Novel laccases of Ganoderma sp. KU-Alk4, regulated by different glucose concentration in alkaline media

Physiological regulation of laccase production from Ganoderma sp. KU-Alk4, isolated in Thailand, was controlled by the initial glucose concentration in liquid culture. Different laccase isozymes were produced using different starting concentrations of glucose. With 1% glucose, two isozymes, KULac 1 and 2 were produced, while with 4% glucose, three different isozymes, KULac 3, 4 and 5, were produced. The KULacs differed in their molecular mass, ranging from 53 to 112 kDa. KULac 2 was a new laccase that had a different N-terminal amino acid sequence from other laccases previously reported. All the isozymes had optimum pH at 3.5 and were stable over the wide range of pH, 3.0–10.0, especially in alkaline pH. It is noteworthy that the activities of the four KULacs with 2,6-dimethoxyphenol were extremely high up to 90°C. They retained 100% of their activities at 60°C for 1 h.     

Presence of an alpha-amylase isozyme with high temperature optima in the wheat variety tolerant to high temperature at juvenile plant stage

In the present study α-amylase was partially purified from detached grains of five day old seedlings of two wheat (Triticum aestivum L.) varieties, showing differential responses to high temperature stress at seedling stage. A three step purification via ammonium sulphate precipitation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150 was employed. A single α-amylase was detected in the high temperature sensitive PBW-175 variety, while two isozymes namely, α-amylase-1 and α-amylase-2 were obtained in the relatively tolerant WL-711 variety. The pH optima of the three α-amylases were in 5.0–5.5 range and comparable to the cereal amylases. The temperature optima of PBW-175 α-amylase and α-amylase-1 of WL-711, which appeared to be the major isozyme of the variety, were same at 45 °C and also comparable to cereal amylases. On the other hand the optimum temperature for α-amylase-2 was high at 70 °C, which is unusual and not reported earlier for cereal amylases. The Km of PBW-175 α-amylase was lower than the Km values of WL-711 isozymes, this was well co-related with an overall high α-amylase activity detected in the detached grains of five day old seedlings of PBW-175 compared to WL-711. However WL-711 variety showed a better inherent seedling growth, vigour and EUE than PBW-175, may be because it had two α-amylase isozymes which could compensate for the higher enzyme activity detected in PBW-175. Moreover, the presence of α-amylase-2 in the grain of WL-711 having temperature optima of 70 °C, possibly rendered its seedlings tolerant to HS of 50 °C, while the seedlings of PBW-175 succumbed to this temperature shock.     

Partial Purification and Some Properties of Polyphenoloxidases from Sago Palm

Two polyphenoloxidases (PPO I and PPO III, EC 1.10.3.1) were extracted and partially purified from sago palm pith by hydroxylapatite chromatography, DEAE-cellulose chromatography and gel filtration. Both purified isozymes gave a single activity band on polyacrylamide gel electrophoresis. The molecular weights of both enzymes were estimated to be 40,000. They had the same pH optima of 6.5 but different temperature optima, 35°C for PPO I and 45°C for PPO III. PPO I was stable at neutral to alkaline pH and PPO III at acidic pH. PPO III was somewhat more stable than PPO I when incubated at various temperatures for 15 min. PPO I and PPO III oxidized well DL-epicatechin and d-catechin, respectively. Both enzymes were strongly inhibited by KCN, Na-diethyldithiocarbamate and NaHSO₃.     

温度对六种外来杂草过氧化物酶同工酶谱的影响

应用聚丙烯酰胺垂直板凝胶电泳 ,比较了加拿大一枝黄花 (Solidagocanadensis)、小飞蓬 (Conyzacanadensis)、野塘蒿 (Conyzabonarinsis)、钻形紫菀 (Astersublatus)、一年蓬 (Erigeronannuus)和马缨丹 (Lan tanacamara)等 6种外来杂草在 3 8、2 5、5°C处理 60h后的过氧化物酶同工酶谱差异 ,分析了酶谱差异与对温度适应的关系。结果表明 ,温度变化对一年蓬、小飞蓬、钻形紫菀过氧化物酶同工酶谱的影响相对较小 ,野塘蒿通过增加酶带、调整同工酶的组成来适应温度的变化 ,反映出这 4种杂草对温度变化具有较强的适应能力 ;加拿大一枝黄花表现出对高温的适应能力较弱 ,对低温适应性较强 ,马缨丹对温度变化表现出相反的适应特点。     

Structural stability and unfolding transition of β-glucosidases: a comparative investigation on isozymes from a thermo-tolerant yeast

