Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.     

Enzymatic cellulose hydrolysis in an attrition bioreactor combined with an aqueous two-phase system

Cellulose was hydrolyzed in the attrition bioreactor (ABR) with enzyme recycling by employing an aqueous two-phase system (composed of dextran and polyethylene glycol) and an ultrafiltration unit. The ABR combines wet ball milling and enzymatic hydrolysis in one process step. The cellulase enzymes were more stable in the two-phase system than in the normal buffer solution. With the initial substrate concentration (Solka Floe BW200) of 40 g/L and intermittent addition of cellulose, sugar was semicontinuously produced at dilution rates of 0.06 h(-1) and productivities of 2.1 g/L h, which is approximately a 10-fold increase of the previously reported values performed in a regular stirred reactor with an aqueous two-phase system. The conversion of the substrate was 86%.     

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。     

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. I. Partition of cellulases from Trichoderma reesei

The partitioning of endo-beta-glucanase, exo-beta-glucanase, and beta-glucosidase from Trichoderma reesei QM 9414 in aqueous two-phase systems has been studied with the object of designing a phase system for continuous bioconversion of cellulose. The partitioning of the enzymes in two-phase systems composed of various water soluble polymeric compounds were studied. Systems based on dextran and polyethylene glycol (PEG) were optimal for one-sidedly partitioning the enzymes to the bottom phase. The influence of polymer molecular weights, polymer concentration, ionic composition of the medium, pH, temperature, and adsorption of the enzymes to cellulose on the enzyme partition coefficients (K) were studied. By combining the effects of polymer molecular weight and adsorption to cellulose, K values could be reduced for endo-beta-glucanase to 0.02 and for beta-glucosidase to 0.005 at 20 degrees C in a phase system of Dextran 40-PEG 40000 in the presence of excess cellulose, At 50 degrees C, K values were increased by a factor of two. In a phase system based on inexpensive crude dextran and PEG, the partition coefficient for endo-beta-glucanase was 0.16 and for beta-glucosidase was 0.14 at 20 degrees C with excess cellulose present.     

Strategies for the design and operation of enzymatic reactors for the degradation of highly and poorly soluble recalcitrant compounds

The presence of recalcitrant compounds in both wastewaters and soils is an important environmental problem. Oxidative enzymes from white-rot fungi have been successfully utilised for the in vitro degradation of xenobiotics, such as the azo dye Orange II and the polycyclic aromatic hydrocarbon anthracene (compounds with high and low solubilities, respectively). Two different reactor configurations are proposed: (i) an enzymatic membrane reactor for the treatment of soluble compounds, consisting of a continuous stirred tank reactor coupled to an ultrafiltration membrane to facilitate the retention and recycling of enzyme; and (ii) a two-phase enzymatic reactor for the degradation of poorly soluble compounds, consisting of an immiscible solvent, which contains the contaminant at high concentrations, and the aqueous phase containing the enzyme and cofactors involved in the catalytic cycle. In this paper, factors affecting the design and operation of both systems are discussed, and experimental results concerning the efficiency and stability of the processes are presented.     

Liquid-liquid partitioning of some enzymes, especially phosphofuctokinase, from Saccharomyces cerevisiae at subzero temperature

The effects of low temperature (−18°C) on the stability and partitioning of some glycolytic enzymes within an aqueous two-phase system were studied. The enzymes were phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase present in a crude extract of bakers' yeast. The partitioning of pure phosphofructokinase, isolated from bakers' yeast, was also examined. The two-phase systems were composed of water, poly(ethylene glycol), dextran, and ethylene glycol and buffer. The influence on the partitioning of the presence of ethylene glycol, phenylmethylsulfonyl fluoride and poly(ethylene glycol)-bound Cibacron Blue F3G-A was investigated at −18, 0 and (in some cases) 20°C. The presence of ethylene glycol, phase polymers and low temperature stabilized all three enzyme activities. Cibacron Blue, an affinity ligand for phosphofructokinase, increased its partitioning into the upper phase with decreasing temperature. Depending on the conditions, various amounts of the enzymes were recovered at the interface, also in systems not containing ethylene glycol. The implications of the observed effects on the use of aqueous two-phase systems for the extraction and fractionation of proteins are discussed.     

