混合溶剂体系中牛肝β-半乳糖苷酶催化5-氟-2'-脱氧尿苷区域选择性半乳糖基化反应

研究了混合溶剂体系中有机溶剂种类和含量对牛肝β-半乳糖苷酶催化5-氟-2'-脱氧尿苷区域选择性半乳糖基化反应的影响.结果表明,在含10%(体积分数)有机溶剂体系中,该酶稳定性差,失活严重.减少有机溶剂含量可显著减轻其对该酶的毒害作用.然而,有机溶剂的添加对酶促糖基化反应的区域选择性的影响很小(均保持在99%以上).牛肝...     

Rapid determination of immobilized ConA lectin activity

ConA was immobilized on an epoxy-activated copolymer of 2-hydroxyethyl-methacrylate and ethylene-dimethacrylate and commercially available high-pressure liquid chromatography (HPLC) sorbents Separon HEMA 1000 EL, Separon HEMA 1000 E, and Separon HEMA 1000 EH (Tessek, Prague, CSFR Denmark). Specific, sensitive, and rapid method for determination of immobilized ConA lectin activity was developed. β-Galactosidase fromAspergilus oryzae oligomannosyl residues was used as specific affinant. After separation of bound and unbound β-galactosidase, enzyme activity was measured in supernatant and thus immobilized ConA lectin activity was calculated easily. The use of the method for evaluating the properties of immobilized ConA, efficiency of immobilization, specific activity, and thermostability is shown. The method developed could be generalized by using artificially glycosylated enzyme for any lectin.     

High Level Production of β-Galactosidase Exhibiting Excellent Milk-Lactose Degradation Ability from Aspergillus oryzae by Codon and Fermentation Optimization

A β-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL^?1 with o-nitrophenyl-β-d-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L^?1 in which the target protein (6.24 g L^?1) occupied approximately 86 %. The recombinant β-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the β-galactosidase may have a good application potential in whey and milk industries.     

Enhanced Properties and Lactose Hydrolysis Efficiencies of Food-Grade β-Galactosidases Immobilized on Various Supports: a Comparative Approach

In this study, a fungal and two yeast β-galactosidases were immobilized using alginate and chitosan. The biochemical parameters and lactose hydrolysis abilities of immobilized enzymes were analyzed. The pH optima of immobilized fungal β-galactosidases shifted to more acidic pH compared to free enzyme. Remarkably, the optimal temperature of chitosan-entrapped yeast enzyme, Maxilact, increased to 60 °C, which is significantly higher than that of the free Maxilact (40 °C) and other immobilized forms. Chitosan-immobilized A. oryzae β-galactosidase showed improved lactose hydrolysis (95.7%) from milk, compared to the free enzyme (82.7%) in 12 h. Chitosan-immobilized Maxilact was the most efficient in lactose removal from milk (100% lactose hydrolysis in 2 h). The immobilized lactases displayed excellent reusability, and chitosan-immobilized Maxilact hydrolyzed >?95% lactose in milk after five reuses. Compared to free enzymes, the immobilized enzymes are more suitable for cost-effective industrial production of low-lactose milk due to improved thermal activity, lactose hydrolysis efficiencies, and reusability.

     

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties

A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50?°C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30?°C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg²⁺ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55?°C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K _m and V _max values of β-galactosidase using ONPG as substrate at 50?°C were 2.805 mM and 37.45?×?10^?3?mM/min/mg, respectively.     

Oil-sealed femtoliter fiber-optic arrays for single molecule analysis

This paper describes a novel method for fabricating and sealing high-density arrays of femtoliter reaction chambers. We chemically etch one end of a 2.3 mm diameter glass optical fiber bundle to create an array of microwells. We then use a contact printing method to selectively modify the surface of the material between microwells with a hydrophobic silane. This modification makes it possible to fill the wells with aqueous solution and then seal them with a droplet of oil, forming an array of isolated reaction chambers. Individual β-galactosidase molecules trapped in these reaction chambers convert a substrate into a fluorescent product that can be readily detected because a high local concentration of product is achieved. This binary readout can be used for ultra-sensitive measurements of enzyme concentration. We observed that the percentage of wells showing enzyme activity was linearly dependent on the concentration of soluble β-galactosidase in the picomolar range. A similar response was also observed for streptavidin-β-galactosidase captured by biotinylated beads. These arrays are also suitable for performing single-molecule kinetics studies on hundreds to thousands of enzyme molecules simultaneously. We observed a broad distribution of catalytic rates for individual β-galactosidase molecules trapped in the microwells, in agreement with previous studies using similar arrays that were mechanically sealed. We have further demonstrated that this femtoliter fiber-optic array can be integrated into a PDMS microfluidic channel system and sealed with oil on-chip, creating an easy to use and high-throughput device for single-molecule analysis.     

