Continuous production of glucose oxidase with Aspergillus sp. under ultrasound waves

Glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4, GOD) was continuously released from Aspergillus sp. under mild ultrasound waves (20 kHz, 15 W). However, GOD was not released from the cells under normal conditions because of their thick wall. GOD production under ultrasound waves was optimum at pH 7.5 and 30°C and decreased with increasing ultrasonic frequency. Ultrasonic cavitation accelerated GOD release from the cells. Microscopic observation and determination of ATP and nucleic acids in the broth revealed that the mycelia were not broken during a 5 h reaction under ultrasound waves (15 W). About 10% of GOD produced in cells was released during the reaction.     

Effect of ultrasound on enzymatic acylation of konjac glucomannan

A comparative study was made of enzymatic acylation of konjac glucomannan with vinyl esters under ultrasonic irradiation and shaking in organic solvent tert-butanol. Among the 13 enzymes selected, Novozym 435 exhibited the highest acylation activity towards KGM whether under ultrasonic irradiation or shaking. The application of ultrasonic irradiation instead of shaking during the acylation led to improvement in the initial reaction rate, yield and degree of substitution of the modified KGM. Appropriate ultrasound power (100 W) and water activity (0.75) were found to accelerate enzymatic reaction. The acceleration effect of ultrasound on Novozym 435-catalyzed acylation decreased with an increase in the chain length of the acyl donors from C2 to C18. Moreover, the acylation of KGM in tert-butanol was proved to be a regioselective one, with C6-OH being acylated. Compared with shaking, ultrasound did not change regioselectivity of Novozym 435 in the acylation.     

Using acoustic cavitation to improve the bio-activity of activated sludge

This paper studied a new method to improve the microbial activity of the activated sludge for wastewater treatment. Concentrated sludge was sonicated in an extra chamber for short period and then returned to the activated sludge system. The results showed that the bio-activity of the activated sludge, expressed as oxygen utilization rate (OUR), could be enhanced by ultrasonic irradiation. Powerful ultrasound (in the magnitude of W/ml) was much more effective than weak ultrasound (in the magnitude of W/L) in stimulating the activated sludge, but too strong sonication (power density higher than 0.5 W/ml) disintegrated the sludge and thus decreased the sludge activity. Low frequency (25 kHz) was more effective than higher ones (80 kHz and 150 kHz), indicating that mechanical effects, instead of free radicals, were responsible for the bio-activity enhancement. The optimal sonication conditions were sound frequency of 25 kHz, power density of 0.2 W/ml and duration of 30s; under which the sludge OUR increased by 28%, the bio-mass growth rate increased by 12.5%, and the wastewater chemical oxygen demand (COD) and total nitrogen removal efficiency increased by 5-6%.     

响应面优化超声辅助法提取拐枣种子中二氢杨梅素的工艺

二氢杨梅素和杨梅素是拐枣种子中的重要成分。通过超声辅助法从拐枣种子中提取二氢杨梅素，采用单因素法考察乙醇体积分数、超声辐照功率、提取温度、液料比和超声辐照时间影响参数的基础上，并选用Box-Behnken响应面设计法分析建立了超声辐照功率、超声辐照时间和液料比的二次多项式模型，优化提取工艺。结果得到超声辅助法提取拐枣种子中二氢杨梅素的最佳工艺参数为：乙醇体积分数为60%、超声辐照功率140 W、超声辐照时间30 min、液料比20.5 mL·g^-1，提取温度40℃。在此最佳条件下，二氢杨梅素得率为2.14±0.09 mg·g^-1。本提取方法简单快速，效率高，有利于拐枣资源的综合加工利用。     

Immobilization of glucose oxidase with Bombyx mori silk fibroin by only stretching treatment and its application to glucose sensor

