the enzyme transmission was maintained at a high level with a bubble flow pattern but it decreased by 70% with a slug flow, whatever the flow rate ratio. Identical results were obtained with flat sheet membranes: for the highest flow rate ratio, the enzyme transmission was reduced by 70% even though the permeate flux was improved by 240%. During diafiltration experiments with the tubular membrane, it was found that a bubble flow pattern led to a 13% higher enzyme recovery compared to single-phase flow conditions, whereas with a slug flow the enzyme recovery was strongly reduced. With bubble flow conditions, energy consumption was minimal, confirming that this flow pattern was the most suitable for enzyme recovery.     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

生淀粉糖化酶催化位点氨基酸及酶合成调控的初步研究

通过对Rhizopus OR-1UVN菌种所产生淀粉糖化酶在不同底物不同缓冲溶液条件下酶最适pH的测定,推测出该生淀粉糖化酶活力中心催化位点氨基酸是天冬氨酸(Asp)和谷氨酸(Glu)。实验证明5～50mg/mL浓度葡萄糖对生淀粉糖化酶没有抑制作用。分别以浓度<5mg/mL葡萄糖和淀粉为碳源的培养基进行不同碳源发酵实验,发现以淀粉为碳源的培养基Ⅰ发酵15h开始产生淀粉糖化酶,以葡萄糖为碳源的培养基Ⅱ发酵35h开始产酶(葡萄糖浓度<8mg/mL),而且前者菌体较后者少,由此可知葡萄糖对产酶有阻遏作用。实验还发现解阻遏熟淀粉糖化酶的葡萄糖浓度(15mg/mL)比生淀粉糖化酶的要高。由于葡萄糖的阻遏作用不发生在翻译水平,而发生在转录水平上,而且生淀粉糖化酶(G1)与熟淀粉糖化酶(G2)来自同一条DNA链,可以推测存在mRNA的拼接。通过以生淀粉为碳源的比较实验,发现生淀粉对生淀粉糖化酶形成的诱导作用可能主要是通过mRNA拼接的调节来实现的。     

Hyper production of enzyme penicillin amidase by locally isolated thermo-tolerantBacillus sp. MARC-0103 from rice starch in cheese whey

The present study concern with the extracellular production of penicillin amidase in a cost-effective cheese whey medium under submerged fermentation. ABacillus sp. MARC-0103 producing a high level of extra cellular penicillin G amidase was isolated from rice starch by heat shock method. The penicillin G amidase production in the strain was induced by phenyl acetic acid. The culture medium was optimized by using Plackett-Burman and central composite experimental designs for enhanced production of penicillin amidase. The factorial design indicated that the main factors that positively affect penicillin amidase production were casein hydro-lysate, CaCl2·2H2O, FeCI3·6H2O, Na2SO4 and cheese whey, whereas the presence of calcium carbonate and magnesium chloride in the medium had no effect on enzyme production. Phenyl acetic acid concentration and time of addition was found critical for enzyme pro duction. Enzyme production was enhanced very much by multiple addition of inducer. Other cultural condition such as pH, temperature, inoculum size and age were also optimized. More than two fold increase in enzyme production (40.7 U/ml/min) was observed under optimized cultural conditions. The molecular mass was estimated to be 40.0 kDa by SDS-PAGE.     

Novel reactive perstraction system applied to the hydrolysis of penicillin G

The activity of penicillin acylase has been studied in aqueous and organic solvents, as free enzyme as well as immobilized within the membrane of liquid-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely phenyl acetic acid (PAA). In order to overcome this inhibition a range of organic solvents were tested for use in in situ product recovery. Of these solvents dibutyl sebacate (DBS) was chosen due to the rapid extraction rate, the high logP and to facilitate capsule production. The extraction efficiency at pH 3.5 for PAA was >80% for phase ratios of >50% free solvent with partition coefficients of 8 and 0.7 for PAA and penicillin G (PenG), respectively, thereby showing that PAA could be selectively extracted at pH 3.5 and 25 degrees C. Liquid-core capsules containing DBS were shown to efficiently remove PAA selectively and the PAA could be effectively back-extracted and the capsules re-used in a three-stage process resulting in high product separation. Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity. Over 53.3% of the PAA formed could be recovered in the capsule core with a concentration over sevenfold higher than in the aqueous phase. Higher extraction efficiencies could be obtained by varying the substrate concentration and number of capsules. The enzyme immobilized on capsules could be stored for over 4 months at pH 8 and 4 degrees C with no loss of activity. Over 80% of the initial activity could be recovered over five repeated batch cycles of the bioconversion process. The importance of capsular perstraction and reactive capsular perstraction has been clearly demonstrated.     