In vitro assessment of the enzymatic degradation of several starch based biomaterials

The susceptibility of starch-based biomaterials to enzymatic degradation by amylolytic enzymes (glucoamylase and alpha-amylase) was investigated by means of incubating the materials with a buffer solution, containing enzymes at different concentrations and combinations, at 37 degrees C for 6 weeks. Two polymeric blends of corn starch with poly(ethylene-vinyl alcohol) copolymer and poly(epsilon-caprolactone), designated by SEVA-C and SPCL, respectively, were studied. The material degradation was characterized by gravimetry measurements, tensile mechanical testing, scanning electron microscopy (SEM), and Fourrier transform infrared-attenuated total reflectance (FTIR-ATR). The degradation liquors were analyzed for determination of reducing sugars, as a result of enzyme activity, and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used to identify the degradation products. All of the analysis performed showed that starch polymeric blends are susceptible to enzymatic degradation, as detected by increased weight loss and reducing sugars in solution. alpha-Amylase caused significant changes on the overall mechanical properties of the materials, with a decrease of about 65% and 58% being observed in the moduli for SEVA-C and SPCL, respectively, when compared with the control (samples incubated in buffer only). SEM analysis detected the presence of fractures and pores at the material's surface as a result of starch degradation by amylolytic enzymes. FTIR spectra confirmed a decrease on the band corresponding to glycosidic linkage (-C-O-C-) of starch after incubation of the materials with alpha-amylase. In contrast, the incubation of the polymers in buffer only, did not cause significant changes on the material's properties and morphology. Comparing the two materials, SEVA-C exhibited a higher degradability, which is related to the physicochemical structure of the materials and also to the fact that the starch concentration is higher in SEVA-C. The identification of the degradation products by HPAEC-PAD revealed that glucose was the major product of the enzymatic degradation of starch-based polymers. alpha-Amylase, as expected, is the key enzyme involved in the starch degradation, contributing to major changes on the physicochemical properties of the materials. Nevertheless, it was also found that starch-based polymers can also be degraded by other amylolytic enzymes but in a smaller extent.     

Association of rabbit muscle glycolytic enzymes with filamentous actin. A counter-current distribution study at high ionic strength

The association between purified glycolytic enzymes and filamentous actin from rabbit muscle has been studied by counter-current distribution. The co-distribution of a glycolytic enzyme and filamentous actin leads to a significant change in the counter-current distribution profile of the enzyme whereas that of actin is unaffected. The changes in the distribution profiles clearly demonstrated that all glycolytic enzymes studied, though to different extents, bind to filamentous actin. The aqueous two-phase system used for the studies contained dextran, poly(ethyleneglycol) and 150 millimolal potassium phosphate buffer, pH 7.0. Since the ionic strength of the two-phase system is determined mainly by the buffer, the glycolytic enzymes are evidently able to associate with filamentous actin, at least in the presence of neutral polymers, at ionic strengths comparable to or higher than those assumed to prevail in vivo.     

Bioconversion of hydrophobic compounds in a continuous closed-gas-loop bioreactor: feasibility assessment and epoxide production

