One-pot fluorescent labeling of xyloglucan oligosaccharides with sulforhodamine

Xyloglucan oligosaccharides fluorescently labeled with sulforhodamine have proved to be a valuable tool in the assessment of transglycosylating activity of plant xyloglucan endotransglucosylase/hydrolase (XTH; EC 2.4.1.207). Here we describe a simple and fast procedure for their preparation. Accordingly, the starting xyloglucan-derived oligosaccharides are in the first step converted to their corresponding 1-amino-1-deoxyalditols (glycamines) by incubation with ammonium acetate and NaCNBH(3) at 80 degrees C for 2-4 h, and in the second step, the glycamines are reacted with Lissamine rhodamine B sulfonyl chloride to obtain fluorescently labeled derivatives of the oligosaccharide glycamines. All operations are carried out in a single centrifuge tube and the products from the individual reaction steps are isolated on the basis of their differential solubility in organic solvents. Using the described protocol, the whole procedure can be accomplished in less than 24 h. The sulforhodamine-labeled xyloglucan oligosaccharides thus obtained proved suitable as substrates for a sensitive fluorescence assay of the transglycosylating activity of XTH.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Influence of borate complexation on the electrophoretic behavior of 2-AA derivatized saccharides in capillary electrophoresis

A complete separation with baseline resolution of the 2-AA derivatized saccharides, including mono-, di-, and oligosaccharides, was achieved using 50 mM sodium phosphate-150 mM borate solution, pH 7.0 as running buffer by capillary electrophoresis. It was thought to be a result of the inclusion of 150 mM borate in the running electrolyte solution. The formation of borate complexes was observed by means of ¹¹B and ¹³C NMR spectroscopy and the electrophoretic mobilities of the various derivatives were calculated. It was found that steric factors play an important role in the stability of the formed borate complexes, which depends strongly on the configuration of the three vicinal hydroxyl groups at C-2, C-3, and C-4. 2-AA-Glc mainly forms stable 1,2-diester complexes with borate and 2-AA-Mal can form stable 1,2-monoesters. In turn, for 2-AA-Rib the formation of complexes is difficult to take place. The results implied that the configurational difference between the hydroxyl groups could cause the difference in formation of borate complexes leading to significant difference among saccharide molecules in their migration time on CE analysis.     

Transformation of 2-aminoacetophenone to (S)-2-amino-1-phenylethanol by Arthrobacter sulfureus

A new isolate of Arthrobacter sulfureus , when incubated at 50 g resting cells (dry cell wt) l(-1) with 50 g glucose l(-1) and 1 g 2-aminoacetophenone l(-1) in 50 mm potassium buffer (pH 7, 4 ml) at 30 degrees C, produced ( S )-2-amino-1-phenylethanol (e.e. >99%) with 75% yield in 6 h.     

Single particle analysis of thylakoid proteins from Thermosynechococcus elongatus and Synechocystis 6803: localization of the CupA subunit of NDH-1

The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from Synechocystis DeltacupA and DeltacupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO(2) or 3% CO(2) indicates that the number of NDH-1MS particles is 30-fold higher under low-CO(2).     

Capillary electrophoresis-based nanoscale assays for monitoring ecto-5'-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT.     

1-Aminocyclopropane-1-carboxylic-acid-dependent ethylene production during re-formation of vacuoles in evacuolated protoplasts of Petunia hybrida

Summary Ethylene formation from 1-aminocycloprane-1-carboxylic acid (ACC) was studied in whole protoplasts, evaluolated protoplasts and isolated vacuoles from mesophyll cells of Petunia hybrida L. cv. Pink Magic. The re-formation of the large, central vacuole in evacuolated protoplasts and morphological characteristics of both types of protoplasts were examined by electron microscopy. Both the normal, whole protoplasts and vacuoles isolated from them produced ethylene from ACC at similar rates. Freshly-prepared evacuolated protoplasts had lost the capacity to produce ethylene. Re-formation of the central vacuole in these evacuolated protoplasts occurred between 14 to 17 h of incubation in the recovery medium and was followed by the development of ethyleneforming activity. Both these processes were inhibited by cycloheximide, indicating a requirement for new protein synthesis. Light stimulated the conversion of ACC to ethylene in both the regenerating, whole protoplasts and the evacuolated protoplasts that had re-formed the central vacuole.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CHI cycloheximide     

Do polyamines contribute to plant cell wall assembly by forming amide bonds with pectins?

