A monte carlo simulation of the depolymerization of linear homopolymers by endo-enzymes exhibiting random-attack probability and single-attack mechanism: application to the (1-->3), (1-->4)-beta-D-glucan/endo-(1-->3),(1-->4)-beta-D-glucanase system

A Monte Carlo simulation of the depolymerization of linear homopolymers by specific endo-enzymes exhibiting random-attack probability and a single-attack mechanism has been developed. The program simulates the "real" depolymerization versus time of a polydisperse sample of substrate by a specific endo-enzyme. Given the initial mass distribution and concentration of the substrate, the initial concentration of the enzyme, and its Michaelis-Menten constant, the program simulates the evolution of the mass distribution of the substrate with the depolymerization time. When tested against experimental data from the depolymerization of barley (1-->3),(1-->4)-beta-D-glucan by malt endo-(1-->3), (1-->4)-beta-D-glucanase, monitored using the Calcofluor-FIA method with fluorescent detection, excellent results were obtained. Copyright 1998 John Wiley & Sons, Inc.     

Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.     

Accuracy of laying hens in jumping upwards and downwards between perches in different light environments

Eighty Lohmann Brown layer hens were reared in floor pens fitted with perches from 1 day of age. After the peak of lay (25 weeks of age), their ability to jump to and from perches in different light environments was assessed in two experiments using digital video techniques that allowed detailed measurements of take-off, flight and landing behaviours.In Experiment 1, birds jumped up or down (angles of 10 and 18°) between two horizontal perches that were separated by a gap of 60 cm under different lighting conditions (5, 10 or 20 lux; incandescent or high- or low-frequency fluorescent). In Experiment 2, the horizontal distance was increased to 80 cm and contrast between perch and background was varied. Fifty-two hens (65%) achieved the training criterion for Experiment 1: jumping a 60 cm gap five times of which three in a row. Thirty-two of these hens (62%) subsequently failed to achieve the 80 cm jump criterion for Experiment 2.In Experiment 1, birds took off sooner (P<0.001), turned their heads further downward relative to the landing perch (P<0.001), beat their wings more often during flight (P<0.001), and took longer to achieve balance (P<0.001) when jumping downward rather than upward. In Experiment 2, refusals to jump, and clumsy or missed landings, occurred more frequently on downward than upward jumps (P<0.001). These behaviours were unaffected by lighting conditions, or by the amount of contrast between the perch and its background.In conclusion, behaviours indicating inaccurate control of landing were more frequent on downward than on upward jumps (P<0.001). However, these behaviours were not affected by light intensity, light type or contrast. The results suggest that there is a difference between the two types of jumps in the risk of injury to hens housed in aviaries where perches must be used to gain access to resources.     

Synthesis, (1-->3)-beta-D-glucanase-binding ability, and phytoalexin-elicitor activity of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides

We describe a approach for the synthesis of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. The particular (1-->3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method, and it was characterized by FTIR spectroscopy. The acid-solubilized (1-->3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan particles, which were qualitatively analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). The peracetylated 3-butenyl (1-->3)-beta-D-oligoglucosides were synthesized by treating peracetylated (1-->3)-beta-D-oligoglucosides with the 3-butenyl alcohols and a Lewis acid (SnCl4) catalyst. Epoxidation of the peracetylated 3-butenyl oligoglucosides took place with m-chloroperoxybenzoic acid (m-CPBA). NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture in an overall yield of 21%. The sample was analyzed by positive-ion electrospray ionization mass spectrometry (ESIMS). In a 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside-binding (1-->3)-beta-D-glucanase assay, we found that the (1-->3)-beta-D-glucanase was obviously inactivated by the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. At the same time, we found the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture was more active as compared to the underivatized oligoglucoside mixture in eliciting phytoalexin accumulation in tobacco cotyledon tissue. Furthermore, it could be kept for a longer time than a (1-->3)-beta-D-oligoglucoside mixture, which indicated it is much more stable than (1-->3)-beta-D-oligoglucosides.     

Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear.     

Analysis and quantification of a mixed exo-acting and endo-acting polysaccharide depolymerization system

A new mathematical model has been proposed based on a model presented by Suga, van Dedem, and Moo-Young.(10) The model requires a separate differential equation for each polymeric species (differentiated by degree of polymerization) in the reaction mixture. The main contribution of this model is the incorporation of experimental molecular weight distributions as the initial conditions. These molecular weight distributional as the initial conditions were obtained using modern analytical equipment previouly unknown for this application. The equipment, SEC/LALLS, measures relative concentrations of specific molecular weight species along with the corresponding molecular weights, thus yielding (through some mathematical manipulation) the absolute concentration of each molecular weight species. The concentration at each molecular weight can then be incorporated as the initial condition for that equation. Theoretically, the system of differential equations can be solved to give a more realistic time course of reaction.Synergism between endo-acting and exo-acting enzymes was examined theoretically using the mathematical model. Through model predictions, it was found that synergy is based on two fundamental parameters: (1) each enzyme's activity relative to the sum of enzyme activities and, (2) overall substrate concentration relative to the exo-acting enzyme's Michaeiis kinetic constant K(m). Theoretically, synergism increases as a function of reaction time. Intermediate endo fractions (ratio of endo-acting enzyme activity to the sum of endo-acting and exo-acting enzyme activity) from 0.3 to 0.7 exhibit the most synergism. Values of k[log(K(m, exo)/S(0))] above about zero also exhibits the most synergism.An examination of experimental data obtained both by SEC/LALLS and by reducing sugar measurements shows that the model is inadequate for successfully predicting quantities associated with the substrate during reaction. This is especially true for synergism predictions. At short reaction times, the model predicts the data fairly well, but at longer times the predictions are inconsistent with experimental data. These inconsistencies may be due to complicating phenomena such as enzyme inhibitions.     

Determination of beta-amylase activity by a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate flow-injection analysis (2, 6-TNS-FIA) method, using amylose and amylopectin as substrates

2-p-Toluidinylnaphthalene-6-sulfonate (2,6-TNS) is a compound which is barely fluorescent in pure water but whose fluorescence can be strongly enhanced if the environment becomes hydrophobic, i.e. by the addition of suitable substrates such as proteins or 1, 4-alpha-D-glucans. The enhancement of fluorescence results from the formation of a 2,6-TNS/substrate complex. For linear and ramified 1, 4-alpha-D-glucans, the fluorescence intensities of the complexes depend linearly on their concentrations but nonlinearly on their average molecular weights (AMW). Thus, the fluorescence detector acts simultaneously as a linear detector concerning the concentration of 1,4-alpha-D-glucan and as a nonlinear mass-selective detector concerning its AMW. These properties have been used for the development of a fluorimetric 2,6-TNS-FIA methodology for the determination of beta-amylase activity, using amylose and amylopectin as substrates. The experimental data points, corresponding to the concentration of "detectable" substrate vs depolymerization time, were fitted using a two-parameter exponential decay curve, and the depolymerization rates at time zero were calculated. The depolymerization rates at time zero vs the corresponding initial substrate concentrations were fitted using the Michaelis-Menten hyperbola and the enzymic constants k(3) and K(m) for amylose (5.93 x 10(-3) g/microKat. min and 1.49 g/L, respectively) and for amylopectin (7.40 x 10(-3) g/microKat+. min and 1.65 g/L, respectively) were determined.     

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillus phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5, a K(m) of 14 mM with alpha-D-Manp-(1-->6)-D-Manp as substrate. It was strongly inhibited by Mn(2+) and did not need Ca(2+) or any other metal cofactor of those tested. The enzyme cleaves specifically (1-->6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1-->3)-D-Manp 10 times faster than alpha-D-Manp-(1-->6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1-->2)-D-Manp. The activity towards (1-->3)-linked mannobiose is strongly activated by 1mM Ca(2+) and inhibited by 10mM EDTA, while (1-->6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities.     

Effect of seat positions on discomfort,muscle activation,pressure distribution and pedal force during cycling

The aim of this study was to measure and analyse discomfort and biomechanics of cycling, i.e., muscle activation, centre of pressure of seat pressure profiles and pedal forces as a function of seat position. Twenty-one recreationally active individuals cycled for 10 min at 100 W on an ergometer cycle using five different seat positions. The neutral position was considered as basic seat position and was compared with upward, downward, forward and backward seat positions. The initial bout was repeated at the end of the recording session. Discomfort increased for upward and backward condition compared with neutral (P < 0.05). Normalized surface electromyography from gastrocnemius decreased in the downward and forward position but increased in the upward and backward position. The minimum force became less negative for forward position compared with neutral seat position (P < 0.05). The degree of variability of centre of pressure increased in the upward and backward position and the entropy of the centre of pressure of sitting posture for backward position decreased compared with neutral seat position (P < 0.05). The present study revealed that consecutive changes of seat position over time lead to increase in discomfort as well as alterations of the biomechanics of cycling.     