Microorganisms can be used as catalysts to produce organic compounds in a highly chemo-, regio- and enantioselective manner, and whole cells do not require the costly addition of cofactors for redox reactions. However, bioconversions are slow compared to alternative chemical reactions, and the biocatalyst works at its best in an aqueous medium, while the transformations of interest frequently involve compounds with a low-aqueous solubility and that are toxic to microorganisms. This results in low-volumetric productivity in classical bioreactors. The Continuous Closed-Gas-Loop Bioreactor is described here-a reactor system with high productivity, but without the problems associated with two-phase systems, such as an emulsified product stream and phase toxicity. Its working principle is to recirculate a gas phase through a bioreaction compartment and a saturator/absorber module where the product accumulates as a clear organic solution. A wide range of bioconversions should be possible in this set-up, and proof of concept was established for the epoxidation of 1,7-octadiene to (R)-1,2-epoxyoct-7-ene by a native strain of Pseudomonas oleovorans. This reaction represents a group of terminal alkene epoxidations where the bioconversion substrate does not support growth of the microorganism. Practical results at a 5l-scale are presented for this bioconversion for both batch and continuous operation with respect to the aqueous phase, showing continuous stable epoxidation at productivities >14 micromol min(-1) L(-1) (U L(-1)). The results confirm that the metabolism does not allow a simple optimization strategy, because growth and biotransformation substrates compete for the same enzyme sites, and conversely growth on a substrate using this very enzyme system is necessary for longterm bioconversion. Integrated removal of the CO(2) formed via the liquid overflow was estimated from theory and verified in experimental work.     

Studies on enzymatic continuous production of cyclodextrins in an ultrafiltration membrane bioreactor

Studies on simultaneous hydrolysis of starch and synthesis of cyclodextrins by Thermo-aerobacter cyclodextrin glucosyltransferase were conducted in an ultrafiltration membrane bioreactor, allowing enzyme recovery and reduction of product inhibition. The influence of various reaction parameters like starch concentration, enzyme dosage and residence time on cyclodextrin composition was tested. A comparison of batch and continuous cyclodextrin production indicates that employing an ultrafiltration membrane bioreactor increases process efficiency.     

Application of ultrafiltration for enzyme retention during continuous enzymatic reaction

In most enzymatic reactions, batch or continuous, separation of the enzyme for reuse is difficult if not impossible. A process will be presented in which an Ultrafiltration membrane serves to separate the reaction products from the enzyme and the substrate. In this manner the enzyme may be retained and re-used. Furthermore, under these conditions, the enzyme need only be present in catalytic amounts regardless of the amount of product produced. Under proper operating conditions and proper ultrafiltration membrane selection, a pure solution of α-amylase from Bacillus subtilis may be retained with no loss in enzyme activity over a test period of 30 hr after steadystate has been achieved. In the presence of substrate, the membrane support and ultrafiltration cell serve as the reaction vessel for the hydrolysis of starch. The substrate is continuously pumped into the cell under constant ultrafiltration pressure. The di-, oligo-, and polysaccharides formed from the enzyme reaction then either pass through the membrane as products or are retained. The molecular weight distribution of the products is dependent on the nominal molecular weight cut-off of the membrane, absolute ultrafiltration pressure, enzyme-to-substrate ratio, temperature, and residence time of the substrate in the reactor. In addition to the partial hydrolysis of starch by α-amylase, some preliminary findings on the complete hydrolysis of starch by glucoamylase will also be presented. In these latter studies, the substrate may be completely hydrolyzed to glucose units.     