Two new reducing glycoconjugates [N-D-galacturonoyl-putrescinamide (GalA-Put) and N,N'-di-D-galacturonoyl-putrescinamide (GalA-Put-GalA)] and homogalacturonan-putrescine (GalAn-Put) conjugates were synthesised as model compounds representing possible amide (isopeptide) linkage points between a polyamine and either one or two pectic galacturonate residues. The amide bond(s) were stable to cold acid and alkali (2M TFA and 0.1M NaOH at 25 degrees C) but rapidly hydrolysed by these agents at 100 degrees C. The amide bond(s) were resistant to Driselase and to all proteinases tested, although Driselase digested GalAn-Put, releasing fragments such as GalA3-Put-GalA3. To trace the possible formation of GalA-polyamine amide bonds in vivo, we fed Arabidopsis and rose cell-cultures and chickpea internodes with [14C]Put. About 20% of the 14C taken up was released as 14CO2, indicating some catabolism. An additional approximately 73% of the 14C taken up (in Arabidopsis), or approximately 21% (in rose), became ethanol-insoluble, superficially suggestive of polysaccharide-Put covalent bonding. However, much of the ethanol-inextractable 14C was subsequently extractable by acidified phenol or by cold 1M TFA. The small proportion of radioactive material that stayed insoluble in both phenol and TFA was hydrolysable by Driselase or hot 6M HCl, yielding 14C-oligopeptides and/or amino acids (including Asp, Glu, Gly, Ala and Val); no free 14C-polyamines were released by hot HCl. We conclude that if pectin-polyamine amide bonds are present, they are a very minor component of the cell walls of cultured rose and Arabidopsis cells and chickpea internodes.     

Oligomerization of the SPP1 scaffolding protein

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His₆) and purified. The protein is an α-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His₆ is structurally a dimer of dimers. Incubation at temperatures above 37 °C correlated with a reduction of its α-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 °C. Refolding of gp11-His₆ thermally denatured at 65 °C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (∼ 90%) and a smaller fraction of 2.4 S dimers (∼ 10%). This population of gp11-His₆ was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His₆ showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His₆.     

High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes--cellulase, pepsin and lipase A--were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a 20h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 degrees C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1h.     

A novel 5-fluorouracil prodrug using hydroxyethyl starch as a macromolecular carrier for sustained release

The objective of this study was to develop a sustained-release drug delivery system for 5-fluorouracil (5-FU) to improve its short half-life. 5-Fluorouracil-1-acetic acid (FUAC) was prepared and then conjugated to hydroxyethyl starch (HES) through ester bonds. The conjugates were relatively stable in acidic buffer solution at pH 5.8 and slowly released FUAC but became more sensitive to hydrolysis with an increase in the pH and temperature. The conjugates were degraded to FUAC both in human and rat plasma with half-time life of 20.4 h and 24.6 h, respectively. Both 5-FU and FUAC were released in a rat liver homogenate following a 12 h incubation of the conjugates. The pharmacokinetic behavior was evaluated in rats after intravenous injection of 5-FU, FUAC and the conjugates. The drug release data in vitro and in vivo indicated that HES is a promising carrier for the sustained-release of antitumor drugs.     

Contributions in astrocytes of SMIT1/2 and HMIT to myo-inositol uptake at different concentrations and pH