The separation of aryl acylamidase by cross flow microfiltration and the significance of enzyme/cell debris interaction

The use of cross flow microfiltration for the separation of aryl acylamide amidohydrolase (amidase) from cell lysate has been investigated. The transmembrane pressure, the feed velocity and the membrane configuration were all found to have significant influence on the transmission of the enzyme through the membrane. Other factors which affect flux and transmission severely are the physical and biochemical properties of the lysate which control the rate of fouling. These, however, are as yet not well characterised. It has been shown that a large amount of amidase is adsorbed to the cell debris which results in a poor enzyme yield. In some cases the apparent membrane transmission of enzyme reaches 154%; this was ascribed to the desorption of the enzyme in a shear field. A model is offered to account for the mechanism of enzyme transmission enhancement.     

Permeabilization of Kluyveromyces marxianus with Mild Detergent for Whey Lactose Hydrolysis and Augmentation of Mixed Culture

Cheese whey is a by-product of cheese-manufacturing industries, and the utilization of whey is a challenging problem either to use it or dispose it, because only few microorganisms can metabolize the whey lactose. Enzymatic hydrolysis of whey lactose to glucose and galactose by β-galactosidase is the approach for biotechnological application. Kluyveromyces marxianus cells were permeabilized with non-toxic, biodegradable, anionic detergent N-lauroyl sarcosine (N-LS) for the enzyme activity. The permeabilization process parameters (N-LS concentration, solvent volume, temperature and incubation time) were optimized. The maximum β-galactosidase activity of 1,220 IU/g dry weight was obtained using permeabilized cells under optimized conditions. Moreover, viability of the permeabilized cells was also evaluated, which showed that cells were alive; however, viability was reduced by two log cycles. The permeabilized cells were evaluated for whey lactose hydrolysis. The maximum lactose hydrolysis of 91 % was observed with 600 mg (dry cell weight/100 mL) in whey powder (5 % w/v) solution at 180-min incubation, pH 6.5 and 30 °C. Further, the hydrolyzed whey was evaluated for amelioration of growth of non-lactose-consuming yeast Saccharomyces cerevisiae. S. cerevisiae was able to grow in hydrolyzed whey simultaneously with K. marxianus. The study confirmed that N-LS could be used to permeabilize K. marxianus cells to make available the enzyme activity.     

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cells in Dual Matrices

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.     

Variation of β-galactosidase expression from Mudlac elements during the development of Escherichia coli colonies

Bacterial colonies reveal the action of systems for multicellular regulation of genetic activity during growth and development. Colonies produced on agar indicator medium by Escherichia coli strains carrying the transposable genetic fusion element Mudlac displayed organized patterns of differential β-galactosidase expression. One feature of these patterns was the presence of phenotypically distinct concenric rings containing cells that were not genetically distinct. A second feature of the patterns was the appearance of sectorial populations with novel phenotypes, frequently displaying coincident variation in several characters, such as enzyme activity, multicellular aggregation and rate of spread over the agar substrate. Subcloning analysis of sectors with more expansive growth phenotypes revealed that they contained bacteria expressing novel developmental sequences on more than one kind of medium. These novel developmental sequences could be transmitted to progeny bacteria but were reversible during growth in liquid medium. More stable clonal variation in patterns of β-galactosidase expression arose during storage in liquid medium. Most of these changes correlated with transpositions or rearrangments of Mudlac sequences. Some changes in β-galactosidase expression involved interactions between Mudlac elements and unlinked Mu derivatives. These results revealed the operation of novel control systems regulating β-galactosidase expression from the lacZ sequences in a chromosomal Mudlac element and demonstrated at least two different kinds of clonal variation events which affected pattern formation in bacterial colonies.     

Bio-solvents change regioselectivity in the synthesis of disaccharides using Biolacta β-galactosidase

Bio-solvents are good alternative solvents that avoid the use of classical organic solvents when performing enzymatic reactions. A noticeably change in regioselectivity was observed in the synthetic behaviour of Biolacta β-galactosidase using bio-solvents derived from dimethylamide and glycerol as co-solvents. Under these conditions, the enzyme changes its well known tendency to produce β-(1→4) to β-(1→6) disaccharides. An evaluation of the bio-solvent concentration and the effects of the non proteic additives in commercially available Biolacta β-galactosidase was undertaken in order to optimize the reaction conditions to improve the yield of the β-(1→6) product.     