Glucose oxidase (GOD) was immobilized in Bombyx mori silk fibroin membrane by only physical treatment, i.e., stretching without any chemical reagents. This is due to the structural transition of the silk fibroin membrane from random coil to antiparallel beta-sheet (Silk II) induced by the stretching treatment. Permeability coefficients of glucose and oxygen through the fibroin membrane were determined; the permeability of glucose decreased with increasing degree of stretching. The immobilized enzyme activity was characterized with apparent Michaelis constant K(m) (app) and maximal activity V(m). Optimum pH of the activity of the immobilized enzyme was shifted to the value around neutrality, and the activity was maintained to the higher values on both sides of the optimum pH compared with the case of free enzymes. Thermal stability was scarcely lost even at 50 degrees C, although the free enzyme lost about 70% of the original activity. Thus, the stabilities of the enzyme vs. pH and heat were much improved by the immobilization with silk. Glucose sensor prepared with this GOD-immobilized fibroin membrane was developed; the capabilities such as the response time, calibration curve, and repeating usage were determined.     

Enhanced production of fructose palmitate by lipase-catalyzed esterification in ionic liquids

For the enhancement of enzyme activity, application of ultrasound irradiation on lipase-catalyzed esterification of fructose with palmitic acid in ionic liquids (ILs) mixture containing supersaturated fructose solution was investigated. In the mixture of [Bmim][TfO] and [Omim][Tf₂N] (1:1, v/v), 1.44 times higher enzyme activity (29.2 μmoL/min/g) was achieved under ultrasound irradiation. Besides, ultrasound irradiation enhanced enzyme stability in viscous ILs mixture. After 5 times reuse of Novozym 435 and ILs mixture, 84.4% of initial enzyme activity was remained under ultrasound irradiation, while the residual activity using magnetic stirring only method was 76.2%. These results show that enzymatic reaction in viscous ILs mixture under ultrasound irradiation is an effective method for enzyme activity, as well as, enzyme stability resulting in economic competitiveness of green process.     

Enhanced co-production of pectinase,cellulase and xylanase enzymes from Bacillus subtilis ABDR01 upon ultrasonic irradiation

The effect of low-intensity ultrasound irradiation was studied to improve the co-production for pectinase, cellulase, and xylanase enzymes using Bacillus subtilis ABDR01. Different parameters such as ultrasonic irradiation at the different growth phases of the bacterial strain, ultrasound power, irradiation duration, and irradiation duty cycle were assessed. Sonication with 90 W ultrasound power, 25 kHz frequency with 70 % duty cycle for 5 min at 6 h of bacterial growth phase gave the maximum productions of 87.82 U/ mL pectinase 22.17 U/ mL cellulase and 137.95 U/ mL xylanase respectively. The enzyme activity of pectinase, cellulase, and xylanase was enhanced by about 38.15 %, 53.77 %, and 24.59 %, respectively, compared to non-sonicated control cultivation. This optimized low-frequency ultrasound irradiation to bacterial cells enhanced the nutrient uptake rate and increased the cell wall permeability, which results in higher enzyme productivity. Our results signify the effectiveness of low-frequency ultrasound irradiation for improved enzyme yields and hyperactivation during microbial fermentation.     

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes

The effect of regucalcin, a regulatory protein of Ca²⁺ signaling, on guanosine-5-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca²⁺ (25–100 M) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni, and Mn) with 50 M. The activatory effect of calcium (50 M) was significantly decreased by calmodulin (2.5 and 5 g/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1–0.5 M) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 M). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca²⁺ in rat liver plasma membranes.     

Oxygenation of Native or UV-Modified Human Hemoglobin in the Presence of Nitric Oxide

The effect of 0.1–10% nitrosohemoglobin (HbNO) on the functional properties of human oxyhemoglobin (HbO₂) was studied before and after UV irradiation at 151–453 J/m². Oxygen binding analysis showed that HbNO intensified the first stage of oxygenation and weakened the cooperative interactions in the tetramer, decreasing the affinity of hemoglobin for oxygen at physiologically important oxygen partial pressures (40–100 mm Hg). Mixtures of HbO₂ and HbNO were highly resistant to therapeutic doses of UV irradiation. Since the functional activity of hemoglobin depended nonlinearly on the concentration of HbNO in the mixture, it was assumed that sophisticated interactions of HbO₂ and HbNO yielded a new product differing in properties from the initial components.     