Structural analyses of two arabinose containing oligosaccharides derived from olive fruit xyloglucan: XXSG and XLSG

Xyloglucan oligosaccharides were prepared by endo-(1-->4)-beta-D-glucanase digestion of alkali-extractable xyloglucan from olive fruit and purified by a combination of gel-permeation (Bio-Gel P-2) chromatography and high-performance anion-exchange chromatography. The two most abundant oligosaccharides were converted to the corresponding oligoglycosyl alditols by borohydride reduction and structurally characterised by NMR spectroscopy and post-source decay (PSD) fragment analysis of matrix-assisted laserinduced desorption/ionisation time-of-flight (MALDI-TOF) mass spectra. The results revealed that olive fruit xyloglucan is mainly built from two novel oligosaccharides: XXSG and XLSG. The structure of the oligosaccharides confirmed the presence of a specific xyloglucan in olive fruit with alpha-L-Araf-(1-->2)-alpha-D-Xylp sidechains as was suggested previously. The presence of such sidechains is a common feature of xyloglucans with an XXGG core produced by solanaceous plants but has not been demonstrated for other dicotyledonous plants, which have in general an XXXG core. Direct treatment of cell wall material from olive fruit with pectin degrading enzymes in combination with endo-(1-->4)-beta-D-glucanase revealed that some of the arabinose residues of the oligosaccharides XXSG and XLSG are substituted with either 1 or 2 O-acetyl groups.     

Improved reproducibility in the determination of the molecular weight of chitosan by analytical size exclusion chromatography

The reproducibility of the determination of the molecular weight of chitosans in the 90–210 kDa range (M_n) by analytical size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) was improved by reducing the salt concentration in the mobile phase from (0.3 M acetic acid, 0.2 M sodium acetate, and 0.8 mM sodium azide) to (0.15 M acetic acid, 0.1 M sodium acetate, and 0.4 mM sodium azide) using Tosoh TSKgel G6000PW_XL and G5000PW_XL columns in series. The variability of measured molecular weight was significantly reduced by lowering the acetate concentration in the mobile phase, while the average molecular weight did not change significantly. The coefficient of variation of the number-average molecular weight, CV(M_n), decreased from 7–12% to 3–6% upon mobile phase dilution. This reduced variability in molecular weight of chitosans obtained from SEC is a significant improvement when precise values of chitosan molecular weight are required, for example in stability studies where viscosity changes in concentrated chitosan solutions are assessed, and in gene delivery applications.     

The effect of agitation and aeration on the synthesis and molecular weight of gellan in batch cultures of Sphingomonas paucimobilis

The effects of agitation and aeration upon synthesis and molecular weight of the biopolymer gellan were systematically investigated in batch fermenter cultures of the bacterium, Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced growth of S. paucimobilis. Although gellan formation occurred mainly in parallel with cell growth, the increase in cells able to synthesise gellan did not always lead to high gellan production. For example, at very high agitation rates (1000 rpm) growth was stimulated at the expense of biopolymer synthesis.Maximal gellan concentration was obtained at 500 rpm agitation and either 1 or 2 vvm aeration (12.3 and 12.4 g/l gellan, respectively). An increase in aeration (from 1 to 2 vvm) enhanced gellan synthesis only at low agitation rates (250 rpm). However, high aeration or dissolved oxygen was not necessary for high gellan synthesis, in fact oxygen limitation always preceded the phase of maximum gellan production and probably enhanced polysaccharide biosynthesis.Some gellan was formed even after glucose exhaustion. This was attributed to the intracellular accumulation of polyhydroxyalkanoates, (such as polyxydroxybutyrate) which were found in S. paucimobilis cells indicating the existence of a carbon storage system, which may contribute to gellan biosynthesis under glucose-limiting conditions.The autolysis of the culture, which occurred at the late stages of the process, seemed to be triggered mainly by limitations in mass (nutrient) transfer, due to the highly viscous process fluid that gradually develops. Rheological measurements generally gave a very good near real time estimate of maximum biopolymer concentration offering the possibility of improved process control relative to time consuming gravimetric assay methods.While mechanical depolymerisation of gellan did not occur, high aeration rates (2 vvm) led to production of gellan of low molecular weight (at either 250 or 500 rpm). This effect of aeration rate upon gellan molecular weight is reported here for the first time, and is important for the properties and applications of gellan. Mechanisms which may have led to this are discussed, but control of molecular weight of the biopolymers is clearly an area needing further research.     