myo-Inositol is important for cell signaling both in cytoplasm and in intracellular organelles. It is required in the plasma membrane and cytoplasm for maintained synthesis of the second messengers, inositoltrisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol bisphosphate (PIP(2)), and in organelles as precursor for synthesis of complex signaling phospholipids and inositolphosphates from IP(3) and PIP(2). myo-Inositol must be taken up into the cell where its is used, because neither neurons nor astrocytes synthesize it. It is also an osmolyte, taken up in response to surrounding hyperosmolarity and released during hypo-osmolarity. There are three myo-inositol transporters, the Na(+)-dependent SMIT1 and SMIT2, and HMIT, which co-transports myo-inositol with H(+). Their relative expressions in astrocytes and neurons are unknown. Uptake kinetics for myo-inositol in astrocytes has repeatedly been determined, but always on the assumption of only one component, leaving kinetics for the individual transporters unknown. This paper demonstrates that astrocytes obtained directly from the brain express SMIT1 and HMIT, but little SMIT2, and that all three transporters are expressed in neurons. Cultured mouse astrocytes show a high-affinity/low-capacity myo-inositol uptake (V(max): 60.0 ± 3.0 pmol/min per mg protein; K(m): 16.7 ± 2.6 μM), mediated by SMIT1 and perhaps partly by SMIT2. It was determined in cells pre-treated with HMIT-siRNA and confirmed by specific inhibition of SMIT. However at physiologically relevant myo-inositol concentrations most uptake is by a lower-affinity/higher-capacity uptake, mediated by HMIT (V(max): 358 ± 60 pmol/min per mg protein; K(m): 143 ± 36 μM) and determined by subtraction of SMIT-mediated from total uptake. At high myo-inositol concentrations, its uptake is inhibited by incubation in medium with increased pH, and increased during intracellular acidification with NH(4)Cl. This is in agreement with literature data for HMIT alone. At low concentration, where SMIT1/2 activity gains importance, myo-inositol uptake is reduced by ammonia-induced intracellular acidification, consistent with the transporter's pH sensitivity reported in the literature.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Biotransformation of Acetamide to Acetohydroxamic Acid at Bench Scale Using Acyl Transferase Activity of Amidase of <Emphasis Type="Italic">Geobacillus pallidus</Emphasis> BTP-5x MTCC 9225

The bioprocess employing acyl transferase activity of intracellular amidase of Geobacillus pallidus BTP-5x MTCC 9225 was harnessed for the synthesis of pharmaceutically important acetohydroxamic acid. G. pallidus BTP-5x exhibited highest acyl transferase activity with acetamide: hydroxylamine in ratio of 1:5 in 0.1 M NaH₂PO₄/Na₂HPO₄ buffer (pH 7.5) at 65°C. In one liter fed-batch reaction containing 1:5 ratio of two substrates total of eight feedings of 0.05 M/20 min of acetamide were made and it was found that maximum acetohydroxamic production was achieved at 3:5 ratios of substrate and cosubstrate. In 1 l bench scale batch reaction containing 0.3 M acetamide, 0.5 M hydroxylamine in 0.1 M NaH₂PO₄/Na₂HPO₄ buffer (pH 7.5, 50°C, 400 rpm) and 0.5 mg/ml (dry cell weight) of whole cells of G. pallidus BTP-5x (as biocatalyst) resulted in an yield of 0.28 M of acetohydroxamic acid after 20 min reaction time at 50°C. The acetamide bioconversion rate was 90–95% (mol mol⁻¹) and 51 g powder containing 40% (w/w) acetohydroxamic acid was recovered after lyophilization.     

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).     

Enzymatic synthesis of two novel non-reducing oligosaccharides using transfructosylation activity with β-fructofuranosidase from Arthrobacter globiformis

Two novel non-reducing oligosaccharides together with tri- and tetra-saccharides were synthesized by transfructosylation activity from sucrose as a donor and cellobiose or cellotriose as an acceptor with a purified beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and these oligosaccharides were identified as O-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside and O-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->2)-alpha,beta-D-fructofuranoside by spectrometric analyses. Both oligosaccharides were stable under condition at 100 degrees C for 30 min, and showed no degradation at pH 2.     

Drought stress increases the production of 5-hydroxynorvaline in two C4 grasses

Plants produce various compounds in response to water deficit. Here, the presence and identification of a drought-inducible non-protein amino acid in the leaves of two C₄ grasses is first reported. The soluble amino acids extracted from the leaves of three different species were measured by high-performance liquid chromatography of derivatives formed with o-phthaldialdehyde and β-mercaptoethanol. One amino acid that increased in amount with drought stress had a retention time not corresponding to any common amino acid. Its identity was determined by metabolite profiling, using ¹H NMR and GC-MS. This unusual amino acid was present in the dehydrated leaves of Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel, but was absent from Paspalum dilatatum Poir. Its identity as 2-amino-5-hydroxypentanoic acid (5-hydroxynorvaline, 5-HNV) was confirmed by synthesis and co-chromatography of synthetic and naturally occurring compounds. The amount of 5-HNV in leaves of the more drought tolerant C₄ grasses, C. dactylon and Z. japonica, increased with increasing water deficit; therefore, any benefits from this unusual non-protein amino acid for drought resistance should be further explored.     