Optimization of the multienzyme system for sucrose biosensor by response surface methodology

An immobilized multienzyme- and cathodic amperometry-based biosensor for sucrose was constructed for the analysis of food and fermentation samples. The multienzyme system, comprising invertase, mutarotase and glucose oxidase (GOD), was immobilized by using glutaraldehyde as cross-linking agent. Operating parameters of the biosensor for the estimation of sucrose in the range 1–10% were standardized. Response surface methodology (RSM) based on three-factor, three-variable design was used to evaluate the effect of important variables (concentration of enzymes, (varied in the range invertase (10–50 IU), mutarotase (5–105 IU) and GOD (1–9 IU)) on the response of biosensor. In the range of parameters studied, response time decreased with decrease in the invertase and with increase in mutarotase and GOD. Mutarotase concentration above 75 IU was found to result in an increased response time due to inhibition of mutarotase by its product -D-glucose. The optimal conditions achieved for the analysis of sucrose were: invertase 10 IU, mutarotase 40 IU, and GOD 9 IU. With these conditions, the predicted and actual experimental response time values were 2.26 and 2.35 min respectively, showing good agreement.     

The Effects of Low Doses of Gamma-Radiation on Growth and Membrane Activity of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> GRP3 and <Emphasis Type="Italic">Escherichia coli</Emphasis> M17

Microorganisms are part of the natural environments and reflect the effects of different physical factors of surrounding environment, such as gamma (γ) radiation. This work was devoted to the study of the influence of low doses of γ radiation with the intensity of 2.56?μW (m²?s)^?1 (absorbed doses were 3.8 mGy for the radiation of 15?min and 7.2 mGy—for 30?min) on Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild type cells. The changes of bacterial, growth, survival, morphology, and membrane activity had been studied after γ irradiation. Verified microbiological (specific growth rate, lag phase duration, colony-forming units (CFU) number, and light microscopy digital image analysis), biochemical (ATPase activity of bacterial membrane vesicles), and biophysical (H⁺ fluxes throughout cytoplasmic membrane of bacteria) methods were used for assessment of radiation implications on bacteria. It was shown that growth specific rate, lag phase duration and CFU number of these bacteria were lowered after irradiation, and average cell surface area was decreased too. Moreover ion fluxes of bacteria were changed: for P. aeruginosa they were decreased and for E. coli—increased. The N,N′-dicyclohexylcarbodiimide (DCCD) sensitive fluxes were also changed which were indicative for the membrane-associated F₀F₁-ATPase enzyme. ATPase activity of irradiated membrane vesicles was decreased for P. aeruginosa and stimulated for E. coli. Furthermore, DCCD sensitive ATPase activity was also changed. The results obtained suggest that these bacteria especially, P. aeruginosa are sensitive to γ radiation and might be used for developing new monitoring methods for estimating environmental changes after γ irradiation.     

Stimulatory effect of regucalcin on proteolytic activity is impaired in the kidney cortex cytosol of rats with saline ingestion

The effect of regucalcin (RC) on neutral proteolytic activity in the cytosol of rat kidney cortex was investigated. Proteolytic activity was significantly increased by the presence of RC (0.01 + 0.10 M) in the enzyme reaction mixture. This increase was completely abolished by the addition of anti-RC monoclonal antibody (150 ng/ml). When the renal cortex cytosol was incubated without RC addition, the degradation of globin of substrate was demonstrated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This degradation was clearly inhibited by the addition of anti-RC antibody (150 ng/ml), indicating that protein degradation results partly from the cytosolic endogenous RC. Meanwhile, proteolytic activity was significantly decreased in the renal cortex cytosol of rats with saline ingestion for 2, 7, and 14 days. The effect of RC (0.1 M) in increasing proteolytic activity was weakened in the kidney cortex cytosol of saline-ingested rats. The present study suggests that endogenous RC plays a role in the activation of proteases in the renal cortex cytosol, and that the RC effect is impaired in saline-ingested rats.     