A novel alpha-glucosidase from the moss Scopelophila cataractae

Scopelophila cataractae is a rare moss that grows on copper-containing soils. S. cataractae protonema was grown on basal MS medium containing copper. A starch-degrading activity was detected in homogenates of the protonema, after successive extraction with phosphate buffer and buffer containing 3 M LiCl. Buffer-soluble extract (BS) and LiCl-soluble extract (LS) readily hydrolyzed amylopectin to liberate only glucose, which shows that alpha-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The K(m) value of BS for maltose was 0.427. The K(m) value of BS for malto-oligosaccharide decreased with an increase in the molecular mass of the substrate. The value for maltohexaose was 0.106, which is about four-fold lower than that for maltose. BS was divided into two fractions of alpha-glucosidase (BS-1 and BS-2) by isoelectric focusing. The isoelectric points of these two enzymes were determined to be 4.36 (BS-1) and 5.25 (BS-2) by analytical gel electrofocusing. The two enzymes readily hydrolyzed malto-oligosaccharides. The two enzymes also hydrolyzed amylose, amylopectin and soluble starch at a rate similar to that with maltose. The two enzymes readily hydrolyzed panose to liberate glucose and maltose (1 : 1), and the K(m) value of BS for panose was similar to that for maltotriose, whereas the enzymes hydrolyzed isomaltose only weakly. With regard to substrate specificity, the two enzymes in BS are novel alpha-glucosidases. The two enzymes also hydrolyzed beta-limit dextrin, which has many alpha-1,6-glucosidic linkages near the non-reducing ends, more strongly than maltose, which shows that they do not need a debranching enzyme for starch digestion. The starch-degrading activity of BS was not inhibited by p-chloromercuribenzoic acid or alpha-amylase inhibitor. When amylopectin was treated with BS and LS in phosphate buffer, pH 6.0, glucose, but not glucose-1-phosphate, was detected, showing that the extracts did not contain phosphorylase but did contain an alpha-glucosidase. These results show that alpha-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.     

Enzyme purification using temperature-induced phase formation.

A new type of aqueous two-phase system composed of an ethylene oxide and propylene oxide random co-polymer, UCON 50-HB-5100, as the upper phase polymer and either dextran or hydroxypropyl starch as the lower phase polymer has been characterized and used to purify 3-phosphoglycerate kinase (EC 2.7.2.3) and hexokinase (EC 2.7.1.1) from bakers' yeast. The UCON 50-HB-5100 polymer has a cloud point of 55 degrees C at which temperature it phase separates from water. This cloud point can be lowered to 40 degrees C by the addition of 0.2 M sodium sulfate salt. The low cloud point of this UCON polymer makes it possible to obtain the target enzymes in a water and buffer solution, and to recover and recycle the UCON 50-HB-5100 polymer. The phase diagrams for the systems UCON 50-HB-5100/Dextran T500 and UCON 50-HB-5100/hydroxypropyl starch have been determined. Yeast homogenate was first partitioned in a system composed of a top phase containing UCON 50-HB-5100 and a bottom phase containing either dextran or hydroxypropyl starch. The top phase containing the enzyme free of cell debris was removed and the temperature increased above the cloud point of the UCON until a new two phase system composed of water as the top phase and a concentrated liquid UCON 50-HB-5100 bottom phase was formed. The water phase containing the enzyme was removed and the bottom phase containing the UCON 50-HB-5100 could be recycled to perform a second extraction.     

Strategies for the design and operation of enzymatic reactors for the degradation of highly and poorly soluble recalcitrant compounds

The presence of recalcitrant compounds in both wastewaters and soils is an important environmental problem. Oxidative enzymes from white-rot fungi have been successfully utilised for the in vitro degradation of xenobiotics, such as the azo dye Orange II and the polycyclic aromatic hydrocarbon anthracene (compounds with high and low solubilities, respectively). Two different reactor configurations are proposed: (i) an enzymatic membrane reactor for the treatment of soluble compounds, consisting of a continuous stirred tank reactor coupled to an ultrafiltration membrane to facilitate the retention and recycling of enzyme; and (ii) a two-phase enzymatic reactor for the degradation of poorly soluble compounds, consisting of an immiscible solvent, which contains the contaminant at high concentrations, and the aqueous phase containing the enzyme and cofactors involved in the catalytic cycle. In this paper, factors affecting the design and operation of both systems are discussed, and experimental results concerning the efficiency and stability of the processes are presented.     