Putative TRP channel antagonists, SKF 96365, flufenamic acid and 2-APB, are non-competitive antagonists at recombinant human α1β2γ2 GABA(A) receptors

Although transient receptor potential (TRP) channel biology research has expanded rapidly in recent years, the field is hampered by the widely held, but relatively poorly investigated, belief that most of the pharmacological tools used to investigate TRP channel function may not be particularly selective for their intended targets. The objective of this study was therefore to determine if this was indeed the case by systematically evaluating the effects of three routinely used putative TRP channel antagonists, SKF 96365, flufenamic acid (FF) and 2-aminoethoxydiphenyl borate (2-APB) against one of the most widely expressed CNS receptor subtypes CNS, the human α1β2γ2 GABA(A) receptor. Using whole cell patch-clamp recording to record responses to rapidly applied GABA in the absence and presence of the three putative antagonists in turn we found that SKF 96365 (1-100 μM) and FF (1-100 μM) significantly inhibited GABA responses of recombinant human α1β2γ2 GABA(A) receptor stably expressed in HEK293 cells with IC(50) values of 13.4 ± 5.1 and 1.9 ± 1.4 μM, respectively, suppressing the maximal response to GABA at all concentrations used in a manner consistent with a non-competitive mode of action. SKF 96365 and FF also both significantly reduced desensitisation and prolonged the deactivation kinetics of the receptors to GABA (1mM; P<0.05). 2-APB (10-1000 μM) also inhibited responses to GABA at all concentrations used with an IC(50) value of 16.7 ± 5.4 μM (n=3-5) but had no significant effect on the activation, desensitisation or deactivation kinetics of the GABA responses. Taken together this investigation revealed that these widely utilised TRP channel antagonists display significant 'off-target' effects at concentrations that are routinely used for the study of TRP channel function in numerous biological systems and as such, data which is obtained utilising these compounds should be interpreted with caution.     

Molecular properties of yeast glyceraldehyde-3-phosphate dehydrogenase in the presence of ATP and KCl.

The influence of ATP and KCl on the quaternary structure and the enzymatic activity of D-glyceraldehyde-3-phosphate dehydrogenase from yeast(Y-GAPDH) has been studied by ultracentrifugation, gel chromatography and standard optical tests. In 0.1 M imidazole buffer pH 7.0, at low temperature (0°C) both complete deactivation and dissociation to dimers occur in the presence of 2 mM ATP and 0.1 M 2-mercaptoethanol. In 0.067 M phosphate buffer pH 7.0, containing 2 mM ATP and 1 mM dithiothreitol, only slight deactivation paralleled by minor changes of the native quaternary structure is observed. In this same buffer, increasing temperature leads to stabilization of both the tetrameric state and the catalytic activity of the enzyme. Deactivation and dissociation in the presence of 0.15 M KCl (in 0.2 M glycine buffer 9.1 ≥ pH ≥ 8.0) is a function of pH rather than electrolyte concentration; at neutral pH the enzyme is stabilized in its native state. Contrary to earlier assumptions in the literature, ATP and KCl under the above experimental conditions do not appear to play an important role in the $\text{in vivo}$ regulation of Y-GAPDH.     

Optimization of Process Variables for Lipase Biosynthesis from <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> NRRL 5905 Using Evolutionary Operation Factorial Design Technique

Extracellular lipase was produced from Rhizopus oligosporus NRRL 5905 through solid state fermentation (SSF). To provide an optimum fermentation conditions for maximum lipase yield, five process variables (temperature, liquid–solid ratio, pH, incubation period and spore concentration) were optimized using evolutionary operation (EVOP) factorial design technique taking into account the interaction between the process variables. Optimization through EVOP resulted in around 3 fold increase in lipase activity (77 U gds⁻¹) at a liquid–solid ratio of 1.5:1, fermentation temperature of 35°C, initial fermentation pH 6, incubation period 5 days and a spore concentration of 10⁸ spores ml⁻¹.