Multifunctional carbon nanotubes for direct electrochemistry of glucose oxidase and glucose bioassay

Polydopamine (Pdop) has recently been shown to adsorb to a wide variety of surfaces and serves as an adhesion layer to immobilize biological molecules. In this work, the multifunctional carbon nanotube (CNT) composites were prepared though the oxidation of dopamine at room temperature and subsequent electroless silver deposition by mildly stirring. The stable immobilization and direct electron transfer of glucose oxidase were achieved on the composite film modified glassy carbon electrode. The resulting electrode gave a well-defined redox peaks with a formal potential of about −482 mV (vs. SCE) in pH 7.0 buffer. The electron transfer rate constant was estimated to be 3.6 s⁻¹, due to the combined contribution of Pdop, CNTs and Ag nanoparticles with the help of Nafion. Furthermore, the method for detecting of glucose was proposed based on the decrease of oxygen caused by the enzyme-catalyzed reaction between glucose oxidase (GOD) and glucose. The linear response to glucose ranging from 50.0 μM to 1.1 mM (R² = 0.9958), with a calculated detection limit of 17.0 μM at a signal-to-noise ratio of 3. The low calculated apparent Michaelis–Menten constant was 5.46 mM, implying the high enzymatic activity and affinity of immobilized GOD for glucose. It can reasonably be expected that this observation might hold true for other noble metal nanostructure-electroactive protein systems, providing a promising platform for the development of biosensors and biofuel cells.     

Multiple forms of acetylcholinesterase from rat erythrocytes. Effect of fat-free diet.

Electrophoretic patterns of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from rat erythrocyte were studied. The enzyme was solubilized by the following treatments: a) Triton X-100, b) sodium deoxycholate, or c) ultrasonic irradiation. When the erythrocyte membrane was solubilized by Triton X-100 at concentrations higher than 0.3%, by 10 mM sodium deoxycholate, or by ultrasonic irradiation for more than 5 min, a single band of acetylcholinesterase activity appeared in the gel. Two bands of activity were stained in the gel when the membrane was solubilized by Triton X-100 at concentrations between 0.1--0.2%, or by ultrasound for 5 min. Electrophoretic patterns of acetylcholinesterase from rats fed a fat-sufficient diet were similar to those for the enzyme from animals fed a fat-free diet. The recombination of lipids with the enzyme eluted from the gels confirmed the "phenotypic allosteric desensitization phenomenon".     

Synthesis of L-tyrosine glyceryl ester catalyzed by α-chymotrypsin in water-miscible organic solvents: A possible sun-tan accelerator product

Summary The synthesis of L-tyrosine glyceryl ester, from glycerol and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by -chymotrypsin. Values of 60 % (v/v) for glycerol and 200 mM for L-tyrosine methyl ester were optimal for the transesterification reaction. Additionally to glycerol, several other water miscible cosolvents (acetonitrile, N,N'-dimetyl formamide and tetrahydrofurane) were tested in the reaction media, but their presence did not give an enhancement on the transesterification activity with respect to the glycerol/water medium. However, increasing the hydrophobicity of the cosolvent resulted in a reduction of the enzyme activity, the water:glycerol mixture being the best reaction media.     

