An immobilized two-enzyme system for the activation of the lactoperoxidase antibacterial system in milk

Lactoperoxidase catalyzes the oxidation of thiocyanate by hydrogen peroxide and an intermediary product is formed with antibacterial properties. The components of this system, with the exception of hydrogen peroxide, are present in milk. H₂O₂ may be introduced by means of enzymatic, generation and thus make the system complete. A two-enzyme system consisting of β–galactosidase and glucose oxidase has been developed for this purpose. The coupled enzyme reaction is shown to work with high efficiency at the neutral pH of milk although the enzymes as such, particularly lactases suitable for immobilization, have optimal activities at much lower pH values. The results indicate that the lactoperoxidase system may in this way be employed to inactivate bacteria present in milk.     

Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase

Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.     

Concurrent saccharification/fermentation of enzymatic corn syrups in light beer production

The high degree of fermentability required in light beer production can be achieved by concurrent saccharification and fermentation of a wort containing an enzyme prepared corn syrup adjunct and glucoamylase. Traditional acid or acid-enzyme syrups used as adjuncts in regular beer production are not effective in a concurrent saccharification/fermentation process due to the presence of oligosaccharides that are resistant to the action of glucoamylase.     

Construction of an alpha-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae.

A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.     

A one-pot two-enzyme system on the production of high value-added D-allulose from Jerusalem artichoke tubers

A one-pot two-enzyme reaction system was developed to produce high-fructose syrup (HFS) containing healthy rare sugar d-allulose from Jerusalem artichoke (JA). Inulin in JA was converted through the cascade reaction of a novel exo-inulinase from Bacillus velezensis (BvInu) and D-allulose 3-epimerase from Ruminococcus sp. (RDAE). BvInu and RDAE were expressed in Bacillus subtilis and successfully secreted into the supernatant, which decrease the production cost and avoid enzyme purification. The optimal temperature and stability of extracellular BvInu significantly increased, thereby promoting the catalytic activity of the two enzymes for the cascade reaction in one pot. Inulin in JA powder was almost completely converted into monosaccharides in the treatment with the optimal ratio of BvInu : RDAE (80 : 40 U/g inulin) at 50 °C for 2 h. The ratio of D-glucose, D-fructose, and D-allulose in the product was approximately 1:3:1, and the yield on JA powder was 67%. The system exhibits potential high-valued application of non-grain crops on producing HFS with D-allulose.     

Preparation of an immobilized two-enzyme system, Beta-amylase--pullulanase, to an acrylic copolymer for the conversion of starch to maltose. III. Process kinetic studies on continuous reactors

Kinetic studies on the parameters influencing the potential industrial application of an immobilized two-enzyme system of β-amylase and pullulanase for conversion of starch to a product with high maltose content, have been performed. The apparent Michaelis constant, the apparent product inhibitor constant, and the activation energy have been determined for the immobilized preparation and compared to the values for the corresponding soluble enzyme system. The catalytic activity of the immobilized enzymes was studied in a plug-flow reactor and a continuous feed stirred tank reactor. Mathematical models for these reactors have been formulated and adapted to fit the experimental data. Comparisons of the reactor efficiencies were made and the conditions were found to be such as to favor the plug-flow reactor. Results on operational stability tests at different temperatures and substrate concentrations are given.     

Preparation of an immobilized two-enzyme system, beta-amylase-pullulanase, to an acrylic copolymer for the conversion of starch to copolymer for the conversion of starch to maltose. I. Preparation and stability of immobilized beta-amylase

β-Amylase (EC 3.2.1.2), obtained from barley, was chemically attached to a crosslinked copolymer of acrylamide-acrylic acid using a water-soluble carbodiimide. The derivative showed 23% β-amylase activity in relation to that of free enzyme with a coupling yield of 40% based on the amount of added β-amylase. In order to find optimal coupling conditions, the effect of pH and different carbodiimide concentrations was investigated. The enzymic activity associated with different β-amylase concentrations was further outlined. A slightly increased operational stability for the enzyme upon immobilization was observed. Markedly improved operational stability has been obtained by coupling in the presence of reduced glutathione of bovine serum albumin.     

Preparation of an immobilized two-enzyme system, beta-amylase-pullulanase, to an acrylic copolymer for the conversion of starch to maltose. II. Cocoupling of the enzymes and use in a packed bed column

β-Amylase (EC 3.2.1.2) and pullulanase (EC 3.2.1.9) have been covalently bound in a two-enzyme system to a crosslinked copolymer of acrylarmide-acrylic acid by using a water-soluble carbodiimide. The coupling yields based on the amounts of added β-amylase and pullulanase were 40% and 38%, respectively, with residual enzymic activities of 22% and 32% of those of free enzymes. A markedly increased operational stability was observed for the immobilized two-enzyme system compared to the free enzymes in solution. In order to find optimal operational conditions the influence of different pH values and temperatures on the conversion process was investigated. The action of the immobilized β-amylase-pullulanase derivative on partially hydrolyzed starch (DE 3.4–10.7) in a packed bed column was studied. Analysis of the product was performed using gas-liquid chromatography.     

The separation of solids from the liquefied mash of cassava tuber and continuous saccharification by immobilized glucoamylase

Summary The solid material in liquefied mash of cassava tuber was very efficiently separated by a mixture of Trichoderma cellulase and Aspergillus niger pectinase. The solid content of the residue after the treatment and centrifugation decreased from 29.5% to 7.0%. The transparent digested solution from cassava tuber after centrifugation was continuously saccharified by glucoamylase immobilized in a gel which was prepared using polyvinylpyrrolidone and -ray irradiation. The addition of 50 ppm of sulfite ion completely prevented microbial contamination during the 18 days of operation. The final DE (dextrose equivalent), glucose content and disaccharide content in the hydrolyzate were 98, 94.4 and 3.3%, respectively.     

Substrate-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments

The ragi alpha-amylase/trypsin bifunctional inhibitor (RATI) from Indian finger millet, Ragi (Eleucine coracana Gaertneri), represents a new class of cereal inhibitor family. It exhibits a completely new motif of trypsin inhibitory site and is not found in any known trypsin inhibitor structures. The alpha-amylase inhibitory site resides at the N-terminal region. These two sites are independent of each other and the inhibitor forms a ternary (1:1:1) complex with trypsin and alpha-amylase. The trypsin inhibition follows a simple competitive inhibition obeying the canonical serine protease inhibitor mechanism. However, the alpha-amylase inhibition kinetics is a complex one if larger (> or =7 glucose units) substrate is used. While a complete inhibition of trypsin activity can be achieved, the inhibition of amylase is not complete even at very high molar concentration. We have isolated the N-terminal fragment (10 amino acids long) by CNBr hydrolysis of RATI. This fragment shows a simple competitive inhibition of alpha-amylase activity. We have also synthesized various peptides homologous to the N-terminal sequence of RATI. These peptides also show a normal competitive inhibition of alpha-amylase with varying potencies. It has also been shown that RATI binds to the larger substrates of alpha-amylase. In light of these observations, we have reexamined the binding of proteinaceous inhibitors to alpha-amylase and its implications on the mechanism and kinetics of inhibition.     

Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor

Summary Glucoamylase production by Aureobasidium pollulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads. Offprint requests to: M. Petruccioli     

Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.     

Preparation of a nuclease-free alpha-amylase and its use in DNA purification

Summary A thermostable alpha-amylase fromBacillus licheniformis when heated to 80°C for 20 min retains >90% of its activity but loses all DNAase activity. This allows the amylase to be used to purify yeast DNA.     

Efficient production of (R)-(−)-mandelic acid in biphasic system by immobilized recombinant E. coli

The recombinant Escherichia coli M15/BCJ2315 which harbored a mandelonitrilase from Burkholderia cenocepacia J2315 was immobilized via catecholic chitosan and functionalized with magnetism by iron oxide nanoparticles. The immobilized cells showed high activity recovery, enhanced stability and good operability in the enantioselective hydrolysis of mandelonitrile to (R)-(−)-mandelic acid. Furthermore, the immobilized cells were reused up to 15 cycles without any activity loss in completely hydrolyzing mandelonitrile (100 mM) within 1 h in aqueous solution. The ethyl acetate–water biphasic system was built and optimized. Under the optimal conditions, as high as 1 M mandelonitrile could be hydrolyzed within 4 h with a final yield and ee value of 99% and 95%, respectively. Moreover, the successive hydrolysis of mandelonitrile was performed by repeated use of the immobilized cells for 6 batches, giving a final productivity (g L⁻¹ h⁻¹) and relative production (g g⁻¹) of 40.9 and 38.9, respectively.     

An investigation of the properties of glucoamylase immobilized on glass beads involving 5-diazosalicylic acid bonded to a titanium (IV) oxide film

Optimum conditions have been determined for the immobilization of glucoamylase on glass involving diazotized 5-aminosalicylic acid bonded to a deposited imperfectly crystallized film of TiO₂. The changes in the kinetic and thermodynamic characteristics of the enzyme on immobilization have been determined. There are significant differences in the behaviour of the immobilized enzyme towards its substrates, maltose and starch. The apparent K_m for starch increased on immobilization whereas that for maltose decreased. The pH optimum for the immobilized preparation showed a shift to acid pH relative to that of the free enzyme.     

Horizontal reactor for the continuous production of ethanol by yeasts immobilized in pectin

Summary Yeast cells (Saccharomyces cerevisiae) were immobilized in pectin gel, incubated 12 h at 30°C and then used for the continuous production of ethanol employing a wedge-shaped horizontal reactor and sugar cane molasses as the carbon source. Under steady state conditions the mean residence time was 1.6 h and the volumetric productivity 40 g EtOH/hl. The gas evolved was easily released. Successive batch incubation in a synthetic medium substantially restored the fermentative capacity of the beads already used in the continuous assay.Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, México D.F.Member of the Scientific Researcher's Career of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

APSIM模型对华北平原小麦-玉米连作系统的适用性

利用中国科学院禹城试验站1999—2001年大田试验及2002—2003年水分池处理数据进行APSIM模型参数的调试及验证,检验其对华北地区冬小麦-夏玉米连作系统的适用性.模型调试和验证结果表明:禹城1999—2000年大田试验的作物叶面积指数、生物量和土壤含水量模拟结果的平均误差分别为27.61%、24.59%和7.68%,2000—2001年分别为32.65%、35.95%和10.26%;2002—2003年高水分处理的作物叶面积指数和生物量模拟结果的平均误差分别为26.65%和14.52%,低水分处理分别为23.91%和27.93%.叶面积指数、生物量的模拟值和实测值拟合较好,除2000—2001年叶面积指数的决定系数为0.78外,其他处理均大于0.85.表明APSIM模型在模拟华北地区小麦-玉米连作系统的作物生物量和土壤水分方面具有较好的准确性,对叶面积指数模拟误差稍大.