Photocontrol of affinity chromatography. I. Trypsin purification by photosensitive soybean trypsin inhibitor (STI) gel

Soybean trypsin inhibitor (STI) was immobilized on the agarose gel modified with spiropyran compound (spiropyran gel), and photocontrolled binding and releasing of trypsin was examined. The STI-spiropyran gel showed reverse photochromism. Trypsin was bound on the STI-spiropyran gel in the dark and released with visible light irradiation. The optimum conditions for photocontrolled binding and releasing of trypsin were pH 6.6 and the buffer concentration of 0.05 m. Approximately 60–80% of bound trypsin was released with visible light irradiation. The activity of released trypsin was the same as that of native trypsin. Approximately 21-fold purification of trypsin was performed with the STI-spiropyran gel column.     

Photocontrol of immobilized trypsin activity

Trypsin was coupled on an agarose gel which was modified with a spiropyran compound. The trypsin–spiropyran (agarose) gel showed reverse photochromism. The activity of the trypsin–spiropyran gel in the dark was 12% of that of native trypsin, and it was higher than that under visible light. The apparent Michaelis constant of the trypsin–spiropyran gel in the dark was larger than that under visible light. On the other hand, the maximum velocity in the dark was higher than that under visible light. The optimum pH of the trypsin–spiropyran gel in the dark was the same as that under visible light. Immobilized trypsin was stable in the pH range from 3 to 9. The trypsin–spiropyran gel was more stable against heat than the native trypsin.     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Light-induced enzyme activity changes associated with the photoisomerization of bound spiropyran

A spiropyran compound which undergoes photoisomerization was covalently attached to several enzymes in order to alter their activity by imposing light on them. The enzyme-bound spiropyran exhibited normal photochromism when the matrix enzyme was hydrophobic in nature, whereas reverse photochromism was evident when the spiropyran was attached to hydrophilic enzymes. The activity of the modified enzymes was found to change because of photoisomerization of the bound spiropyran. A fluorescent probe for hydrophobic sites revealed that the modified enzymes remained hydrophobic when the bound spiropyran closed and turned hydrophilic when the spiropyran opened. A mechanism for the photoinduced activity changes is postulated in relation to the enzyme-substrate affinity in the open- and closed-ring states of spiropyran.     

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.     

Photocontrol of affinity chromatography: Purification of asparaginase by photosensitive AHA-gel

An agarose gel modified with N-(ω-aminohexytl)-L -aspartic acid (AHA) and spiropyran compound (AHA–spiropyran gel) was prepared and the photocontrolled binding and releasing of asparaginase were investigated with the AHA–spiropyran gel. Asparaginase was bound on the AHA–spiropyran gel under visible light and was released in the dark. The optimum conditions for photocontrolled binding and releasing of asparaginase were a 0.05M phosphate buffer concentration and pH 7.0. Seventy-five percent of the bound asparaginase was released from the AHA–spiropyran gel column in the dark. Ninetyfold purification of asparaginase was performed with the AHA–spiropyran gel Column.     

Amino acid esterification by alpha-chymotrypsin immobilized in spiropyran membrane.

α-Chymotrypsin was immobilized in a collagen membrane modified with a spiropyran compound. The immobilized chymotrypsin was used for the esterification of N-acetyl-l-tyrosine (AT). N-Acetyl-l-tyrosine ethyl ester (ATEE) was synthesized from AT and ethanol by immobilized chymotrypsin under visible light. The optimum pH for the esterification was 7. An increase of the chymotrypsin content in the spiropyran-collagen membrane increased the rate and the yield of ATEE. The yield of ATEE reached 40% under visible light. Initially, ATEE was synthesized in the dark. However, the ATEE synthesized was gradually hydrolyzed in the dark. The amount of ATEE in the reaction mixture increased with irradiation by visible light and decreased in the dark. Therefore, the esterification of N-acetyl-l-tyrosine was controlled by light irradiation.     

Dark-mediated dormancy release in stratified Lolium rigidum seeds is associated with higher activities of cell wall-modifying enzymes and an apparent increase in gibberellin sensitivity

Dormancy release in freshly matured, imbibed annual ryegrass (Lolium rigidum) seeds is inhibited by light and involves a decrease in seed sensitivity to abscisic acid. Other processes involved in dormancy release in the dark were investigated by measuring seed storage compound mobilisation and the activity of cell wall-degrading enzymes. Activities of endo-β-mannanase and total peroxidase were higher in dark-stratified compared to light-stratified seeds, indicating that weakening of the structures constraining the embryo was accelerated in the dark. A dramatic degradation of storage proteins in light-stratified seeds, accompanied by induction of a high molecular mass protease, suggests that maintenance of storage(-like) proteins is also important in dark-mediated dormancy release. α-Amylase activity was induced in dark-stratified seeds at least 48 h prior to radicle emergence upon transfer to conditions permitting germination, or in light-stratified seeds supplied with exogenous gibberellin A₄. This suggests that (a) α-amylase is involved in stimulation of germination of non-dormant L. rigidum seeds, and (b) dark-stratified seeds have an increased sensitivity to gibberellins which permits the rapid induction of α-amylase activity upon exposure to germination conditions. Overall, it appears that a number of processes, although possibly minor in themselves, occur in concert during dark-stratification to contribute to dormancy release.     

Photoresponsive peptide and polypeptide systems: 10. Synthesis and reversible photochromism of azo aromatic poly(L-alpha,beta-diaminopropionic acid)

Poly-L-alpha,beta-diaminopropionic acid) having azo aromatic side chain was synthesized by the water-soluble carbodiimide procedure. The photochemical properties of the azo polypeptide poly[N beta-p-(phenylazo)benzoyl-L-alpha,beta-diaminopropionic acid] (PPABLDPA) was investigated by absorption and circular dichroism (c.d.) spectroscopy in hexafluoro-2-propanol (HFIP) and dimethylformamide. The photochromism of the absorption band in the visible and ultraviolet wavelength regions was found to be mostly reversible as a function of irradiation time at different wavelengths due to the photostationary state (88% trans)-cis photoisomerization of the azo aromatic moieties. The c.d. spectra exhibited two and three-stage photochromism on irradiation by light. The reversible photo-induced solubility change was also studied. On irradiation PPABLDPA is soluble under ultraviolet light (cis) and precipitates under visible light (88% trans) in HFIP-water. A discussion was presented that includes our previous results on this azo aromatic polylysine homologue series.     

Effects of poly(ethylene glycol) and salt on the binding of α-amylase from the fermentation broth of Bacillus amyloliquefaciens by Cu2+-β-CD affinity adsorbent

α-Amylase from Bacillus amyloliquefaciens was purified by the immobilized metal ion affinity adsorbent, β-CD_cl-IDA-Cu²⁺. The adsorbent was prepared by reacting the cross-linked β-cyclodextrin (β-CD) with the ligand, iminodiacetic acid (IDA). The copper ion was further linked to the adsorbent. Poly(ethylene glycol) (PEG) was added to the fermentation broth to improve the adsorption efficiency of the adsorbent toward α-amylase. The effort was to provide hydrophobic interactions with the impurities which might interfere with the adsorption of α-amylase. It also provided a polymer shielding effect to prevent non-specific interactions. With the addition of PEG, the adsorption efficiency could be increased to 98%. Imidazole containing a phosphate buffer and NaCl was used to elute the bound α-amylase. By consecutive adsorption/desorption steps, up to 81% of the α-amylase activity could be recovered. Regarding the reutilization of the affinity adsorbents, α-amylase could be adsorbed and desorbed six times consecutively without a significant loss of α-amylase activity.     

Photoswitching of ligand association with a photoresponsive polymer-protein conjugate

Light-regulated molecular switches that reversibly control biomolecular function could provide new opportunities for controlling activity in diagnostics, affinity separations, bioprocessing, therapeutics, and bioelectronics applications. Here we show that site-specific conjugation of light-responsive polymers near the biotin-binding pocket of streptavidin provides control of ligand binding affinity in response to UV and visible light irradiation. Two different light-responsive polymers were utilized that display opposite photoresponsive solubility changes under UV or visible (vis) light irradiation in aqueous solutions. At 40 degrees C, the N,N-dimethylacrylamide (DMA)-co-4-phenylazophenyl acrylate (AZAA) copolymer (DMAA) was soluble under UV irradiation and precipitated under visible light, while the DMA-co-N-4-phenylazophenyl acrylamide (AZAAm) copolymer (DMAAm) was soluble under visible irradiation and precipitated under UV light. Both polymers were synthesized with a vinyl sulfone terminus and conjugated to the Glu116Cys (E116C) streptavidin mutant via thiol coupling. The DMAA-streptavidin conjugate bound biotin efficiently when the polymer was in the soluble state under UV irradiation, but under visible irradiation, the polymer collapsed and blocked free biotin association. Furthermore, if biotin was allowed to bind when the polymer was in the soluble state under UV irradiation, then when the polymer was collapsed by visible light, the streptavidin released the bound biotin. The DMAAm-streptavidin conjugate showed the opposite response, with association of biotin allowed under visible light irradiation and blocked under UV irradiation. The photoresponses of the streptavidin conjugates thus correspond to the original photoresponsive phase transition properties of the polymer switches triggered by the cis-trans isomerization of the diazo chromophores.     

Effects of Pulsed Electric Field (PEF) Treatment on Enhancing Activity and Conformation of α-Amylase

To explore an efficient, safe, and speedy application of pulsed electric field (PEF) technology for enzymatic modification, effects of PEF treatment on the enzymatic activity, property and kinetic parameters of α-amylase were investigated. Conformational transitions were also studied with the aid of circular dichroism (CD) and fluorescence spectra. The maximum enzymatic activity of α-amylase was obtained under 15 kV/cm electric field intensity and 100 mL/min flow velocity PEF treatment, in which the enzymatic activity increased by 22.13 ± 1.14 % compared with control. The activation effect could last for 18 h at 4 °C. PEF treatment could widen the range of optimum temperature for α-amylase, however, it barely exerted any effect on the optimum pH. On the other hand, α-amylase treated by PEF showed an increase of V _max, t_1/2 and ΔG, whereas a decrease of K _m and k were observed. Furthermore, it can be observed from fluorescence and CD spectra that PEF treatment had increased the number of amino acid residues, especially that of tryptophan, on α-amylase surface with enhanced α-helices by 34.76 % and decreased random coil by 12.04 % on α-amylase when compared with that of untreated. These changes in structure had positive effect on enhancing α-amylase activity and property.     

Effect of light on phosphatidate phosphohydrolase activity of retina rod outer segments: the role of transducin

The aim of the present paper is to evaluate the modulation of phosphatidate phosphohydrolase (PAPase) and diacylglyceride lipase (DGL) activities in bovine rod outer segment (ROS) under dark and light conditions and to evaluate the role of transducin (T) in this phenomenon. In dark-adapted ROS membranes exposed to light, PAPase activity is inhibited by 20% with respect to the activity found under dark conditions. To determine whether the retinal G protein, T, participates in the regulation of PAPase activity in these membranes, the effects of GTPgammaS and GDPbetaS on enzyme activity were examined. Under dark conditions in the presence of GTPgammaS, which stabilizes T in its active form (Talpha + Tbetagamma), enzyme activity was inhibited and approached control values under light conditions. GDPbetaS, on the other hand, which stabilizes the inactive state of T (Talphabetagamma), stimulated PAPase activity by 36% with respect to control light conditions. ADP-ribosylation by cholera and pertussis toxin was also studied. In ADP-rybosilated ROS membranes with pertussis toxin under dark conditions, PAPase activity was 36% higher than the activity found under control light conditions. ADP-ribosylation by CTx, on the other hand, inhibited PAPase activity by 22%, with respect to dark control conditions, mimicking light effect. The effects of GTPgammaS and GDPbetaS and conditions of ADP-ribosylation by PTx and CTx on DGL activity were similar to those of PAPase activities. Based on NEM sensitivity we have also demonstrated that the PAPase present in ROS is the PAP 2 isoform. Our findings therefore suggest that light inhibition of PAP 2 in ROS is a transducin-mediated mechanism.     