Green and efficient production of octyl hydroxyphenylpropionate using an ultrasound-assisted packed-bed bioreactor

A solvent-free system to produce octyl hydroxyphenylpropionate (OHPP) from p-hydroxyphenylpropionic acid (HPPA) and octanol using immobilized lipase (Novozym^® 435) as a catalyst in an ultrasound-assisted packed-bed bioreactor was investigated. Response-surface methodology (RSM) and a three-level-three-factor Box-Behnken design were employed to evaluate the effects of reaction temperature (x ₁), flow rate (x ₂) and ultrasonic power (x ₃) on the percentage of molar production of OHPP. The results indicate that the reaction temperature and flow rate were the most important variables in optimizing the production of OHPP. Based on a ridge max analysis, the optimum conditions for OHPP synthesis were predicted to consist of a reaction temperature of 65°C, a flow rate of 0.05 ml/min and an ultrasonic power of 1.74 W/cm² with a yield of 99.25%. A reaction was performed under these optimal conditions, and a yield of 99.33 ± 0.1% was obtained.     

Direct gene transfer to plant protoplasts by mild sonication

Summary A novel procedure employing mild sonication for transformation of plant protoplasts is described. Transient expression of a chloramphenicol acetyltransferase (CAT) gene in protoplasts of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) was obtained by a brief exposure of the protoplasts to 20 kHz ultrasound in the presence of plasmid DNA. Maximum levels of CAT activity were achieved by sonication for 500–900 ms at 30–70 W electric power (0.65–1.6 W/cm2 acoustic power). This reduced the viability to 15–20 % and 60 % for sugar beet and tobacco protoplasts, respectively. Up to 12 % (sugar beet) and 81 % (tobacco) of maximum transient expression could be achieved with no significant loss of viability. Protoplasts surviving exposure to ultrasound were found to have a similar long-term viability and to regenerate to micro-calli as untreated protoplasts. Plasmid DNA concentrations of 80–110 g/ml and sucrose concentrations of 21–28 % in the sonication medium were found to be optimal for transient expression.Abbreviations CAT chloramphenicol acetyltransferase     

Tonometric biosensor with a differential pressure sensor for chemo-mechanical measurement of glucose

A tonometric biosensor for glucose was constructed using a chemo-mechanical reaction unit and a differential pressure sensor. The reaction unit was fabricated by using both liquid and gas cells separated by an enzyme diaphragm membrane, in which glucose oxidase was immobilized onto the single (gas cell) side of the dialysis membrane. By applying glucose solution (0, 25.0, 50.0, 100, 150 and 200 mmol/l) into the liquid cell of the chemo-mechanical reaction unit, the pressure in the gas cell decreased continuously with a steady de-pressure slope because the oxygen consumption in the gas cell was induced by the glucose oxidase (GOD) enzyme reaction at the enzyme side of the porous diaphragm membrane. The steady de-pressure slope in the gas cell showed the linear relationship with the glucose concentration in the liquid cell between 25.0 and 200.0 mmol/l (correlation coefficient of 0.998). A substrate regeneration cycle coupling GOD with l-ascorbic acid (AsA: 0, 1.0, 3.0, 10.0 and 50.0 mmol/l; as reducing reagent system) was applied to the chemo-mechanical reaction unit in order to amplify the output signal of the tonometric biosensor. 3.0 mmol/l concentration of AsA could optimally amplify the sensor signal more than 2.5 times in comparison with that of non-AsA reagent.     

High-content screening of <Emphasis Type="Italic">Aspergillus niger</Emphasis> with both increased production and high secretion rate of glucose oxidase

Objectives

To develop a rapid, dual-parameter, plate-based screening process to improve production and secretion rate of glucose oxidase simultaneously in Aspergillus niger.

Results

A morphology engineering based on CaCO₃ was implemented, where the yield of GOD by A. niger was increased by up to 50%. Analysis of extracellular GOD activity was achieved in 96-well plates. There was a close negative correlation between the total GOD activity and its residual glucose of the fermentation broth. Based on this, a rapid, plate-based, qualitative analysis method of the total GOD activity was developed. Compared with the conventional analysis method using o-dianisidine, a correlation coefficient of ?0.92 by statistical analysis was obtained.

Conclusion

Using this dual-parameter screening method, we acquired a strain with GOD activity of 3126 U l^?1, which was 146% higher than the original strain. Its secretion rate of GOD was 83, 32% higher than the original strain.