Dual lifetime referencing enables pH‐control for oxidoreductions in hydrogel‐stabilized biphasic reaction systems

pH‐shifts are a serious challenge in cofactor dependent biocatalytic oxidoreductions. Therefore, a pH control strategy was developed for reaction systems, where the pH value is not directly measurable. Such a reaction system is the biphasic aqueous‐organic reaction system, where the oxidoreduction of hydrophobic substrates in organic solvents is catalysed by hydrogel‐immobilized enzymes, and enzyme‐coupled cofactor regeneration is accomplished via formate dehydrogenase, leading to a pH‐shift. Dual lifetime referencing (DLR), a fluorescence spectroscopic method, was applied for online‐monitoring of the pH‐value within the immobilizates during the reaction, allowing for a controlled dosage of formic acid. It could be shown that by applying trisodium 8‐hydroxypyrene‐1, 3, 6‐trisulfonate as pH indicator and Ru(II) tris(4, 7‐diphenyl‐1, 10‐phenantroline) (Ru[dpp]) as a reference luminophore the control of the pH‐value in a macroscopic gel‐bead‐stabilized aqueous/organic two phase system in a range of pH 6.5 to 8.0 is possible. An experimental proof of concept could maintain a stable pH of 7.5 ± 0.15 during the reaction for at least 105 h. With these results, it could be shown that DLR is a powerful tool for pH‐control within reaction systems with no direct access for conventional pH‐measurement.     

Intraparticle concentration gradients for substrate and acidic product in immobilized cephalosporin C amidase and their dependencies on carrier characteristics and reaction parameters

Cephalosporin C amidase was covalently attached using a protein loading of 7.0–200 mg protein/g dry carrier on four epoxy‐activated Sepabeads differing in particle size and pore diameter. Initial‐rate kinetic analysis showed that for Sepabeads with small pore diameters (30–40 nm), the apparent K_M of the amidase for hydrolysis of cephalosporin C at 37°C and pH 8.0 increased ～3‐fold in response to increased particle size (～120–400 µm) and increased amount of immobilized enzyme (7.0–70 mg protein/g dry carrier) while maximum specific activity (3.2 U/mg protein; 25% of free amidase) was affected only by particle size. In contrast, for Sepabeads with wide pores (150–250 nm), the K_M was independent of the enzyme loading. Internal effectiveness factors calculated from observable Thiele modulus reflected the dependence of K_M on geometrical parameters of the particles. A new method for determination of the overall intraparticle pH was developed based on luminescence lifetime measurements in the frequency domain. Sepabeads were doubly labeled using a lipophilic variant of the pH‐sensitive dye fluorescein, and Ru(II) tris(4,7‐diphenyl‐1,10‐phenantroline) whose phosphorescence properties are independent of pH. Luminescent lifetime measurements of doubly labeled particle suspensions showed superior signal‐to‐noise ratio compared to fluorescence intensity‐based measurements using singly labeled particles. The difference at apparent steady state (ΔpH) between bulk (external pH) and intraparticle pH (internal pH) was as large as ～0.6 units. The ΔpH was dependent on substrate concentration, particle size, and pore diameter. Therefore, these results characterize the role of carrier characteristics and reaction parameters in the formation of concentration gradients for substrate and acidic product during hydrolysis of cephalosporin C by immobilized amidase. The strong pH dependence of the immobilized amidase underscores the importance of considering intraparticle pH gradients in the design of an efficient carrier‐bound biocatalyst. Biotechnol. Bioeng. 2010;106: 528–540. © 2010 Wiley Periodicals, Inc.     