固定化青霉素酰化酶在光-pH敏感可回用两水相中裂解青霉素G为6-APA

两水相体系在发展中存在的关键问题是相体系回收困难.由于生产成本及降低污染的原因, 用过的相体系需要回收和重复使用.用环境敏感型溶解可逆聚合物形成可回用两水相体系是当前是为可行的回收方法。本文在光敏感可回用高聚物PNBC与pH敏感型可回用高聚物PADB形成的两水相体系中进行固定化青霉素酰化酶的相转移催化青霉素G产生6-APA的反应。在这个两水相体系中,通过优化,在1% NaCl 存在下,６－ＡＰＡ的分配系数可达5.78。催化动力学显示,达平衡的时间近7h,反应最高得率约85.3%（pH 7.8, 20℃）。较相近条件下的单水相反应得率提高近20%。在反应过程中,通过底物及产物的分配系数检测,发现底物分配系数变化不大,而产物6-APA及苯乙酸的分配系数发生很大变化,从而引起产物的得率变化。在两水相中,底物及产物主要分配在上相,固定化酶分配在下相,底物青霉素G进入下相经酶催化产生的6-APA及苯乙酸又转入上相,从而解除了青霉素酰化酶催化反应的底物及产物抑制作用,达到提高产物得率的效果。此外,采用固定化酶较固定化细胞效率高,占用下相体积小,较游离酶稳定性高,且完全单侧分配在下相。因此,在两水相中进行固定化酶的催化反应具有明显的优越性。形成两水相的高聚物PNBC通过488 nm 的激光照射或经滤光的450nm 光源照射得到回收;pH敏感型成相聚合物PADB可通等电点 4.1沉淀可实现循环利用,高聚物的回收率在95%-98%之间,按此回收率计算,聚合物可使用60次以上。     

Bioconversion with whole cell penicillin acylase in aqueous two-phase systems

The deacylation of Pen G was carried out by using recombinant E. coli in an aqueous two-phase system consisting of polyethylene glycol and potassium phosphate solution, which partitions the cells to the bottom phase and the products to the top phase. Bioconversion and product separation were carried out in the same reactor. Repeated batch conversion was employed ten times and enzymic activity showed only a slight decline. When pure enzyme was used for bioconversion in an aqueous two-phase system, the decline was fast and bioconversion using whole cell penicillin acylase was better than that obtained using the pure acylase.     

Extractive fermentation for improved production of endoglucanase by an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae using aqueous two-phase system

Extractive aqueous two-phase fermentation of endoglucanase, a key enzyme for the conversion of cellulosic substances to fermentable sugars, from an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae is a meaningful approach for better production and simple recovery of this enzyme. A phase composition of 6.5% (w/w) dextran and 7.5% (w/w) polyethylene glycol 6000, having a partition coefficient of 2.89 and 1.31 for endoglucanase from an intergeneric fusant of T. reesei/S. cerevisiae and T. reesei (WT) (being a control in this study), respectively, was chosen for extractive fermentation of the enzyme. Endoglucanase production is higher in medium containing polyethylene glycol (PEG) 6000 than in medium without PEG 6000. Comparative analysis of endoglucanase fermentation by fusant and T. reesei was carried out in shake culture and environment-controlled bioreactor conditions. The fusant produced 0.43U of endoglucanase (overall production: 0.34U) in the top phase of an aqueous two-phase system (ATPS), compared to 0.3U in medium without the phase system in shake culture. In a batch reactor, the endoglucanase level for the fusant in the top phase of ATPS was 0.49U (overall production: 0.40U), compared to 0.38U produced in medium without aqueous two-phase components. To corroborate this study, T. reesei produced 8.41U of endoglucanase (overall production: 5.96U) in the top phase of ATPS, compared to 7.18U in the medium without the phase system in shake culture. On the other hand, in a batch bioreactor, T. reesei produced 10.13U of endoglucanase (overall production: 6.90U) in the top phase of ATPS, compared to 8.56U of the enzyme in medium without aqueous two-phase components. The lower overall enzyme production by T. reesei in the two-phase system might be due to limitation in oxygen transfer to the dispersed phase where the enzyme is produced. A higher cell concentration and a reduced lag phase was obtained in ATPS, compared to a similar medium without phase forming polymers for both the intergeneric fusant of T. reesei/S. cerevisiae and T. reesei.     

Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction

A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35 degrees C at 5% phosphate, and increased linearly to 63 degrees C at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems.