Photocontrol of urease activity in spiropyran collagen membrane.

1. Collagen fibrils were modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyran)] propionic anhydride. 2. Urease (urea amidohydrolase, EC 3.5.1.5) was immobilized in spiropyran collagen membrane. The activity of the urease-spiropyran collagen membrane was found to increase in the dark and then decrease with visible light irradiation. 3. The optimum pH of the urease-spiropyran collagen membrane under visible light was lowered in the dark. 4. The apparent Michaelis constant (K'm) of the urease-spiropyran collagen membrane in the dark was almost the same as that under visible light. The apparent maximum velocity was increased in the dark. 5. The diffusion coefficient of urea through the spiropyran collagen membrane in the dark was 1.4 times that under visible light. However, the increase of the diffusion rate was not responsible for the activity increase of the urease-spiropyran collagen membrane.     

Chymotrypsin adsorbed to a polyethylene terephtalate support (SORSILEN) as a suitable model for the investigation of enzymatically catalyzed organic syntheses in nonaqueous media

Summary The kinetics of acetyl-L-tryptophan esterification by ethanol in an organic solvent immiscible with water (chloroform) and catalyzed by chymotrypsin adsorbed from aqueous solution to a polyethylene terephthalate copolymer (SORSILEN) was examined by HPLC. The esterification yield (in %) increased with the decreasing concentration of the substrate and with the increasing activity of immobilized chymotrypsin. It has been shown that there was no chymotrypsin leakage from the aqueous phase on the support surface during the catalysis.     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Cerebral proteinases in the growing rat

—The proteolytic activity of brain homogenates obtained from 1-, 5-, 14-, 60-, 150-, and 300-day-old rats was assayed with urea-denatured haemoglobin and casein, endogenous tissue proteins, Nα-benzoyl-dl-arginine 2-naphtylamide (BANA), N^α-benzoyl-dl-arginine methyl ester (BAME), N^α-toluene p-sulphonyl-dl-arginine methyl ester (TAME), N^α-benzoyl-dl-phenylalanine 2-naphthyl ester (BPANE), and N^α-acetyl-dl-tyrosine ethyl ester (ATEE) as substrates. Several peaks of activity were detected with all these substrates in different pH ranges. Activity was highest with protein substrates at pH 3·0-4·0, with smaller peaks of activity at pH 5·5-6·5 and 8·0-9·0. At pH 3·0 the activity with trypsin substrates, viz. BANA, BAME and TAME, was also relatively high, but much less with chymotrypsin substrates, ATEE or BPANE. With BAME, TAME, BPANE and ATEE the hydrolysis rate was highest at neutral or slightly alkaline pH. During postnatal development the hydrolysis of protein substrates increased three-fold at pH 3·0 and about two-fold at pH 6·5 and 8·5. The rate of hydrolysis of BANA, BAME and TAME generally increased during the first 2 postnatal weeks and thereafter decreased, whereas no marked increase in the rate of hydrolysis of BPANE and ATEE occurred until the age of about 2 weeks. The results were less consistent with synthetic substrates than with protein substrates, indicating the existence of non-uniform alterations during development in the activity of the individual hydrolytic enzymes participating in the breakdown of brain proteins.     

Evidences for the presence of chymotrypsin-like activity in human spermatozoa with a role in the acrosome reaction

The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 10⁷ cells/ml, 37°C, and 5% CO₂, After 4.5 hr, the chymotrypsin inhibitor TPCK (N-Tosyl-L-Phenylalanine-Chloromethyl Ketone) or the substrate ATEE (N-Acetyl-L-Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome-reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida-induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester), Succinyl-Ala-Ala-Phe-7-Amido-4-Methyl-Coumarin (Suc-Ala-Ala-Phe-AMC), and Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methyl-Coumarin (Suc-Leu-Leu-Val-Tyr-AMC) inhibited the human follicular fluid-induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca²⁺, and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin-like activity with a possible role in the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Substrate activation of rat trypsins 1 and 2

Qualitative differences in the active center of rat trypsins 1 and 2 resulted in different ratios of K_cat for N¹-tosyl-l-arginine methyl ester vs K_cat for N¹-benzoyl-l-arginine ethyl ester. These ratios were 2.5 for trypsin 1 and 1.2 for trypsin 2.Substrate activation with N¹-tosyl-l-arginine methyl ester enhanced the catalytic rate constant of rat trypsin 1 2.5-fold and that of rat trypsin 2 only 1.5-fold. The increase in the catalytic rate constant found with N¹-benzoyl-l-arginine ethyl ester was the same (1.5-fold) for both trypsins. Consequently, at 20 mm substrate concentration, trypsin 1 catalyzed the esterolysis of N¹-tosyl-l-arginine methyl ester 4.5 times faster than that of N¹-benzoyl-l-arginine ethyl ester, while trypsin 2 was only 1.3 times more efficient with the first substrate.Furthermore, the activation of both rat enzymes by N-acetyl-l-tyrosine ethyl ester was even more effective than that obtained with the two cationic esters; the maximum rates of hydrolysis of this neutral substrate by trypsins 1 and 2 were enhanced 120- and 50-fold, respectively, by high concentrations of N-acetyl-l-tyrosine ethyl ester.     

Photosystem II Cyclic Heterogeneity and Photoactivation in the Diazotrophic, Unicellular Cyanobacterium Cyanothece Species ATCC 51142

The unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 demonstrated important modifications to photosystem II (PSII) centers when grown under light/dark N₂-fixing conditions. The properties of PSII were studied throughout the diurnal cycle using O₂-flash-yield and pulse-amplitude-modulated fluorescence techniques. Nonphotochemical quenching (q_N) of PSII increased during N₂ fixation and persisted after treatments known to induce transitions to state 1. The q_N was high in cells grown in the dark, and then disappeared progressively during the first 4 h of light growth. The photoactivation probability, ε, demonstrated interesting oscillations, with peaks near 3 h of darkness and 4 and 10 h of light. Experiments and calculations of the S-state distribution indicated that PSII displays a high level of heterogeneity, especially as the cells prepare for N₂ fixation. We conclude that the oxidizing side of PSII is strongly affected during the period before and after the peak of nitrogenase activity; changes include a lowered capacity for O₂ evolution, altered dark stability of PSII centers, and substantial changes in q_N.     

Chemically modified porous silica gel as a bioadsorbent and a biocatalyst.

Aminopropyl silica gel was prepared from porous silica gel by reaction with γ-aminopropyltrimethoxysilane in toluene and was used for immobilizing chymotrypsin (EC 3.4.4.5) and human serum albumin. Immobilized chymotrypsin was used for the resolution of N-acetyl-dl-phenylalanine and immobilized human serum albumin was used for the purification of goat anti-human serum albumin. Epoxy silica gel, prepared by reaction of porous silica gel with γ-glycidoxypropyltriethoxysilane, was coupled with m-aminobenzamidine and the resulting matrix was used for trypsin purification.     

Synthesis of 2-ethyl hexanol fatty acid esters in a packed bed bioreactor using a lipase immobilized on a textile membrane

An enzymatic process using a packed bed bioreactor with recirculation was developed for the scale-up synthesis of 2-ethylhexyl palmitate with a lipase from Candida sp. 99–125 immobilized on a fabric membrane by natural attachment to the membrane surface. Esterification was effectively performed by circulating the reaction mixture between a packed bed column and a substrate container. A maximum esterification yield of 98% was obtained. Adding molecular sieves and drying the immobilized lipase both decreased the water content at the reactor outlet and around the enzyme, which led to an increase in the rate of esterification. The long-term stability of the reactor was tested by continuing the reaction for 30 batches (over 300 h) with an average esterification yield of about 95%. This immobilized lipase bioreactor is scalable and is thus suitable for industrial production of 2-ethylhexyl palmitate.     

Effect of Na(3)VO(4) on the P State of Nitella translucens

The capacity of sodium orthovanadate to inhibit the plasmalemma H⁺ ATPase of Nitella translucens internodal cells in vivo was tested. Here we show that 1 millimolar vanadate added externally depolarizes strongly and permanently the membrane potential, both in dark and light, to the Nernst potential for potassium consistent with pump inhibition by vanadate. From the results it is clear that the H⁺ ATPase is always active, under light or dark conditions, in contradiction with the widespread idea of pump inactivation by darkness. The changes in conductance for light, dark, and vanadate-induced conditions are analyzed. The effect of dark on membrane passive permeabilities and on the possibility that some plasmalemma channels could be regulated by a phosphorylation-dephosphorylation process is discussed.     