Application of ammonium bicarbonate buffer as a smart microenvironmental pH regulator of immobilized cephalosporin C acylase catalysis in different reactors

In a stirred tank reactor, during catalysis with immobilized cephalosporin C acylase (CCA), the microenvironmental pH dropped to 7.2 in a nonbuffered system (with the pH maintained at 8.5 by adding alkali) due to the existence of diffusional resistance. Moreover, the immobilized CCA only catalyzed five batch reactions, suggesting that the sharp pH gradient impaired the enzyme stability. To buffer the protons produced in the hydrolysis of cephalosporin C by CCA, phosphate and bicarbonate buffers were introduced. When CCA was catalyzed with 0.1 M ammonium bicarbonate buffer, no obvious gradient between the bulk solution and intraparticle pH was detected, and the catalysis of 15 batch reactions was achieved. Accordingly, with 0.2 M ammonium bicarbonate buffer in a packed bed reactor, the immobilized CCA exhibited continuous catalysis with high conversion rates (≥95%) for 21 days. Reactions with ammonium bicarbonate buffer showed significant increases in the stability and catalytic efficiency of the immobilized CCA in different reactors compared to those in nonbuffered systems.     

氨基化二氧化硅颗粒固定木瓜蛋白酶研究

采用正硅酸乙酯与N-(β-氨乙基)氨丙基三乙氧基硅烷在油包水形成的微胶囊中同步水解的方法,一步法制备了氨基化的二氧化硅颗粒,得到的颗粒粒径在0.3～0.5μm之间,平均大小为0.37μm, 氨基含量和颗粒大小可控,氨基含量高达56mmol/g。此颗粒经戊二醛处理后,采用共价法固定木瓜蛋白酶,固定化最适pH6.5,最佳给酶量为15mg/g载体,固定化酶的最适反应温度为70℃,最适反应pH为6.5,固定化酶热稳定性,pH耐受性,贮存稳定性都明显高于游离酶,表明此颗粒可作为一种优良的酶固定化载体。     

Immobilized CALB Catalyzed Transesterification of Soybean Oil and Phytosterol

Candida antarctica lipase B (CALB) was immobilized on Fe₃O₄/SiO_x-g-P(GMA) polymer carrier to catalyzed the transesterification of soybean oil and phytosterol. The enzyme loading of the obtained particles was 98.7 mg/g supports and the enzyme activity was 1226.5 U/g. The average particle size was 100.5?±?1.30 nm and the magnetization was 15.80 emu/g. The immobilized enzyme showed higher activities at a wider range of pH and temperatures. Its optimum reaction temperature was up to 50 °C; increased by 5 °C compared to the free enzyme. The obtained magnetic immobilized Fe₃O₄/SiO_x-g-P(GMA) lipase was nanoscale. First-grade soybean oils were used as a substrate. System pH was adjusted to 7.0. The optimal reaction temperature was 50 °C and the reaction time was 3 h. The phytosterol concentration of 5% and immobilized CALB of 2% were obtained. The conversion rate of transesterification reaction between soybean oil and phytosterol was 86.2%. The use of magnets can quickly separate the immobilized enzymes from the substrates. The relative activity of the immobilized enzymes was 83.0% when reused seven times. The prepared immobilized CALB can improve efficiently enzyme activity and reutilization.     

Immobilization of glucoamylase and glucose oxidase in activated carbon: effects of particle size and immobilization conditions on enzyme activity and effectiveness

Glucoamylase and glucose oxidase have been immobilized on carbodiimide-treated activated carbon particles of various sizes. Loading data indicate nonuniform distribution of immobilized enzyme within the porous support particles. Catalysts with different enzyme loading and overall activities have been prepared by varying enzyme concentration in the immobilizing solution. Analysis of these results by a new method based entirely upon experimentally observable catalyst properties indicates that intrinsic catalytic activity is reduced by immobilization of both enzymes. Immobilized glucoamylase intrinsic activity decreases with increasing enzyme loading, and similar behavior is suggested by immobilized glucose oxidase data analysis. The overall activity data interpretation method should prove useful in other immobilized enzyme characterization research, especially in situations where the intraparticle distribution of immobilized enzyme is nonuniform and unknown.     

