Characterisation of biocatalysts immobilised in niobium—a new inorganic solid support

A new support for enzyme immobilisation, an alloy of niobium ore and graphite, was tested with: invertase from baker's yeast and inulinase from Aspergillus niger. The efficiency of immobilisation was about 30% for invertase and 72% for inulinase. The maximum activities values were observed for both enzymes at: pH 4.5, 50°C and 500 g/L of sucrose. The hydrolysis of inulin (5% w/v) by the free inulinase and invertase presented specific productivity of reducing sugars lower after immobilisation, about 15 and 1.5 times, respectively; with the hydrolysis of sucrose (50% w/v) the reductions observed were of 14 and 3.5 times, respectively. © 2012 Canadian Society for Chemical Engineering     

Modeling and Application of UF-Stirred Cell Reactors in Hydrolysis of Saccharides

Hydrolysis of sucrose by invertase as a typical example of saccharides enzymatic hydrolysis has been conducted in a UF-stirred cell reactor. A model has been derived by which the effect of some pertinent variables, such as membrane pore size, applied pressure, substrate concentration and stirring speed, on the performance capability of such systems may be predicted. A close correlation has been found between the experimental results obtained under a wide range of operating conditions and those calculated from the model.     

Invertase immobilised on activated clay: Properties and kinetic study

Invertase was immobilised on activated ball clay. The immobilised enzyme showed maximum activity at pH 4.8–6. The thermal deactivation was significant at temperatures above 30°C. The kinetics of sucrose hydrolysis with free invertase showed substrate and product inhibition. Immobilised enzyme kinetics followed the same mechanisms. Modelling curves were plotted to explain the experimental results. The thermal deactivation followed first order kinetics. The characteristics of thermal deactivation of immobilised enzyme were studied in the range 25–50°C.     

Diffusion and adsorption phenomena in an immobilized enzyme reactor using adsorbed polymer for attachment of the enzyme in porous alumina particles

Experiments were performed to study the effects of an immobilized enzyme on the adsorption capacity and on the diffusion in a porous solid. The enzyme invertase was immobilized by covalently binding it to a polymer matrix adsorbed in the pores of alumina. The enzyme was found to hydrolyse sucrose to glucose plus fructose with an initial activity of 60 μmol/min-g alumina. Data on the long-term stability showed a 14% decrease in initial activity in 22 days. The adsorption of glucose, fructose, or sucrose alone on porous alumina and on porous alumina containing polymer and immobilized invertase was investigated. The presence of immobilized invertase and polymer matrices decreased the adsorption capacity by about 75%. The presence of immobilized invertase on the adsorbed polymer matrices also raised the experimental tortuosity factor for diffusion in the pores from 2.0 for plain alumina to 2.75 for invertase immobilized on alumina. The decrease of particle void fraction, the enzyme blockage effect, and an increase in pore restriction effect were used to explain the increase of the tortuosity factor.     

SIMULTANEOUS ENZYMATIC HYDROLYSIS OF CELLULOSE AND SUCROSE

The kinetics of simultaneous hydrolysis of cellulose and sucrose by cellutase and invertase were investigated. Sucrose and/or invertasc was found to exert no inhibition or activation effect on the cellulose/cellulase system. The reverse was also true. The use of a hollow-fibre ultrafiltration cartridge to allow continuous removal of products was found to enhance the hydrolysis.     

SIMULTANEOUS ENZYMATIC HYDROLYSIS OF CELLULOSE AND SUCROSE

The kinetics of simultaneous hydrolysis of cellulose and sucrose by cellutase and invertase were investigated. Sucrose and/or invertasc was found to exert no inhibition or activation effect on the cellulose/cellulase system. The reverse was also true. The use of a hollow-fibre ultrafiltration cartridge to allow continuous removal of products was found to enhance the hydrolysis.     

Entrap-immobilization of invertase on composite gel fiber of cellulose acetate and zirconium alkoxide by sol–gel process

We prepared a composite gel fiber by the gel formation of cellulose acetate and zirconium tetra-n-butoxide. Gel fiber is stable in common solvents, phosphate solution, and electrolyte solution. Invertase was entrap-immobilized on the gel fiber. The immobilization was easily performed under the mild conditions. The apparent Michaelis constant (K_m) and maximum reaction velocity (V_max) were estimated from Eadie–Hofstee plot for immobilized invertase. The K_m of immobilized invertase was larger than that of native invertase, while the opposite tendency was observed for the V_max. The activity for the immobilized invertase became higher with increasing fiber diameter. It indicates that the hydrolysis of sucrose occurs in the neighborhood of the fiber surface. The thermal stability of the immobilized invertase was higher than those of its native counterpart. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 2084–2088, 2001     

Continuous hydrolysis of olive oil by lipase in microporous hydrophobic membrane bioreactor

Continuous hydrolysis of olive oil byCandida cylindracea’s lipase was studied in a microporous hydrophobic membrane bioreactor. Olive oil and buffer solution, fed continuously through two compartments partitioned by membrane, caused reaction at the interface of lipase-adsorbed membrane and buffer solution. Fatty acid was obtained in a single phase without being mixed with components of other phases. At all mean residence times, countercurrent flow mode was superior to cocurrent one. The lipase was adsorbed onto the membrane, and its adsorption was suggested to be partially specific from the experiments with enzymes having various levels of purity. The percent hydrolysis depended hyperbolically on the interfacial enzyme concentration. The hydrolysis seemed to be limited by diffusion of fat or fatty acid through the micropores of the membrane at higher interfacial enzyme concentrations. The lipase was stabilized significantly by glycerol added to the buffer solution. Satisfactory performance of the membrane bioreactor was obtained in a longterm continuous operation which lasted for 24 days by feeding buffer-glycerol (18.0%) solution over the adsorbed lipase. The operational half-life of the adsorbed enzyme was 15 days at 40 C.     

Transport of Eu3+ through a Bis(2-ethylhexyl)-phosphoric Acid,n-Dodecane Solid Supported Liquid Membrane

Abstract

The coupled transport of Eu³⁺ and H⁺ ions through a solid supported liquid membrane consisting of a porous polypropylene film immobilizing an HDEHP solution in n-dodecane has been studied as a function of the membrane area, stirring speed of the aqueous solutions, membrane composition, and acidity of the feed solution. The experimental results are in agreement with predictions derived from a theoretical permeability coefficient equation which assumes that membrane diffusion and aqueous film diffusion are the only rate-controlling factors.     

The enzymatic hydrolysis and tissue oxidation of fatty acid esters of sucrose

Summary Various preparations of sucrose fatty acid esters were hydrolyzed by wheat germ or pancreatic lipase, pancreatin, pancreatic juice, α-amylase, invertase, or liver homogenates to yield sucrose and free fatty acids as products. The greatest activity was observed with the liver homogenate. None of the enzymes studied cleaved the glycosidic linkage as indicated by the lack of appearance of reducing groups and by paper chromatography of the products. The greatest hydrolysis by pancreatic lipase was observed with sucrose esters having a greater preponderance of unsaturated fatty acids, namely, sucrose trilinoleate, sucrose dilinoleate, sucrose tetralinoleate, and “Sequol 260” (69% unsaturated fatty acids). Sodium taurocholate was required for hydrolysis by pancreatic lipase but not by wheat germ lipase. Sucrose ester was inhibitory to the hydrolysis of triolein by all lipolytic preparations. Tetra-ethyl pyrophosphate and cupric ions were not inhibitory to the hydrolysis of sucrose ester. Sucrose fatty acid esters supported respiration by rat liver homogenates and to a much lesser extent by rat intestinal mucosa. The rate of oxidation was greater than that observed with sucrose and the corresponding fatty acid. The greatest activity was observed with esters of fatty acids having a greater preponderance of unsaturated fatty acids, namely, “Sequol 260,” sucrose di-, tri-, and tetralinoleate. This work was carried out under U.S.P.H.S. Grant No. A-1808 and Sugar Research Foundation Grant No. 109.     

Fibrous support for immobilization of enzymes

A poly(vinyl alcohol) (PVA) fiber support incorporating various aminoacetal functional groups has been developed for immobilizing enzymes. The aminated PVA fiber seems to adsorb enzymes with electrostatic force of attraction; thus the immobilization procedure is simple. By the use of this fiber having immobilized enzymes, the reaction between enzymes and substrates is nearly independent of the size of subtstrates. This newly developed type of fiber, which is formed by a mass superfine fibers (SFF), each measuring 1 μm or less in diameter, permits much more increased surface area than the conventional enzyme immobilization supports. Our studies of the properties of the fiber for immobilization of enzymes show the following results: (1) SFF has a greater ability for the immobilization of invertase than ordinary fibers; (2) dimethyl-aminated SFF has the best performance for the immobilization of invertase. From these results, it is concluded that the dimethyl-aminated SFF is an excellent support for the immobilization of invertase.     

Sucrose hydrolysis by invertase immobilized on Duolite A-568 employing a packed-bed reactor

The conversion of sucrose to a highly concentrated commercial syrup by immobilized invertase by combining the processes of adsorption and cross-linking using Duolite A-568 as the carrier was studied. Central Composite Design (CCD) was used to assess the effect of glutaraldehyde concentration and cross-linking reaction time on immobilized enzyme activity throughout the hydrolysis of sucrose in a batch reactor. Cross-linking optimization allowed us to find the optimum conditions for activity with a glutaraldehyde concentration of 0.6?g?·?L^?1 and a cross-linking time of 6?h. The temperature and pH that maximized the activity of the immobilized biocatalyst in the cross-linking process were 50°C and 4.0, respectively. Cross-linking allows the biocatalyst to be active at higher temperatures and lower pH. High-sucrose conversions to invert sugar using a continuous fixed-bed reactor were obtained. The immobilized biocatalyst also demonstrated greater thermal stability at low temperatures.     

Development of polyion complex hollow fiber membrane for separation of water-ethanol mixtures

For the purpose of separating water-ethanol mixtures by the use of the pervaporation technique, a new hollow fiber membrane composed of the polyion complex (PIC) consisting of polyacrylic acid and polycation was developed. To obtain this a new technique was developed instead of coating. That is, polyacrylonitrile membrane was partially hydrolysed to introduce carboxylic groups and then converted to the polyion complex.

The hydrolysis reaction and the formation of a separating layer were analyzed by the use of IR, SEM and EDS. Through these observations it was found that carboxylic groups were uniformly formed by immersing the membrane into a NaOH aqueous solution. Moreover, the pores of less than 0.1 μm in diameter on the inner surface of the membrane disappeared with the proceeding of a hydrolysis reaction. The carboxylic layer was changed into the separating layer by immersing the membrane into an ionene aqueous solution. By varying the hydrolysis conditions and optimizing the post-treatment, the preparation of a hollow fiber having PIC as a separating layer became possible. An excellent module with an effective membrane area of 6 m² showed more than a 5,000 separation factor and approximately 400 g/m²·h in permeation rate for 95 wt% ethanol solution at 60°C.     

Production and properties of Aspergillus niger inulinase

An inulinase producing Aspergillus niger strain was isolated from Compositae rhizosphere soil samples. High inulinase levels were produced on a corn steep liquor (CSL)-maltose medium in the absence of inulin at 28°C within 110 h of fermentation. Media based on CSL-sucrose yielded high cell-bound inulinase activity; on inulin-based media the enzyme was mainly extracellularly produced. Both crude extra-and intracellular inulinase preparations displayed identical pH and temperature optima with maximal activity at pH 4.3–4.4 and at 55–56°C. These properties are favourable in view of large scale inulinase application for pure fructose production. High operation temperatures would avoid microbial contamination of reactors and would allow the use of high inulin-substrate concentrations, a limiting factor in obtaining high conversion ratios. The remarkably low pH optimum prevents colour formation and undesirable chemical side reactions. An advantageous low ratio of invertase to inulinase activity (S/I value) of 0.85 was found for the crude extracellular enzyme preparation. Crude inulin (chicory) extracts are hydrolysed faster than pure inulin. Apart from inulin (100% hydrolysis), sucrose (45%) and raffinose (20%) are also hydrolysed, and no liberation of oligomers or of sucrose from inulin was observed. These facts indicate that the A. niger enzyme is an exo-acting inulinase. The above characteristics make this A. niger inulinase an industrially attractive enzyme for the preparation of pure fructose from inulin-containing agricultural crops.     

