Quartz crystal microbalance study of endothelial cell number dependent differences in initial adhesion and steady-state behavior: evidence for cell-cell cooperativity in initial adhesion and spreading

The quartz crystal microbalance (QCM) technique has been applied to the real time monitoring of endothelial cell (EC) adhesion and spreading on the QCM gold surface. We previously showed that the measured QCM Deltaf and DeltaR shifts were due to cells adhering to the gold crystal surface, requiring proteolytic enzyme treatment to be removed from the surface, in order for the Deltaf and DeltaR shifts to return to zero. In the present report, we demonstrate the quantitative dependence and saturation of the measured Deltaf and DeltaR shifts on the number of firmly attached ECs as measured by electronic counting of the cells. We demonstrate through a light microscope simulation experiment that the different Deltaf and DeltaR regions of the QCM temporal response curve correspond to the incident ECs contacting the surface, followed by their adhesion and spreading, which reflect cellular mass distribution and cytoskeletal viscoelasticity changes. Also, we demonstrate that the dose response curve of Deltaf and DeltaR values versus attached EC number is more sensitive and possesses less scatter for the hydrophilically treated surface compared to the native gold surface of the QCM. For both surfaces, a Deltaf and DeltaR versus trypsinized, attached EC number plot 1 h post-seeding exhibits a sigmoid curve shape whereas a similar plot 24 h post-seeding exhibits a hyperbolic curve shape. This number dependence suggests cell-cell cooperativity in the initial cell adhesion and spreading processes. These QCM data and our interpretation are corroborated by differences in cell appearance and spreading behavior we observed for ECs in a light microscope fluorescence simulation experiment of the cell density effect. For a stably attached EC monolayer at 24 h post-addition, steady-state Deltaf and DeltaR values are higher and exhibit saturation behavior for both the hydrophilically treated gold surface as compared to the untreated surface. The steady-state 24 h Deltaf and DeltaR values of stably attached ECs are shifted from the 1 h attached ECs. The 24 h values are characteristic of a more energy-dissipative structure. This is consistent with the time-dependent elaboration of surface contacts in anchorage-dependent ECs via the attachment of intregrins to underlying extracellular matrix. It is also in agreement with the known energy dissipation function of the ECs that cover the interior of blood vessels and are exposed to continuous pulsatile blood flow.     

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

A piezoelectric quartz crystal impedance study on Cu(2+)-induced precipitation of bovine serum albumin in aqueous solution

Piezoelectric quartz crystal impedance (QCI) technique was used for monitoring the Cu(2+)-induced precipitation of bovine serum albumin onto the gold electrode. The critical precipitate concentration of Cu(2+) reflected by the significant decrease in the resonant frequency was estimated to be 9.98 x 10(-5) mol x l(-1), and the saturated adherence of the precipitate on the electrode occurred when the Cu(2+) concentration was greater than 9.79x10(-3) mol x l(-1). The frequency shift in air was about 85.5% of that in liquid, and the Deltaf(0)/DeltaR(1) ratio found in solution was 82.67 Hz Omega(-1), suggesting that the frequency response was predominated by the mass change due to precipitate adherence to the electrode surface. The response of the resonant frequency was analyzed using an equation Deltaf=a(0) + a(1) e(-t/tau(1)) + a(2) e(-t/tau(2)). The relationship between the total a(0) values and the Cu(2+) concentration was discussed.     

alpha-Amylase immobilized on bulk acoustic-wave sensor by UV-curing coating

A new method for immobilization of alpha-amylase by UV-curing coating is proposed in this paper. The immobilization procedure of UV-curing coating on piezoelectric quartz crystal is simple and convenient, and causes less loss of enzymatic activity. The activity of the immobilized alpha-amylase is monitored by a technique based on bulk acoustic-wave (BAW) sensor. The frequency shift of BAW sensor can reflect the degree of hydrolysis of starch by the immobilized alpha-amylase. It is appropriate for the immobilized alpha-amylase to hydrolyze the soluble starch under pH 7.0 condition, which is similar to that of the free alpha-amylase. Kinetic parameters (the Michaelis constant, K(m), and the maximum initial rate V(max)) of the enzymatic hydrolysis of starch by the immobilized alpha-amylase are estimated by using a linear method of Lineweaver-Burk plot. K(m)=12.7mgml(-1) and V(max)=15.9Hzmin(-1). And the experimental results show that the immobilized alpha-amylase entrapped by the UV-curing coating retains adequate enzymatic activity and can be reused more than 50 times under certain experimental conditions.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Quartz crystal microbalance biosensor study of endothelial cells and their extracellular matrix following cell removal: Evidence for transient cellular stress and viscoelastic changes during detachment and the elastic behavior of the pure matrix

A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.     