The membrane potential of intact Rhodospirillum rubrum cells in the absence of light-dependent and oxygen-linked electron transfer

In the absence of oxygen-linked and light-dependent electron transfer, the steady-state membrane potential of intact Rhodospirillum rubrum cells was usually between 65 and 75% of that of dark aerated cells, as indicated by the relative extent of the bacteriochlorophyll electrochromic changes that were induced by oxygen and by uncouplers. That potential was not due to residual levels of oxygen or light, because its value was not significantly altered by the presence of oxygen-trapping systems or by exhaustive gassing with Ar, and because it was also exhibited by a reaction-center-less mutant. The dark anaerobic potential was unaffected by 0.11 M K⁺; that seemed to exclude a diffusion potential generated by dissipation of a previously built K⁺ gradient. In contrast, it was largely abolished by 0.5 mM N,N′-dicyclohexylcarbodiimide, suggesting its dependence on ATP hydrolysis by the proton-translocating ATPase of the bacterial membrane. That was not expected because R. rubrum did not grow fermentatively under the conditions used. Low concentrations of protonophores were more effective in dissipating the anaerobic than the aerobic membrane potential. That observation indicated a lower activity of the electrogenic system responsible for the anaerobic potential. In consequence, the addition of uncouplers at low levels resulted in a marked enhancement of the membrane potential decrease which followed the transition between the aerobic and the anaerobic steady states.     

Occurrence of cytochrome c-554 in a facultative methylotroph,Protaminobacter ruber strain NR-1, during bacteriochlorophyll formation

A facultative methylotroph, Protaminobacter ruber was grown under two different conditions (aerobically grown under light, and aerobically in the dark after a light period). Bacteriochlorophyll was synthesized inducibly in the cells which were initially grown in the ligt and then grown in the dark, while bacteriochlorophyll was not found in the cells cultured under continuous light. Cytochrome c-554 was solely synthesized parallel to bacteriochlorophyll after switching from light to dark conditions. Both cytochrome c-554 and bacteriochlorophyll levels in the membrane preparation reached to a plateau in 24 h after switching from light and dark conditions. This cytochrome was membrane-bound and its M _r was 45,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis. The midpoint potential was 358 mV at pH 7. Other major membrane-bound cytochromes and two soluble cytochromes were present in both types of cells and their content did not change irrespective of growth conditions.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Bchl bacteriochlorophyll     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

Acclimation-induced changes in cell membrane composition and influence on cryotolerance of in vitro shoots of native plant species

Cell membranes are the primary sites of cryopreservation injury and measuring changes to membrane composition arising from cold acclimation may assist with providing a rationale for optimising cryopreservation methods. Shoot tips from two south-west Western Australian species, Grevillea scapigera and Loxocarya cinerea, and Arabidopsis thaliana (reference species) were subjected to cryopreservation using the droplet vitrification protocol. Two pre-conditioning regimes involving a constant temperature (23 °C, CT with a 12 h light/dark cycle) or an alternating temperature (AT) regime (20/10 °C with a 12 h light/dark cycle) were compared. Soluble sugars, sterols and phospholipids present in the shoot tips were analysed. Use of AT pre-conditioning (acclimation) resulted in a modest decrease in cryotolerance in A. thaliana, increased cryotolerance in G. scapigera, and increased survival in the non-frozen control explants of L. cinerea in comparison to CT pre-conditioning. Increased cryotolerance was accompanied by a higher total sugar sterol and phospholipid content, as well as an increase in strong hydrating phospholipid classes such as phosphatidylcholine. The double bond index of bound fatty acyl chains of phospholipids was greater after AT pre-conditioning, mostly due to a higher amount of monoenes in A. thaliana and trienes in G. scapigera and L. cinerea. These findings suggest that AT pre-conditioning treatments for in vitro plants can have a positive influence on cryotolerance for some plant species and this may be related to observed changes in the overall composition of cell membranes. However, alternative factors (e.g. oxidative stress) may be equally important with other species (e.g. L. cinerea).     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

Pentynyl dextran as a support matrix for immobilization of serine protease subtilisin Carlsberg and its use for transesterification of N-acetyl-l-phenylalanine ethyl ester in organic media