Activity of ligninase peroxidase from Acinetobacter anitratus and the degradation of Pinus pinaster lignin

The ligninase activity of Acinetobacter anitratus cells and enzyme extracted from that bacterium was measured with a model substrate (benzyl alcohol) and two lignin substrates: Pinus pinaster lignin precipitated from kraft black liquor and treated black liquor. The activity of this intracellular enzyme was confirmed on benzyl alcohol, showing a sigmoid Michaelis curve for constant H₂O₂ concentration. Enzymatic activity on treated black liquor leads to the production of low molecular weight compounds and to partial polymerisation of lignin. The evolution of the molecular weight distribution of the original sample with time was followed by high pressure liquid chromatography.     

Responses of non-spiking giant interneurons to substrate tilt in the crayfish, with special reference to multisensory control in the compensatory eyestalk movement system

In the brain of the intact crayfish, three pairs of non-spiking giant interneurons (G1, G2, G3; NGIs) scarcely responded to substrate tilt about the longitudinal axis of the body either in the dark or in the presence of an overhead light. However, when the statolith was removed, these NGIs responded with depolarizing and hyperpolarizing potentials respectively to upward movements of the ipsilateral legs (2nd–5th pereiopods) and upward movements of the contralateral legs produced by substrate tilt. The relationships between the polarity of the potential and the direction of movement in the contralateral legs were opposite to those in the ipsilateral legs. The amplitude of the responses was proportional to the frequency (0.5-0.05 Hz) and amplitude of tilting. When the legs were moved unilaterally, the NGIs responded with depolarizing and hyperpolarizing potentials to upward movements of the ipsilateral legs and to upward movements of the contralateral legs, respectively. When the legs were moved bilaterally in the same direction by upward or downward movement of the substrate, the NGIs scarcely responded to the leg movements. A hypothetical model is presented to account for the pathways of sensory inputs to the NGIs and the role of NGIs in compensatory oculomotor system.     

A gravity-induced regulation of swimming speed in Euglena gracilis

We investigated the autotrophic flagellate Euglena gracilis for gravity-induced modulation of the speed of swimming as previously documented for larger protozoan cells. Methods of video-tracking of swimming and sedimenting cells under 1 g and hypergravity up to 2 g, and computer-assisted data processing were applied. The vertical and horizontal swimming speed, and sedimentation rates of immobilized cells, were found to be linear functions of acceleration. Accounting for sedimentation in the observed upward and downward movements of Euglena, the active component of speed (propulsion) rose in proportion to acceleration. No saturation of gravikinesis was seen within the g-range tested. Gravity-dependent augmentation of speed was maximal in upward swimmers and decreased continuously over horizontal to downward swimmers. Linear extrapolations of the data to zero-g conditions suggest the absence of a threshold of gravikinesis in Euglena. Energetic considerations indicate a high sensitivity of gravitransduction near the level of Brownian molecular motion. Accepted: 22 August 1999     

Effects of light and low temperature on the reciprocal style curvature of Flexistylous Alpinia Species (Zingiberaceae)

The style curvature (flexistyly) of Alpinia species in ginger family is a unique plant organ movement because the style of each flower curves twice during its 1-day anthesis and styles of two phenotypes of each Alpinia species in the same population synchronously curve in opposite directions at the same time. In this study, we investigated the effects of low temperature and light conditions on these reciprocal style movements. Our results indicate that low temperature cannot change the direction of each curvature movement, but can slow down these movements and decrease the curve degrees. Light did not affect the upward curvature of the cataflexistylous morph, but the degrees of downward curvature decreased in darkness. For the anaflexistylous morph, the downward curvature only occurred in darkness, but curved directly upward in light condition; after the first (downward) curvature, the second (upward) movement only occurred in light, but did not occur if styles maintained in darkness. These results suggest that low temperature does not stimulate style curvature; light is the necessary condition for the upward movement of the anaflexistylous morph. The stimuli that induced curvature movements in the two morphs were different. Both two curvatures of the cataflexistylous style and downward movement of the anaflexistylous style were controlled via an endogenous program, while the upward movement of the anaflexistylous style was controlled by light.     

Sequencing of a 1,3-1,4-beta-D-glucanase (lichenase) from the anaerobic fungus Orpinomyces strain PC-2: properties of the enzyme expressed in Escherichia coli and evidence that the gene has a bacterial origin.