固定化β-葡萄糖苷酶双相体系中水解大豆异黄酮

采用共价交联的方法将β-葡萄糖苷酶固定到球形壳聚糖上,并对固定化酶的性质进行表征,得出固定化酶的最佳反应条件：pH=5,温度40℃。据此用乙酸乙酯和pH=5缓冲溶液的双相体系,在40℃条件下水解大豆异黄酮。与游离酶相比,固定化酶在此双相体系中起到了稳定酶的作用;与单相体系相比,双相体系中产物的产率显著提高,反应速率也更快,且能有效去除粗品大豆异黄酮的异味。对于30%左右级别的大豆异黄酮,水解的两个主要水解产物（大豆苷元和染料木素）的产率都能达到70%。     

利用乳液酶膜反应器拆分萘普生甲酯实验研究

利用乳液酶膜反应器进行外消旋萘普生甲酯的水解反应,以制备光学纯对映体(S)-萘普生,反应同时从膜透过侧收集产物,实现反应分离一体化。实验研究了固定酶前后膜传质阻力和反应过程的水相跨膜通量,考察了透过侧的产物浓度、反应器的转化率、产量和对映体选择性。结果表明:固定化酶引起的传质阻力远大于膜本身的阻力;透过侧的产物浓度与水相渗透通量密切相关,通量较低时,产物浓度较高;固定化酶的初始反应速率为3.660μmol/(h.g),为自由酶的20倍以上,固定化酶的对映体过剩值为99%—100%,远高于自由酶的选择性,表明该反应体系为脂肪酶催化拆分反应提供了良好环境。     

Assay-increment and solubilisation of α-glucosidase and invertase in disruption of brewers' yeast

A variety of disruption techniques were used on brewers' yeast with subsequent estimation of the additional (assay-increment) α-glucosidase and invertase made accessible to p-nitrophenylglucoside (PNPG) and sucrose, respectively, in the total disruptate. The extent of actual solubilisation of these two enzymes in the 0.75 M potassium phosphate buffer (pH 6.5) present, was also measured. Extensive release (i.e. solubilisation) of α-glucosidase was achieved using ultrasonication or ethyl acetate autolysis, and here differential release of α-glucosidase over invertase was large. Extensive release of both α-glucosidase and invertase was obtained with snail gut enzyme and toluene (papain) autolysis, while air drying released some invertase but destroyed 80% of the α-glucosidase. The assay-increment, over intact yeast values, for α-glucosidase in total disruptate, which is due to increased accessibility of PNPG to this enzyme, was high for salt autolysis and toluene (papain) autolysis but was low for the Mickle shaker and the Potter-Elvehjem homogeniser. No increment was observed using the X-press, although solubilisation of α-glucosidase was moderately good. Increment, over intact yeast values, for invertase assay in total disruptate was highest with ultra-sonication and air drying, but was nevertheless generally low owing to almost complete availability of this cell wall enzyme for assay—but not for isolation—in intact yeast.     

固定化Aspergillus sp.吸附Cr(Ⅵ)的实验研究

采用包埋法固定Aspergillus sp．菌体制备成生物吸附剂,对水中的Cr^6 进行吸附实验。通过比较几种不同的固定化裁体,发现5％PVA 1％SA为最佳固定裁体。还研究了pH值、Cr^6 初始浓度、菌体浓度、吸附时间、共存离子等因素对Cr^6 吸附的影响,并对Aspergillus sp．菌体去除Cr^6 的吸附机理进行了探讨。     

Photoinduced biohydrogen production from saccharide mixture with the photosensitization of Mg chlorophyll a from green plant

Here, we developed a photoinduced biohydrogen production system coupling saccharide hydrolysis with hydrogen production. A mixture of three different saccharides (sucrose, maltose, and cellobiose) was hydrolyzed by three enzymes (invertase, glucoamylase, and cellulase) and glucose dehydrogenase (GDH). Hydrogen production was performed with colloidal platinum as a catalyst, using visible light-induced photosensitization of Mg chlorophyll a (Mg Chl a). Irradiation of the sample solution containing the saccharides, enzymes, GDH, NAD⁺, Mg Chl a, methyl viologen (MV²⁺), and colloidal platinum resulted in continuous hydrogen production. Approximately 2.9 μmol hydrogen was produced after 2 h of irradiation, and the total yield was approximately 8.8%.     

仿生膜吸附-絮凝组合固定化脂肪酶及其应用

研究了猪胰脂肪酶的仿生膜、吸附-絮凝组合固定化新工艺,考察了该组合固定化脂肪酶体系用于橄榄油水解的性能。最终试验结果表明：该组合固定化脂肪酶水解反应体系的反应转化率达到54.5％,固定化酶质量回收率86,7％,酶活回收率66．7％,远高于单一固定化方法。