In this study, serine protease (subtilisin Carlsberg) was immobilized on pentynyl dextran (PyD, O–alkynyl ether of dextran, 1) and used for the transesterification of N-acetyl-l-phenylalanine ethyl ester (2) with different aliphatic (1-propanol, 1-butanol, 1-pentanol, 1-hexanol) and aromatic (benzyl alcohol, 2-phenyl ethanol, 4-phenyl-1-butanol) alcohols in tetrahydrofuran (THF). The effect of carbon chain length in aliphatic and aromatic alcohols on initial and average transesterification rate, transesterification activity of immobilized enzyme and yield of the reaction under selected reaction conditions was investigated. The transesterification reactivity of the enzyme and yield of the reaction increased as the chain length of the alcohols decreased. Furthermore, almost no change in yield was observed when the immobilized enzyme was repeatedly used for selected alcohols over six cycles. Intrinsic fluorescence analysis showed that the catalytic activity of the immobilized enzyme in THF was maintained due to retention of the tertiary structure of the enzyme after immobilization on PyD (1).     

Rapid homogeneous lauroylation of wheat straw hemicelluloses under mild conditions

Hemicellulose-based hydrophobic biomaterials with degrees of substitution ranging from 0.46 to 1.54 were synthesized under mild conditions in homogeneous media (N,N-dimethylformamide-lithium chloride) by reacting the native wheat straw hemicellulosic polymers with lauroyl chloride using 4-dimethylaminopyridine as a catalyst. Other catalysts such as N-bromosuccinimide, N-methyl pyrrolidine, N-methyl pyrrolidinone, and pyridine were also investigated. Under optimum reaction conditions (2 equiv of lauroyl chloride and triethylamine per hydroxyl group, 5% 4-dimethylaminopyridine, 40 °C, 35 min), a high DS value of 1.54 was obtained. The biomaterials were characterized by FT-IR spectroscopy and ¹³C NMR spectroscopy as well as by thermal analysis. The results showed that the lauroylation occurred preferably at the C-3 hydroxyl group of β-d-Xylp units in the hemicelluloses, and the thermal stability of the hydrophobic polymers increased by esterification.     

Reaction of formaldehyde with the histidine residues of proteins

Imidazoles or imidazoles substituted in the 2 or 4(5) position but with the ring nitrogen free, give a positive Pauly reaction. N-Methylimidazole does not react. In the presence of formaldehyde, all imidazole compounds give a negative Pauly reaction. In agreement with nmr studies, it is concluded that formaldehyde reacts with the imidazole ring nitrogen in acid solution to form N-hydroxymethyl derivatives.The Pauly color yield of various proteins (chymotrypsin, TPCK-chymotrypsin, lysozyme, ribonuclease, and reduced ribonuclease) is reduced 90–95% when the reaction is performed in the presence of formaldehyde. The color yield in water is essentially accounted for by the known reactive histidine and tyrosine content. In the presence of formaldehyde the color yield can be interpreted as arising from the known tyrosine content. It is therefore concluded that the histidine residues of the proteins examined have reacted with formaldehyde to form N-hydroxymethyl derivatives.In contrast to the Pauly color yield of chymotrypsin (A_M, 52 720) which can be accounted for by the contribution of its two histidine and three reactive tyrosine residues, the color yield of TPCK-chymotrypsin (A_M, 47 685) is higher than would be expected on the basis of the reported site of reaction of TPCK with chymotrypsin. The experimental molar extinction coefficient should be close to that calculated (A_M, 31 300) for its presumed one reactive histidine and three tyrosine residues. That it is not is in agreement with a previous report from our laboratories suggesting that His-57 is not the only site of reaction of TPCK with chymotrypsin.     

Immobilization of Rhizopus oryzae lipase on magnetic Fe3O4-chitosan beads and its potential in phenolic acids ester synthesis

A novel and simple method was developed for the preparation of magnetic Fe₃O₄ nanoparticles by chemical co-precipitation method and subsequent coating with 3-aminopropyltrimethoxysilane (APTMS) through silanization process. Magnetic Fe₃O₄-chitosan particles were prepared by the suspension cross-linking and covalent technique to be used in the application of magnetic carrier technology. The synthesized immobilization supports were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). Using glutaraldehyde as the coupling agent, the lipase from R. oryzae was successfully immobilized onto the functionalized magnetic Fe₃O₄-chitosan beads. The results showed that 86.60% of R. oryzae lipase was bound on the synthesized immobilization support. This immobilized lipase was successfully used for the esterification of phenolic acid which resulted in esterification of phenolic acid in isooctane solvent reaction system for 8 consecutive cycles (totally 384 h), 72.6% of its initial activity was retained, indicating a high stability in pharmaceutical and industrial applications.