Characterization of a recombinant l-rhamnose isomerase from Dictyoglomus turgidum and its application for l-rhamnulose production

A putative recombinant enzyme from Dictyoglomus turgidum was characterized and immobilized on Duolite A568 beads. The native enzyme was a 46 kDa tetramer. Its activity was highest for l-rhamnose, indicating that it is an l-rhamnose isomerase. The maximum activities of both the free and immobilized enzymes for l-rhamnose isomerization were at pH 8.0 and 75 °C in the presence of Mn²⁺. Under these conditions, the half-lives of the free and immobilized enzymes were 28 and 112 h, respectively. In a packed-bed bioreactor, the immobilized enzyme produced an average of 130 g l-rhamnulose l^?1 from 300 g l-rhamnose l^?1 after 240 h at pH 8.0, 70 °C, and 0.6 h^?1, with a productivity of 78 g l^?1 h^?1 and a conversion yield of 43 %. To the best of our knowledge, this is the first report describing the enzymatic production of l-rhamnulose.     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

The stabilizing effects of immobilization in D-amino acid oxidase from <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis>

Background  

Immobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications.     

Immobilized d-Amino Acid Oxidase Preparation,Some Enzymatic Properties,and Potential Uses

Immobilization of d-amino acid oxidase was investigated by covalently binding the enzyme to cyanogen bromide activated polysaccharides. Among polysaccharides tested, Sepharose 6B was found to be the best carrier.

Some enzymatic properties of the immobilized enzyme were investigated and compared with those of the native enzyme. The optimum pH of the immobilized enzyme was shifted by 0.5 pH units to the acid side in comparison with that of the native enzyme. With regard to substrate specificity, heat stability and effect of temperature, no significant differences were observed between the immobilized and native enzymes.

The immobilized enzyme was conveniently used for a determination of d-amino acids and an analysis of optical purity of l-amino acids.     

Direct measurement of pH profiles in immobilized enzyme carriers during kinetically controlled synthesis using CLSM

Confocal laser scanning microscopy was applied to measure the pH value in the carrier of immobilized enzymes during the enzyme-catalyzed synthesis. pH profiles with a high resolution are shown, with the pH increasing in the core of the particles. Significant differences occur for different carrier material, particle size, porosity and surface modification. The increased pH value is identified as one of the reasons leading to reduced enzyme selectivity in the penicillin amidase catalyzed synthesis of cephalosporins and penicillins.     

硅藻土固定中性脂肪酶的制备及其性质

以硅藻土为载体,采用吸附法,对脂肪酶进行固定化,研究了固定化条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件：给酶量为33374U/g,固定化温度为35℃,pH值为7.5,时间为4h,此时固定化酶的活力约为5833U/g载体。固定化酶的热稳定性较游离酶有了很大的提高,其在80℃以下能保持80%以上的酶活,而游离酶60℃残余酶活仅为5%。最适反应温度和最适pH值也分别由游离酶的40℃上升至50℃和由7上升到7.5。对固定化中的中性脂肪酶在生物柴油合成中的应用也进行了初步研究。     

Novel approach to quantify immobilized-enzyme distributions

The quantitative intraparticle enzyme distribution of Assemblase, an industrially employed polydisperse immobilized penicillin-G acylase, was measured. Because of strong autofluorescence of the carrier, the generally applied technique of confocal scanning microscopy could not be used; light microscopy was our method of choice. To do so, Assemblase particles of various sizes were sectioned, labeled with antibodies specifically against the enzyme, and analyzed light microscopically. Image analysis software was developed and used to determine the intraparticle enzyme distribution, which was found to be heterogeneous, with most enzyme located in the outer regions of the particles. Larger particles showed steeper gradients than smaller ones. A mathematical representation of the intraparticle profiles, based on in-stationary enzyme diffusion into the particles, was validated successfully for a broad range of particle sizes using data for volume-averaged particle size and enzyme loading. The enzyme gradients determined in this work will be used as input for a physical model that quantitatively describes the complex behavior of Assemblase. Such a physical model will lead to identification of the current bottlenecks in Assemblase and can serve as a starting point for the design of improved biocatalysts that also may be based on intelligent use of enzyme gradients.     