重组毕赤酵母产青霉素G酰化酶的分批发酵动力学研究

构建了重组毕赤酵母产青霉素G酰化酶的分批发酵动力学模型。实验考察了分批发酵过程中甘油消耗、甲醇浓度、菌体浓度、溶氧、补料时间对青霉素G酰化酶活力的影响。应用Matlab软件,对菌体生长、基质消耗和产物生成方程进行最优参数估算和非线性拟合,得到相应的动力学模型。模型的计算值与实验值能较好地拟合,表明所建模型能较好反映重组毕赤酵母产青霉素G酰化酶的分批发酵过程。     

Properties of microfiltration membranes: the effects of adsorption and shear on the recovery of an enzyme

An experimental study of the interaction of the enzyme yeast alcohol dehydrogenase (YADH) with microfiltration membranes has been carried out. Most measurements were made with capillary pore inorganic membranes (Anopore) with some comparative measurements being made with polymeric membranes of low protein affinity (Durapore). It has been shown that the prolonged exposure of the enzyme to the inorganic membrane under low-shear conditions (slow recycle) resulted in a loss of enzyme activity. Under filtration conditions, the membrane permeation rate decreased continuously with time. This decrease could be quantified using the standard blocking filtration law, which describes a decrease in pore volume due to deposition of enzyme on the walls of the pore. No significant loss in activity of permeating enzyme occurred under solution conditions where the enzyme was stable. However, a significant loss of such activity occurred under solution conditions where the enzyme was slightly unstable. The experiments indicate that the likely mechanism for activity loss is a membrane/enzyme interaction resulting from a shear induced deformation of the enzyme structure. Two conclusions of practical importance are drawn from the work. (c) 1992 John Wiley & Sons, Inc.     

Symmetric enzyme distribution in asymmetric UF polysulfone membranes

Invertase as well as as amyloglucosidase were immobilized within asymmetyric ultrafiltration membranes that were prepared from polysulfone or homogeneously modified polysulfone. The chemical modification was carried out by sulfonation and halomethylation. This additional change of the surface properties of the capillaries within the membrane offers the possibilities for various types of enzyme fixation, namely adsorption, charge interactions, or covalent bonding. By variation of the immobilization conditions the distribution of the enzyme could be adjusted over the membrane's cross section. At a distinct enzyme concentration in the loading solution a homogeneous enzyme distribution within the membrane could be verified. This was shown by diffusion experiments. Under ultrafiltration conditions using a solution that contains membrane-impermeable macromolecules as well as a membrane-permeable solute like saccharose the residence time within the membrane was increased due to gel formation atop the membrane yet the kinetic was no affected. The nonpermeable soluble starch was not reacted by the amyloglucosidase membrane, indicating that the skin layer was free of enzymes. (c) 1994 John Wiley & Sons, Inc.     

A scalable membrane gradostat reactor for enzyme production using Phanerochaete chrysosporium

This is the first demonstration of process scale-up of a membrane gradostat reactor for continuous enzyme production using Phanerochaete chrysosporium ME446. The fungus was immobilised by reverse filtration on to externally unskinned, ultrafiltration capillary membranes and then nutrient gradients were induced across the biofilm. A 10-fold scale-up from a single capillary bioreactor to a 2.4 l multi-capillary unit resulted in a 7-fold increase in enzyme productivity with a peak at 209 U l^–1 d^–1. Subsequent scale effects on the spore distribution, continuous manganese peroxidase production profile and biofilm development are discussed.