Conjugation of α-amylase with dextran for enhanced stability: Process details, kinetics and structural analysis

The influence of enzyme polysaccharide interaction on enzyme stability and activity was elucidated by covalently binding dextran to a model enzyme, α-amylase. The conjugation process was optimized with respect to concentration of oxidizing agent, pH of enzyme solution, ratio of dextran to enzyme concentration, temperature and time of conjugate formation, and was found to affect the stability of α-amylase. α-Amylase conjugated under optimized conditions showed 5% loss of activity but with enhanced thermal and pH stability. Lower inactivation rate constant of conjugated α-amylase within the temperature range of 60-80°C implied its better stability. Activation energy for denaturation of α-amylase increased by 8.81kJ/mol on conjugation with dextran. Analysis of secondary structure of α-amylase after covalent binding with dextran showed helix to turn conversion without loss of functional properties of α-amylase. Covalent bonding was found to be mandatory for the formation of conjugate.     

Efficient photochromism from the network of chemical reactions of 7,4'-dihydroxyflavylium in CTAB micelles.

Differently from water, efficient photochromism with a strong colour contrast has been observed for the multistate compound 7,4'-dihydroxyflavylium in the presence of cetyl trimethylammonium bromide (CTAB) micelles. Two states are responsible for the photochromism: trans-chalcone (inside the micelle) in the dark, and flavylium cation, AH(+), (in bulk water) upon irradiation. The kinetics of the system was characterized by flash photolysis and pH jumps. Evidence that the photochemical process leading to AH(+) occurs in the micelle was achieved. The best colour contrast is obtained at pH = 1.5, from a solution practically colourless in the dark, to an intense yellow upon irradiation (quantum yield 0.4). The system is completely reversible with a lifetime of 38 min at room temperature, and exhibits a reasonable stability. A kinetic model capable of fitting the data from thermal entrance of the compound into the micelle, its ejection to bulk water upon irradiation and quantum yields of the photochemical process is proposed.     

The contribution of different α-amylase isoenzymes of the commodity grain spring wheat in the formation of falling number values

The participation of various isoenzymes of α-amylase in the formation of falling number values of the commodity grain of wheat grown in the Republic of Kazakhstan was investigated. It was found that active isoenzymes α-AMY1 and α-AMY2 of the embryonic shield were present in the grain with an index over 200. A significant decrease in the falling number depended mainly on the synthesis of α-AMY1 and α-AMY2 isoenzymes in the aleurone layer. In the grain, isoenzymes with high isoelectric points (p1 ≥ 7.3) were found these isoenzymes belong to α-amylase or late maturing or α-amylase of practically mature grains. It was discovered that the exogenous hormone (gibberellic acid) induced synthesis of α-amylase isoenzymes of scutellum, whole caryopses, and aleurone. It was shown that the impact of exogenous gibberellic acid on the activity and structure of α-amylase is reduced in grain with a low falling number.     

野生苋属植物籽实中新型α淀粉酶抑制剂的分离纯化及其性质研究(英文)

从野生苋属植物 (Amaranthuspaniculatus)籽实中分离纯化出α淀粉酶的一种新型蛋白质类抑制剂 .该抑制剂被命名为WAI 1 .MALDI TOF质谱测得其分子量为 986 5 ,是目前报道的α 淀粉酶的蛋白质类抑制剂中分子量最小的 .初步的组成和结构分析结果表明 ,WAI 1由 9个氨基酸残基组成 ,其N端为焦谷氨酸 .直接用RP HPLC纯化后 ,WAI 1能在弱酸性条件下 ,以非竞争性抑制作用方式有效抑制美洲蜚蠊消化道α淀粉酶的活性 ,最适抑制pH 6 0 ,但对人唾液淀粉酶活性无影响 .WAI 1在 37℃下与酶预温浴约 30min后显示最大抑制活性 .当α淀粉酶用量一定时 ,α淀粉酶活性的抑制率在约 5 0 %的范围内随抑制剂 酶比例的增大而呈线性增加 ,超过 5 0 %后 ,抑制率随抑制剂 酶比例的增大而缓慢上升 ,最终达到最大值 (约 6 5 % ) .     