Immobilization of the β-galactosidase fromAspergillus niger on chitosan

Crude or purifiedAspergillus niger β-galactosidase preparations were immobilized on chitosan (deacetylated chitin, activated with glutaraldehyde). The most active immobilized systems were obtained withcrude enzyme preparations. The immobilized enzyme hydrolyzed lactose in pure lactose solutions, ultrafiltrate whey permeates, or acid wheys at similar rates. The pH activity profiles and K_m values of the chitosan-bound enzyme were not significantly altered on immobilization, and its stability on repetitive use up to 60°C was increased by reduction with NaBH₄. After 8 weeks on discontinuous operation (8 h use per day), β-galactosidase-chitosan columns were found to retain about 90, 50, or 60% of their initial activities with lactose, ultrafiltrate permeate, or acid whey solutions, respectively. The efficiency of the β-galactosidase-chitosan conjugate appears to be comparable or greater than those of other described systems, and its stability should allow its use on an industrial scale. A preliminary report of this work has already been presented at the 4th International Enzyme Engineering Conference (September 25-30, 1977, Bad Neuenahr, FRG).     

Effect of mutations to amino acid A301 and F361 in thermostability and catalytic activity of the β-galactosidase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> VTCC-DVN-12-01

Background

Beta-galactosidase (EC 3.2.1.23), a commercially important enzyme, catalyses the hydrolysis of β-1,3- and β-1,4-galactosyl bonds of polymer or oligosaccharidesas well as transglycosylation of β-galactopyranosides. Due to catalytic properties; β-galactosidase might be useful in the milk industry to hydrolyze lactose and produce prebiotic GOS. The purpose of this study is to characterize β-galactosidase mutants from B. subtilis.

Results

Using error prone rolling circle amplification (epRCA) to characterize some random mutants of the β-galactosidase (LacA) from B. subtilisVTCC-DVN-12-01, amino acid A301 and F361 has been demonstrated significantly effect on hydrolysis activity of LacA. Mutants A301V and F361Y had markedly reduced hydrolysis activity to 23.69 and 43.22 %, respectively. Mutants the site-saturation of A301 reduced catalysis efficiency of LacA to 20–50 %, while the substitution of F361 by difference amino acids (except tyrosine) lost all of enzymatic activity, indicating that A301 and F361 are important for the catalytic function. Interestingly, the mutant F361Y exhibited enhanced significantly thermostability of enzyme at 45–50 °C. At 45 °C, LacA-361Y retained over 93 % of its original activity for 48 h of incubation, whereas LacA-WT and LacA-301Vwere lost completely after 12 and 24 h of incubation, respectively. The half-life times of LacA-361Y and LacA-301 V were about 26.8 and 2.4 times higher, respectively, in comparison to the half-life time of LacA-WT. At temperature optimum 50 °C, LacA-361Y shows more stable than LacA-WT and LacA-301 V, retaining 79.88 % of its original activities after 2 h of incubation, while the LacA-WT and LacA-301 V lost all essential activities. The half-life time of LacA-361Y was higher 12.7 and 9.39 times than that of LacA-WT and LacA-301 V, respectively. LacA-WT and mutant enzymes were stability at pH 5–9, retained over 90 % activity for 72 h of incubation at 30 °C. However, LacA-WT showed a little bit more stability than LacA-301 V and LacA-361Y at pH 4.

Conclusions

Our findings demonstrated that the amino acids A301V and F361 play important role in hydrolysis activity of β -galactosidase from B. subtilis. Specially, amino acid F361 had noteworthy effect on both catalytic and thermostability of LacA enzyme, suggesting that F361 is responsible for functional requirement of the GH42 family.

     

Production of high-content galacto-oligosaccharides mixture using β-galactosidase and <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> entrapped in polyvinylalcohol gel

The production of a high-content galacto-oligosaccharides mixture (GOS) by immobilised β-galactosidase and yeasts in LentiKats® lens-shaped polyvinylalcohol (PVA) capsules was evaluated. Galacto-oligosaccharides were produced from lactose (300 g L^?1) by immobilised fungal β-galactosidase and the yeast Kluyveromyces marxianus in polyvinylalcohol hydrogel in batch mode. The low-content GOS mixture produced by the immobilised enzyme consisted of 71.7 g L^?1 with a final purity of 22.7 % after 30 h of transgalactosylation reaction at 30·C and pH 4.5. The lowcontent GOS mixtures were subsequently used in 20 repeated batch runs with immobilised yeasts for increasing the GOS content. Digestible sugars were fermented to ethanol and the resulting mixture consisted of 88 mass % of GOS after 26 h of fermentation. The PVA lenses exhibited high fermentative stability without any mechanical deformations.     