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

Background

Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R).

Results

Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP) that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc).

Conclusion

Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.     

Establishment of automated culture system for murine induced pluripotent stem cells

Background

Cost-effective production of industrially important enzymes is a key for their successful exploitation on industrial scale. Keeping in view the extensive industrial applications of lignin peroxidase (LiP), this study was performed to purify and characterize the LiP from an indigenous strain of Trametes versicolor IBL-04. Xerogel matrix enzyme immobilization technique was applied to improve the kinetic and thermo-stability characteristics of LiP to fulfil the requirements of the modern enzyme consumer sector of biotechnology.

Results

A novel LiP was isolated from an indigenous T. versicolor IBL-04 strain. T. versicolor IBL-04 was cultured in solid state fermentation (SSF) medium of corn cobs and maximum LiP activity of 592?±?6 U/mL was recorded after five days of incubation under optimum culture conditions. The crude LiP was 3.3-fold purified with specific activity of 553 U/mg after passing through the DEAE-cellulose and Sephadex-G-100 chromatography columns. The purified LiP exhibited a relatively low molecular weight (30?kDa) homogenous single band on native and SDS-PAGE. The LiP was immobilized by entrapping in xerogel matrix of trimethoxysilane (TMOS) and proplytetramethoxysilane (PTMS) and maximum immobilization efficiency of 88.6% was achieved. The free and immobilized LiPs were characterized and the results showed that the free and immobilized LiPs had optimum pH 6 and 5 while optimum temperatures were 60°C and 80°C, respectively. Immobilization was found to enhance the activity and thermo-stability potential of LiP significantly and immobilized LiP remained stable over broad pH and temperature range as compare to free enzyme. Kinetic constants K _m and V _max were 70 and 56???M and 588 and 417 U/mg for the free and immobilized LiPs, respectively. Activity of this novel extra thermo-stable LiP was stimulated to variable extents by Cu²⁺, Mn²⁺ and Fe²⁺ whereas, Cystein, EDTA and Ag⁺ showed inhibitory effects.

Conclusions

The indigenously isolated white rot fungal strain T. versicolor IBL-04 showed tremendous potential for LiP synthesis in SSF of corncobs in high titters (592 U/mL) than other reported Trametes (Coriolus, Polyporus) species. The results obtained after dual phase characterization suggested xerogel matrix entrapment a promising tool for enzyme immobilization, hyper-activation and stabilization against high temperature and inactivating agents. The pH and temperature optima, extra thermo-stability features and kinetic characteristics of this novel LiP of T. versicolor IBL-04 make it a versatile enzyme for various industrial and biotechnological applications.     

On the mechanism of peroxyoxalate chemiluminescence. Quenched chemiluminescence as a detection method in HPLC

Several analytes such as the inorganic anions bromide, iodide, sulphite and nitrite and organic compounds as substituted anilines and sulphur compounds cause quenching of peroxyoxalate chemiluminescence. A detection method for liquid chromatography based on the quenching phenomenon has been developed. It makes use of an immobilized luminophore, i.e. 3-aminofluoranthene covalently bound via an alkyl-spacer on controlled pore glass, packed in the detector cell. The mechanism behind the quenching has been elucidated by investigating the roles of luminophores (both in the liquid and in solid state) and oxalates in peroxylate CL with respect to quenchers. Most probably the quencher destroys the radical ion pair produced after electron transfer in the last stage of the CIEEL reaction scheme, thus preventing the formation of electronically excited luminophore.     

Hydrolysis of lactose by β-galactosidase immobilized in polyvinylalcohol

Summary Fungal -galactosidase was immobilized in polyvinylalcohol gel formed in pores of contton material. Temperature and pH effects on the activity of free and immobilized enzymes were studied. The optimum temperatures of free and immobilized enzymes were 60° C and 55° C respectively. The pH optimum ranged from 4.5 to 5.0 for both enzymes. The thermal stability of the immobilized -galactosidase was slightly higher. The K_m values for soluble and immobilized enzymes were respectively 1.9 mM and 2.5 mM. The optimization of conditions for a highly effective hydrolysis of 4% lactose solution and reusability of the immobilized enzyme resulted in 75% hydrolysis after 5–6 h. The degree of conversion decreased to 50% after 30 repeated runs. The capacity of the immobilized enzyme to hydrolyze lactose in whey was also studied.     