Substrate recognition mechanism of alpha-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans

We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the α-1,6-glucosidic linkage of isomaltooligosaccharides from their non-reducing ends to produce α-glucose. By using the mutant of catalytic acid Glu236→Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 Å resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (β/α)₈-barrel in domain A, which is common to the α-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites −1 to +2. The environment of the glucose residue at subsite −1 is similar to the environment of this residue in the α-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite −1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile α-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile α-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the α-amylase structure from Bacillus subtilis. The comparison with the α-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of α-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes.     

Process parameters study of α-amylase production in a packed-bed bioreactor under solid-state fermentation with possibility of temperature monitoring

Production of α-amylase in a laboratory-scale packed-bed bioreactor by Bacillus sp. KR-8104 under solid-state fermentation (SSF) with possibility of temperature control and monitoring was studied using wheat bran (WB) as a solid substrate. The simultaneous effects of aeration rate, initial substrate moisture, and incubation temperature on α-amylase production were evaluated using response surface methodology (RSM) based on a Box-Behnken design. The optimum conditions for attaining the maximum production of α-amylase were 37°C, 72% (w/w) initial substrate moisture, and 0.15 L/min aeration. The average enzyme activity obtained under the optimized conditions was 473.8 U/g dry fermented substrate. In addition, it was observed that the production of enzyme decreased from the bottom of the bioreactor to the top.     

逆境条件下小麦种子活力与种子萌发相关酶活性及其基因表达的关系

分析不同基因型小麦品种逆境萌发过程中种子萌发相关酶活性及基因表达差异,明确在逆境条件下,种子活力与种子萌发相关酶活性及基因表达量的关系.通过标准发芽试验和逆境(冷浸、人工老化、干旱胁迫)发芽试验,测定4个小麦品种种子活力、萌发过程中可溶性总糖和可溶性蛋白含量、α-淀粉酶活性、半胱氨酸蛋白酶活性及相关基因表达量.结果表明:干旱、人工老化和冷浸胁迫3种逆境对种子活力都有一定影响.不同萌发条件下,可溶性总糖含量呈现先小幅度升高后小幅度降低再迅速升高的趋势;而可溶性蛋白含量随着萌发时间的延长呈现逐渐下降的趋势.α-淀粉酶活性整体呈现逐渐升高的趋势,但在冷浸胁迫处理后,豫农949和轮选061的α-淀粉酶活性在萌发60 h后出现下降.半胱氨酸蛋白酶活性整体呈先降低后升高的趋势,但在干旱胁迫条件下,豫农949、豫麦49-198和轮选061的半胱氨酸蛋白酶活性呈现先升高后降低再升高的趋势.不同逆境萌发条件下,α-AMY(α-淀粉酶基因)表达量整体呈先上升后下降的趋势.冷浸胁迫处理后,轮选061的α-AMY表达量高于对照,在其他逆境萌发条件下,4个品种的α-AMY表达量均低于对照;人工老化处理后,长4738的CP(半胱氨酸蛋白酶基因)表达量与对照差异不显著,在其他逆境萌发条件下,4个品种的CP表达量均高于对照.种子萌发期间,不同萌发条件下α-淀粉酶和半胱氨酸蛋白酶活性与其基因表达并没有直接关系,α-淀粉酶活性与可溶性总糖含量达到显著正相关,半胱氨酸蛋白酶活性与可溶性蛋白含量的相关性不显著.在标准发芽条件下,α-淀粉酶活性与活力指数呈显著正相关,而在逆境萌发过程中,其相关性不显著.冷浸胁迫处理后,半胱氨酸蛋白酶活性与活力指数呈显著正相关,但在标准发芽、干旱胁迫、人工老化处理后,其相关性不显著.