Synergistic action of alpha-amylase and glucoamylase on hydrolysis of starch

Synergistic action of alpha-amylase and glucoamylase on hydrolysis of starch is modeled by the kinetic equations presented in this paper. At the early stage of the reaction alpha-amylase acts as a contributor of newly formed nonreducing ends of starch molecules to glucoamylase by splitting the original starch molecules. This is expressed by the simultaneous differential equations which consist of each rate equation for alpha-amylase and glucoamylase. After the molecular weight of the substrate decreases to the value of about 5000, which is obtained experimentally in this work, the action of alpha-amylase can be neglected and the rate of formation of glucose obeys only the rate equation for glucoamylase.     

Long-term monitoring of biofilm growth and disinfection using a quartz crystal microbalance and reflectance measurements

A biofilm reactor was constructed to monitor the long-term growth and removal of biofilms as monitored by the use of a quartz crystal microbalance (QCM) and a novel optical method. The optical method measures the reflectance of white light off the surface of the quartz crystal microbalance electrode (gold) for determination of the biofilm thickness. Biofilm growth of Pseudomonas aeruginosa (PA) on the surface was used as a model system. Bioreactors were monitored for over 6 days. Expressing the QCM data as the ratio of changes in resistance to changes in frequency (DeltaR/Deltaf) facilitated the comparison of individual biofilm reactor runs. The various stages of biofilm growth and adaptation to low nutrients showed consistent characteristic changes in the DeltaR/Deltaf ratio, a parameter that reflects changes in the viscoelastic properties of the biofilm. The utility of white light reflectance for thickness measurements was shown for those stages of biofilm growth when the solution was not turbid due to high numbers of unattached cells. The thickness of the biofilms after 6 days ranged from 48 mum to 68 mum. Removal of the biofilm by a disinfectant (chlorine) was also measured in real time. The combination of QCM and reflectance allowed us to monitor in real time changes in the viscoelastic properties and thickness of biofilms over long periods of time.     

Dynamic measurement of the surface stress induced by the attachment and growth of cells on Au electrode with a quartz crystal microbalance

The processes of adhesion, spreading and proliferation of human mammary cancer cells MCF-7 on two Au electrodes with different surface roughness (R(f) and R(f)=3.2 or 1.1) were monitored and clearly identified with the quartz crystal microbalance (QCM) technique. Analyses of the QCM responses on the resonant frequency shifts (Deltaf(0)) vs. the motional resistance changes (DeltaR(1)) revealed a significant surface-stress effect in the involved courses, in addition to a viscodensity effect and a relatively small mass effect (especially at the smooth electrode). Experiments of fluorescence microscopy, cyclic voltammetry and electrochemical impedance spectroscopy were conducted to investigate the cell population on the electrode vs. the electrode-surface roughness. Simplified equations are deduced to quantitatively evaluate the surface stress, and a novel QCM method for dynamically measuring the surface stress on an electrode in cell-culture course is thus described. It was found that the smoother surface (R(f)=1.1) gave a higher surface stress during cell attachment and less cell population on it than the rougher surface (R(f)=3.2). In addition, real-time QCM monitoring showed on the same electrode the surface stress induced by hepatic normal cells being notably higher than that caused by hepatic cancer cells at cell-attachment stage, suggesting that the surface-stress measurement can exhibit the difference of adhesion-performance between the healthy and ill-behaved cells.     

A quantitative assessment of the importance of barley seed alpha-amylase, beta-amylase, debranching enzyme, and alpha-glucosidase in starch degradation.

Extracts of germinated barley (Hordeum vulgare L.) seeds of 41 different genotypes were analyzed for their activities of alpha-amylase, beta-amylase, alpha-glucosidase, and debranching enzyme and for their abilities to hydrolyze boiled soluble starch, nonboiled soluble starch, and starch granules extracted from barley seeds with water. Linear correlation analysis, used to quantitate the interactions between the seven parameters, revealed that boiled soluble starch was not a good substrate for predicting activities of enzymes functioning in in vivo starch hydrolysis as the extracts' abilities to hydrolyze boiled soluble starch was not correlated with their abilities to hydrolyze native starch granules. Activities of alpha-amylase and alpha-glucosidase were positively and significantly correlated with the seed extracts' abilities to hydrolyze all three starches. beta-Amylase was only significantly correlated with hydrolysis of boiled soluble starch. No significant correlations existed between debranching enzyme activity and hydrolysis of any of the three starches. Interactions between the four enzymes as they functioned together to hydrolyze the three types of starch were evaluated by path coefficient analysis. alpha-Amylase contributed to hydrolyses of all three starches primarily by its direct effect (noninteractive component). This direct contribution increased as the substrate progressed from the completely artificial boiled soluble starch, to the most physiologically significant substrate, native starch granules. alpha-Glucosidase contributed to the hydrolysis of boiled soluble starch primarily by its direct effect (noninteractive) yet contributed to starch granule hydrolysis primarily via its interaction with alpha-amylase (indirect effect). The contribution of beta-amylase to hydrolysis of boiled soluble starch was direct and it did not contribute significantly to hydrolysis of native starch granules.     