A 971-bp cDNA, designated licA, was obtained from a library of Orpinomyces sp. strain PC-2 constructed in Escherichia coli. It had an open reading frame of 738 nucleotides encoding LicA (1,3-1,4-beta-D-glucanase; lichenase) (EC 3.2.1.73) of 245 amino acids with a calculated molecular mass of 27,929 Da. The deduced amino acid sequence had high homology with bacterial beta-glucanases, particularly in the central regions and toward the C-terminal halves of bacterial enzymes. LicA had no homology with plant beta-glucanases. The genomic DNA region coding for LicA was devoid of introns. More than 95% of the recombinant beta-glucanase produced in E. coli cells was found in the culture medium and periplasmic space. A N-terminal signal peptide of 29 amino residues was cleaved from the enzyme secreted from Orpinomyces, whereas 21 amino acid residues of the signal peptide were removed when the enzyme was produced by E. coli. The beta-glucanase produced by E. coli was purified from the culture medium. It had a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gels. The Km and Vmax values with lichenin as the substrate at pH 6.0 and 40 degrees C were 0.75 mg/ml and 3,790 micromol/min/mg, respectively. With barley beta-glucan as the substrate, the corresponding values were 0.91 mg/ml and 5,320 micromol/min/mg. This enzyme did not hydrolyze laminarin, carboxymethylcellulose, pustulan, or xylan. The main products of lichenin and barley beta-glucan hydrolysis were triose and tetraose. LicA represented the first 1,3-1,4-beta-D-glucanase reported from fungi. The results presented suggest that licA of Orpinomyces had a bacterial origin.     

Effect of calmodulin antagonists on the growth and graviresponsiveness of primary roots of maize

We examined the effect of calmodulin (CaM) antagonists applied at the root tip on root growth, gravity-induced root curvature, and the movement of calcium across the root tip and auxin (IAA) across the elongation zone of gravistimulated roots. All of the CaM antagonists used in these studies delayed gravity-induced curvature at a concentration (1 M) that did not affect root growth. Calmodulin antagonists ( 1M) inhibited downward transport of label from ⁴⁵Ca²⁺ across the caps of gravistimulated roots relative to the downward transport of ⁴⁵Ca²⁺ in gravistimulated roots which were not treated with CaM antagonists. Application of CaM antagonists at the root tip ( 1M) also decreased the relative downward movement of label from ³H-IAA applied to the upper side of the elongation zone of gravistimulated roots. In general, tip application of antagonists inhibited neither the upward transport of ⁴⁵Ca²⁺ in the root tip nor the upward movement of label from ³H-IAA in the elongation zone of gravistimulated roots. Thus, roots treated with CaM antagonists ( 1 M) become less graviresponsive and exhibit reduced or even a reversal of downward polarity of calcium transport across the root tip and IAA transport across the elongation zone. The results indicate that calmodulin-regulated events play a role in root gravitropism.     

Construction of a fusion enzyme of dextransucrase and dextranase: Application for one-step synthesis of isomalto-oligosaccharides

The linear isomalto-oligosaccharides (IMO) with DP2–DP10 were produced by one-step process using engineered fusion enzyme (DXSR) of endo-dextranase and only α-(1–6) glucan synthesizing dextransucrase. The fusion enzyme was successfully expressed in Escherichia coli and characterized. Compared to individual enzymes, DXSR had 150% increased endo-dextranase activity and 98% decreased dextransucrase activity. The partially purified DXSR displayed molecular mass of 240 kDa as analyzed by SDS–PAGE. It showed both enzyme activities on analysis by zymogram. The thermal- and pH-stability of DXSR was around 28 °C and pH at 5.0–6.4, respectively. IMOs production by DXSR was increased by the addition of metal ions such as Fe²⁺, Li⁺, K⁺ and Ni²⁺, but the enzyme was strongly inhibited by Hg²⁺ and Ag⁺. DXSR produced linear IMO with DP2–DP10 using sucrose as a sole substrate. The molecular weight and amount of IMO could be controlled by the sucrose concentration. DXSR gave 30-fold higher production of IMO than that of an equal activity mixture of the two enzymes such as dextranase and dextransucrase.     

Efficient near infrared fluorescence detection of elastase enzyme using peptide-bound unsymmetrical squaraine dye

Extended wavelength analyte-responsive fluorescent probes are highly desired for the imaging applications owing to their deep tissue penetration, and minimum interference from autofluorescence by biomolecules. Near infra-red (NIR) sensitive and self-quenching fluorescent probe based on the dye-peptide conjugate (SQ 1 PC) was designed and synthesized by facile and efficient one-pot synthetic route for the detection of Elastase activity. In the phosphate buffer solution, there was an efficient quenching of fluorescence of SQ 1 PC (86%) assisted by pronounced dye-dye interaction due to H-aggregate formation. Efficient and fast recovery of this quenched fluorescence of SQ 1 PC (> 50% in 30 s) was observed on hydrolysis of this peptide-dye conjugate by elastase enzyme. Presently designed NIR sensitive self-quenching substrate offers the potential application for the detection of diseases related to proteases by efficient and fast detection of their activities.