β-Galactosidase fluorescence probe with improved cellular accumulation based on a spirocyclized rhodol scaffold

We identified a rhodol bearing a hydroxymethyl group (HMDER) as a suitable scaffold for designing fluorescence probes for various hydrolases. HMDER shows strong fluorescence at physiological pH, but phenolic O-alkylation of HMDER results in a strong preference for the spirocyclic form, which has weak fluorescence. As a proof of concept, we utilized this finding to develop a new fluorescence probe for β-galactosidase. This probe has favorable characteristics for imaging in biological samples: it has good cellular permeability, and its hydrolysis product is well-retained intracellularly. It could rapidly and clearly visualize β-galactosidase activity in cultured cells and in Drosophila melanogaster tissue, which has rarely been achieved with previously reported fluorescence probes.     

Characterization and Association Analysis with Litter Size Traits of Porcine Matrix Metalloproteinase-9 Gene (pMMP-9)

During alcoholic fermentation, the products build up and can, ultimately, kill the organism due to their effects on the cell’s macromolecular systems. The effects of alcohols on the steady-state kinetic parameters of the model enzyme β-galactosidase were studied. At modest concentrations (0 to 2 M), there was little effect of methanol, ethanol, propanol and butanol on the kinetic constants. However, above these concentrations, each alcohol caused the maximal rate, V _max, to fall and the Michaelis constant, K _m, to rise. Except in the case of methanol, the chaotropicity of the solute, rather than its precise chemical structure, determined and can, therefore, be used to predict inhibitory activity. Compounds which act as compatible solutes (e.g. glycerol and other polyols) generally reduced enzyme activity in the absence of alcohols at the concentration tested (191 mM). In the case of the ethanol- or propanol-inhibited β-galactosidase, the addition of compatible solutes was unable to restore the enzyme’s kinetic parameters to their uninhibited levels; addition of chaotropic solutes such as urea tended to enhance the effects of these alcohols. It is possible that the compatible solutes caused excessive rigidification of the enzyme’s structure, whereas the alcohols disrupt the tertiary and quaternary structure of the protein. From the point of view of protecting enzyme activity, it may be unwise to add compatible solutes in the early stages of industrial fermentations; however, there may be benefits as the alcohol concentration increases.     

Enzyme-regulated the changes of pH values for assembling a colorimetric and multistage interconnection logic network with multiple readouts

Based on enzymatic reactions-triggered changes of pH values and biocomputing, a novel and multistage interconnection biological network with multiple easy-detectable signal outputs has been developed. Compared with traditional chemical computing, the enzyme-based biological system could overcome the interference between reactions or the incompatibility of individual computing gates and offer a unique opportunity to assemble multicomponent/multifunctional logic circuitries. Our system included four enzyme inputs: β-galactosidase (β-gal), glucose oxidase (GOx), esterase (Est) and urease (Ur). With the assistance of two signal transducers (gold nanoparticles and acid–base indicators) or pH meter, the outputs of the biological network could be conveniently read by the naked eyes. In contrast to current methods, the approach present here could realize cost-effective, label-free and colorimetric logic operations without complicated instrument. By designing a series of Boolean logic operations, we could logically make judgment of the compositions of the samples on the basis of visual output signals. Our work offered a promising paradigm for future biological computing technology and might be highly useful in future intelligent diagnostics, prodrug activation, smart drug delivery, process control, and electronic applications.     

High sensitive competitive immunodetection of 2,4-dichlorophenoxyacetic acid using enzymatic amplification with electrochemical detection

The amplification cycle consisting of NADH independent oligosaccharide dehydrogenase (ODH) and laccase has been recently reported to be highly sensitive to several catecholamines and p-aminophenol. A competitive immunoassay for 2,4-dichlorophenoxyacetic acid has been developed by combining this amplification cycle with β-galactosidase as enzyme label resulting in p-aminophenol as product. The combination of enzymatic amplification cycles with a competitive immunoassay yields a highly sensitive measurement of 2,4-dichlorophenoxyacetic acid. Using a monoclonal antibody the linear range of the assay was between 0.02 and 100 ng/l and the c₅₀ was found at 0.2 ng/l; the detection limit was at 5 pg/l (25 fmol/l) corresponding to 5 amol.     

褶合光谱法测定β-半乳糖苷酶的活性

运用褶合光谱法建立了 β 半乳糖苷酶的最佳褶合光谱以及该酶在最佳测试条件下的线性方程 :Qj=1.90 6× 10 -2 +4 .946× 10 -3 C +1.90 9× 10 -4 C2 ,r =1.0 0 0 0 ;对已瞬时转染 β 半乳糖苷酶表达载体的COS 1细胞提取液样品中的 β 半乳糖苷酶的活性进行了测定 ,并与吸光度值分析法相对照。结果表明 :本法快速、简便、准确 ,在生化分析中有广泛的应用价值