Production of alpha-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

alpha-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55 degrees C and 5.5, respectively, and the apparent K(m) for starch was 0.8 mg ml. The products of alpha-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of alpha-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial alpha-amylase. Activities of alpha-amylase up to 4.4 U ml (1 U represents 1 mumol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml), alpha-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed alpha-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of alpha-amylase (initial concentration, 2.5 mg ml), they were found to be less effective than starch, but maltose was almost as effective. The fungal alpha-amylase was found to be stable at 60 degrees C in the presence of low concentrations of starch (相似文献   

Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase

Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the alpha-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 micromol min(-1) mg(-1)); acarbose is a strong inhibitor of CGTase and of most other alpha-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of alpha-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the alpha-amylase family, changed ATase into an enzyme with 4-alpha-glucanotransferase activity (3.4 micromol min(-1) mg(-1)). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 micromol min(-1) mg(-1)).     

The effect of process conditions on the alpha-amylolytic hydrolysis of amylopectin potato starch: An experimental design approach

The hydrolysis of amylopectin potato starch with Bacillus licheniformis alpha-amylase (Maxamyl) was studied under industrially relevant conditions (i.e. high dry-weight concentrations). The following ranges of process conditions were chosen and investigated by means of an experimental design: pH [5.6-7.6]; calcium addition [0-120 microg/g]; temperature [63-97 degrees C]; dry-weight concentration [3-37% [w/w]]; enzyme dosage [27.6-372.4 microL/kg] and stirring [0-200 rpm]. The rate of hydrolysis was followed as a function of the theoretical dextrose equivalent. The highest rate (at a dextrose equivalent of 10) was observed at high temperature (90 degrees C) and low pH (6). At a higher pH (7.2), the maximum temperature of hydrolysis shifted to a lower value. Also, high levels of calcium resulted in a decrease of the maximum temperature of hydrolysis. The pH, temperature, and the amount of enzyme added showed interactive effects on the observed rate of hydrolysis. No product or substrate inhibition was observed. Stirring did not effect the rate of hydrolysis. The oligosaccharide composition after hydrolysis (at a certain dextrose equivalent) did depend on the reaction temperature. The level of maltopentaose [15-24% [w/w]], a major product of starch hydrolysis by B. licheniformis alpha-amylase, was influenced mostly by temperature.     

Paradoxical effects of substitution and deletion mutation of Arg56 on the structure and chaperone function of human alphaB-crystallin

Human alphaB-crystallin is a small heat-shock protein that functions as a molecular chaperone. Recent studies indicate that deletion of a peptide (54FLRAPSWF61) from its N-terminus makes it a better chaperone, and this particular sequence is thought to participate in substrate interaction and subunit exchange with alphaA-crystallin. To determine whether the positive charge on arginine 56 (R56) influences these functions, we prepared human alphaB-crystallin mutants in which R56 was deleted (DeltaR56) or replaced by alanine (R56A). To determine if the effects are specific to R56, we generated two additional mutant proteins in which the two neighboring amino acids were deleted (DeltaL55 and DeltaA57). Dynamic light scattering studies suggested that none of the mutations affected the oligomeric mass of the protein. Far-ultraviolet circular dichroism (UV CD) spectra revealed greater helicity in the secondary structures of R56A and DeltaR56 compared to that of the wild-type (Wt) protein. Near-UV CD spectra showed that the tertiary structure is perturbed in all mutants. Insulin and citrate synthase aggregation assays showed 38 and 30% improvement of chaperone function in DeltaR56 compared to that of the Wt. In contrast, the R56A mutant lost most of its chaperone function. Deletion mutants, DeltaL55 and DeltaA57, showed no significant changes in the chaperone function compared to that of the Wt. The DeltaR56 mutant had a higher surface hydrophobicity than the Wt, but the R56A mutant had a lower hydrophobicity. Our data show paradoxical effects of the deletion and substitution of R56 and imply that the chaperone function of human alphaB-crystallin is dictated not only by the positive charge on R56 but also by the conformational change that it bestows on the protein.     

Production and partial characterization of thermostable and calcium-independent alpha-amylase of an extreme thermophile Bacillus thermooleovorans NP54

AIM: An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization. METHODS AND RESULTS: The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively. The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C. Both alpha-amylase activity and production were Ca2+ independent. CONCLUSIONS: Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase. SIGNIFICANCE AND IMPACT OF THE STUDY: The calcium-independent and thermostable alpha-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.     

Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum

An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60 degrees C, respectively. In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity. The alpha-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).     

High-efficiency, one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse alpha-amylase.

Transformed, hybrid Saccharomyces strains capable of simultaneous secretion of glucoamylase and alpha-amylase have been produced. These strains could carry out direct, one-step assimilation of starch, with conversion efficiency greater than 93% during a 5-day growth period. One of the transformants converted 92.8% of available starch into reducing sugars in only 2 days. Glucoamylase secretion by these strains resulted from expression of one or more chromosomal STA genes derived from Saccharomyces diastaticus. The strains were transformed by a plasmid (pMS12) containing mouse salivary alpha-amylase cDNA in an expression vector containing yeast alcohol dehydrogenase promoter and a segment of yeast 2 micron plasmid. The major starch hydrolysis product produced by crude amylases found in culture broths was glucose, indicating that alpha-amylase and glucoamylase acted cooperatively.     

High-efficiency, one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse alpha-amylase

Transformed, hybrid Saccharomyces strains capable of simultaneous secretion of glucoamylase and alpha-amylase have been produced. These strains could carry out direct, one-step assimilation of starch, with conversion efficiency greater than 93% during a 5-day growth period. One of the transformants converted 92.8% of available starch into reducing sugars in only 2 days. Glucoamylase secretion by these strains resulted from expression of one or more chromosomal STA genes derived from Saccharomyces diastaticus. The strains were transformed by a plasmid (pMS12) containing mouse salivary alpha-amylase cDNA in an expression vector containing yeast alcohol dehydrogenase promoter and a segment of yeast 2 micron plasmid. The major starch hydrolysis product produced by crude amylases found in culture broths was glucose, indicating that alpha-amylase and glucoamylase acted cooperatively.     

The effect of increased background resistance on the resistive load threshold for eliciting the respiratory-related evoked potential.

The detection threshold (DeltaR(50)) of resistive (R) loads is a function of the total background resistance (R(0)). Increased R(0) increases the DeltaR(50), but the ratio DeltaR(50)/R(0) remains constant. The respiratory-related evoked potential (RREP) is elicited only by R loads greater than the cognitive detection threshold, DeltaR(50). We hypothesized that the RREP Nf, P1, and N1 peaks will be elicited only when the added load DeltaR/R(0) is greater than the normal detection threshold, DeltaR(50)/R(0) = 0.30. We also hypothesized that when the R(0) is increased by adding extrinsic R, the RREP will not be elicited if the DeltaR/R(0) is less than the 0.30 ratio. RREPs were recorded with healthy volunteers (n = 20) respiring through a non-rebreathing valve. Three inspiratory R loads that spanned the DeltaR(50)/R(0) = 0.30 detection threshold were presented in two conditions: 1) no added R(0) (R1 < 0.30, R2 > 0.30, R3 > 0.30); and 2) increased R(0) = 13.3 cmH(2)O.l(-1).s (R1 < 0.30, R2 < 0.30, R3 > 0.30). For the control R(0), P1, Nf, and N1 peaks of the RREP were elicited by both R2 and R3, and not present with R1. The increased R(0) decreased R2/R(0) > 1.5 to R2/R(0) < 0.15. With increased R(0), the R1 and R2 loads did not elicit the RREP, but the Nf, P1, and N1 peaks were present for R3. These results demonstrate that the RREP is present if the DeltaR is above the cognitive detection threshold, and the RREP is absent if the load is below the detection threshold. When the R(0) is increased to make the DeltaR/R(0) less than the detection threshold, the DeltaR no longer elicits the RREP.     

Continuous enzymatic liquefaction of starch for saccharification

A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequently saccharification to glucose. The process appears to be quite efficient for conversion of starch to glucose and enzymatic liquefaction and should be readily adaptable to industrial fermentation processes. Preliminary work indicated that milled corn or other cereal grains also can be suitably converted by such a process. Essentially, the process involved incorporation of a thermostable, bacterial alpha-amylase for liquefaction and, subsequently, of a glucoamylase into the continuous mixer under conditions conductive to rapid enzymatic hydrolyses. Also studied was the effect on substrate liquefaction of variable such as starch concentration (40-70 degrees ), level of alpha-amylase (0.14-0.4%, dry starch basis), temperature (70-100 degrees C), pH (5.8-7.1), and residence time (6 and 12 min). The degree of liquefaction was assessed by determining (1) the Brookfield viscosity, (2) the amount of reducing groups, and (3) the rate and extent of glucose formed after glucoamylase treatment. Best liquefaction process conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6-min residence period in the mixture. Under these conditions, rate and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24h with the use of 0.4% glucoamylase was 86% of theory after a 6-min continuous liquefaction, compared to 90% for a 30-min laboratory batch liquefaction (95 degrees C, 0.4% alpha-amylase).