Mathematical model for internal pH control in immobilized enzyme particles

A mathematical model has been developed for the internal pH control in immobilized enzyme particles. This model describes the kinetics of a coupled system of two enzymes, immobilized in particles of either planar, cylindrical, or spherical shape. The enzyme kinetics are assumed to be of a mixed type, including Michaelis-Menten kinetics, uncompetitive substrate inhibition, and competitive and noncompetitive product inhibition. In a case study we have considered the enzyme combination urease and penicillin acylase, whose kinetics are coupled through the pH dependence of the kinetic parameters. The hydrolysis of urea by urease yields ammonia and carbon dioxide, whereas benzylpenicillin (Pen-G) is converted to 6-amino penicillanic acid and phenyl acetic acid by penicillin acylase. The production of acids by the latter enzyme will cause a decrease in pH. Because of the presence of the ammonia-carbon dioxide system, however, the pH may be kept under control. In order to obtain information about the optimum performance of this enzymatic pH controller, we have computed the effectiveness factor and the conversion in a CSTR at different enzyme loadings. The results of the computer simulations indicate that a high conversion of Pen-G may be achieved (80-90%) at bulk pH values of about 7.5-8.     

Bacterial cellulose as carrier for immobilization of laccase: Optimization and characterization

Bacterial cellulose (BC) has attracted attention as a new functional material due to its excellent mechanical strength, tridimensional nanostructure, high purity, and increased water absorption, compared to plant cellulose. In this work, commercial laccase was immobilized on BC and the influence of enzyme concentration, contact time, and pH was optimized toward the recovery activity of immobilized laccase. This optimization was carried out using a 3³ experimental design and response surface methodology. Enzyme concentration played a critical role in laccase immobilization. Under optimized conditions (0.15 μL L^?1 of enzyme concentration, 4.8 h of contact time, pH 5.4), the predicted and experimental response were equal to 47.88 and 49.30%, respectively. The thermal stability of the immobilized laccase was found to increase notably at 60 and 70°C presenting stabilization factor equal to 1.79 and 2.11, respectively. The immobilized laccase showed high operational stability, since it retained 86% of its initial activity after seven consecutive biocatalytic cycles of reaction with 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid). Kinetic studies showed that the values of Michaelis–Menten constant and maximum reaction rate decreased upon immobilization (9.9‐ and 1.6‐fold, respectively). Globally, the use of immobilized laccase on BC offers an interesting tool for industrial biocatalytic applications.     

Immobilized enzymes: Pectin esterase covalently coupled to dorous glass particles

Pectin esterase (E.C. 3.1.1.11) was covalently immobilized to porous glass particles by reaction of the native protein with pendant, benzoyl azide groups of the carrier. Enzyme loading on the carrier was 0.5 unit per ml as measured by pH stat, assay. Decreasing the size of the immobilized enzyme particles by grinding produced a 12-fold increase in activity suggesting severe internal mass transport restrictions on turnover kinetics, Gross fractionation of the citrus pectin substrate into high and low molecular weight categories and their subsequent use in kinetic characterization shows no effect of molecular weight upon the kinetic behavior of the native enzyme. In contrast the immobilized enzyme displayed a 5-fold increase in the apparent. K_m for the high molecular weight fraction relative to that of the low molecular weight fraction. A striking difference exists in the low pH profile of immobilized pectin esterase relative to the native enzyme. Carrier matrix interactions with the polyelectrolyte substrate are invoked to explain this difference. The thermal stability of the immobilized enzyme is relatively low and displays a half-life of approximately 2 weeks at 25°C.