The folding of proteins in the milieu of the cellular environment involves various interactions among the residues of the polypeptide chain and the microenvironment where it resides. These interactions are responsible for stabilizing the protein molecule, and disruption of the same provides information about the stability of the molecule. β-Glucosidase isozymes, despite having high homology in their primary and tertiary designs, show deviations in their properties such as unfolding, refolding, and stability. In a comparative study on two large cell-wall-bound isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from a thermo-tolerant yeast, Pichia etchellsii, we have investigated guanidine hydrochloride (GdnHCl)-induced, alkali-induced, and thermal-unfolding transitions using CD and fluorescence spectroscopy and high sensitivity differential scanning calorimetry. Using spectral parameters (MRE 222 nm) to monitor the conformational transitions of the GdnHCl-induced unfolding phenomenon, it was observed that the midpoints of unfolding, apparent C _m, occurred at 1.2 M ± 0.05 and 0.8 M ± 0.03 GdnHCl, respectively, for BGLI and BGLII. The alkali-induced unfolding process indicated that BGLI showed a mid-transition point at pH 11 ± 0.17, while for BGLII it was at pH 10 ± 0.40, further indicating BGLI to be more stable to alkali denaturation than BGLII. In the case of thermal unfolding, the midpoint of transition was observed at 63 ± 0.12°C for BGLI and at 58 ± 0.55°C for BGLII. Analysis by high sensitivity differential scanning calorimeter supported the unfolding data in which BGLI showed higher melting temperature, T _m, (56.07°C ± 0.34) than BGLII (54.02°C ± 0.36). Our results clearly indicate that BGLI is structurally more rigid and stable than BGLII.     

Release behavior of allyl sulfide from cyclodextrin inclusion complex of allyl sulfide under different storage conditions

The stability of allyl sulfide, an organosulfur compound present in garlic oil, in its α-, β-, and γ-cyclodextrin inclusion complexes was investigated under various storage conditions. The complexes of cyclodextrins and allyl sulfide were prepared by spray drying. The storage temperature, relative humidity, and initial moisture content of the inclusion complex had different effects on the release rate of allyl sulfide. Allyl sulfide in α-cyclodextrin complexes had a lower release rate than in β- and γ-cyclodextrin complexes at 100 °C and at 50 °C under 6, 40, 54, and 73% relative humidity. The initial moisture content affected only the release rate of allyl sulfide from α-cyclodextrin complexes. The release behavior of allyl sulfide can be correlated with the first-order release rate equation with a normal Gaussian distribution of free energy of activation of release rate constant. The results indicated α-cyclodextrin is a suitable material for controlled release of allyl sulfide.     

Distribution of alpha-amylase isozymes among the varieties of winter wheat in Russia and Ukraine

A catalog of winter wheat varieties bred in Russia and Ukraine for alpha-amylase isozymes is presented. Among them, 11 phenotypic classes were found. It was established that the observed differences in the frequency of specific phenotypes for alpha-amylase in the culture of winter wheat depended on the geographical origin. The percentage of the most widespread cultural phenotype, designated as AbCde, decreased from the south (45°–46° N) to the north (49°–50° N) by 31.0%. At the same time, the frequency of the abCde variant increased by 25.0%. The distribution of the Abcde phenotype changed significantly from 20.7% in the west (30o36′ E) to 0% in the east (40o18′ E). The observed territorial dynamics for alpha-amylase isoenzymes may indicate the adaptive value of the identified phenotypes of common winter wheat for the weather and climatic conditions of the region of their origin.     

The effect of temperature on energy distribution during the last-larval stadium of the female house cricket, Acheta domesticus

The distribution of energy during the last stadium of the house cricket at two temperatures was the main theme of this study. Food consumption, growth, and oxygen consumption were greater in the first half of the stadium at both 25 and 35°C. An RQ > 1 indicated the conversion of carbohydrates to lipids during the first half of the instar at both temperatures. The duration of the stadium increased from 6 days at 35°C to 14 days at 25°C. The same maximal weight, protein content and lipid content were attained at both 25 and 35°C. A weight loss (mostly in stored lipids) after the midstadium peak weight was greater at the lower temperature. The absorption efficiency and the production of metabolic wastes were not affected by temperature, but the metabolic efficiency was much higher at 35 than at 25°C during the first half as well as the latter half of the stadium. Although during the first half of the stadium more energy was ingested, absorbed, and made available for growth at 25 than at 35°C, only slightly more growth occurred at 25°C. During the last half of the stadium less energy was ingested at 25 than at 35°C, and much more growth occurred at 35°C because of the even greater heat loss at 25 than at 35°C. Therefore at a lower temperature cricket larvae eat slightly more and reach the same maximal weight as at a higher temperature, but they end up smaller because they waste more energy during the extended duration of the stadium at the lower temperature.     

Adaptation to fluctuations in temperature by nine species of bacteria

Rapid environmental fluctuations are ubiquitous in the wild, yet majority of experimental studies mostly consider effects of slow fluctuations on organism. To test the evolutionary consequences of fast fluctuations, we conducted nine independent experimental evolution experiments with bacteria. Experimental conditions were same for all species, and we allowed them to evolve either in fluctuating temperature alternating rapidly between 20°C and 40°C or at constant 30°C temperature. After experimental evolution, we tested the performance of the clones in both rapid fluctuation and in constant environments (20°C, 30°C and 40°C). Results from experiments on these nine species were combined meta‐analytically. We found that overall the clones evolved in the fluctuating environment had evolved better efficiency in tolerating fluctuations (i.e., they had higher yield in fluctuating conditions) than the clones evolved in the constant environment. However, we did not find any evidence that fluctuation‐adapted clones would have evolved better tolerance to any measured constant environments (20°C, 30°C, and 40°C). Our results back up recent empirical findings reporting that it is hard to predict adaptations to fast fluctuations using tolerance curves.     

Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum

Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures of Fusarium proliferatum (MUCL 31970). Initial pH value in the cultures was also an overriding factor for increasing its production. By gel permeation high-performance liquid chromatography, the enzyme eluted as an apparently homogeneous peak with a molecular mass of 54 kDa, but by isoelectrofocusing, two proteins with pI values of 5.17 and 5.07 were revealed. Two different phenoloxidase activities were also detected after nondenaturing polyacrylamide gel electrophoresis. By matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF-MS), both proteins showed unique fingerprints, so they were classifiable as isozymes, and were named laccase 1 (Lac1, pI 5.17) and laccase 2 (Lac2, pI 5.07). No clear matches were found when compared with other proteins. The tandem mass spectrometry of some peptides from both isozymes reanalyzed by nanoelectron ionization–ion trap–mass spectrometry (nESI-IT-MS) confirmed their unique character. The following interesting properties, particularly its stability at alkaline pH, make this laccase a promising industrial enzyme for biotechnological applications: maximum activity at 60°C, thermal stability for 2 h at 40°C, optimum pH 3.5 (km=62 μM) measured on 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate), and pH stability 4–8 (75% stability at pH levels 2.2 and 9) for 2 h at 25°C.     

Temperature influence in developing tadpoles. II. Influence of temperature on isozymes and energies of activation of partially purified glucose-6-phosphate dehydrogenase

Isozymic banding patterns and energies of activation were studied in partially puriled G6PDH preparations from spring and fall Rana pipiens eggs and tadpoles raised at 10 °C and 20 °C.G6PDH preparations from spring tadpoles raised at 20 °C demonstrated new isozyme patterns. However, in the spring 10 °C and fall 10 °C and 20 °C raised tadpoles G6PDH preparations conserved isozymes already present.The energies of activation of G6PDH determined from the maximum velocity constants in the spring groups are lower than in the corresponding fall groups.     

Rat liver phosphofructokinase isozymes

The labile phosphofructokinase activity of rat liver was found to be stabilized and efficiently extracted in 50 mm Tris-HCl, pH 8.0, 50 mm NaF, 10 mm dithiothreitol, and 1.0 mm ATP. By the method of DEAE-cellulose chromatography liver phosphofructokinase activity could be resolved into two isozymes. The major isozyme which was 85% of the total isolated activity was purified to homogeneity. This 15,000-fold purified isozyme had a specific activity of about 90 IU/mg protein with 25–30% recovery of the total activity. Sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of the sodium dodecyl sulfate-treated isozyme indicated a subunit molecular weight of 65,000. Antiserum to the major isozyme was obtained from rabbits, and immunotitration of the two isozymes indicated that they were immunologically different. Kinetic properties of the two isozymes indicated that the major isozyme was more susceptible to ATP and citrate inhibition as well as relief of ATP and citrate inhibition by fructose-6-P, AMP, and ammonia. With the use of DEAE-cellulose chromatography and antiserum titration of 100,000g supernatant fluids, it was shown that the two hepatic isozymes were always found together in adult, embryonic, and neoplastic liver and in kidney.     

Effect of gibberellic acid on starch degrading enzymes in leaves of Digitaria decumbens

Tropical ‘Pangola’ digitgrass was treated with gibberellic acid (GA) and subjected to 30° or 10° nights. The starch degrading enzymes of the leaves were separated by polyacrylamide gel electrophoresis. Densitometer tracings of gels negatively stained by starch-iodine showed the presence of seven bands regardless of treatment of plants. GA treatment increased starch degrading enzyme activity in plants subjected to 10° to the level of activity found in 30° untreated (control) plants and, additionally, enhanced enzyme activity in plants at 30°. GA treatment of 10° plants decreased sucrose and starch levels when compared to levels found in untreated 10° plants. The action of GA in reversing the effects of 10° nights on ‘Pangola’ leaves was found to be the result of a quantitative increase in activity of existing enzyme forms rather than production of isozymes.     

Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector

Summary We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34°C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca⁺⁺-resistant cells did not proliferate at the nonpermissive temperature (40°C), indicating that they depended on T antigen for their proliferation. The temperaturesensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40°C. The serum/Ca⁺⁺-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca⁺⁺-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.     

Properties of membrane-bound α-glucosidases: Possible sugar receptor protein of the blowfly, Phormia regina

Previously reported PII-type α-glucosidase located in the precipitate of the labellar homogenate of the blowfly Phormia regina was solubilized by sodium deoxycholate (DOC) and further separated into three isozymes with different molecular weight: PII-M (mol. wt 9 × 10⁴). PII-D (mol. wt 2 × 10⁵) and PII-T (mol. wt 8 × 10⁵) by molecular sieve chromatography on Biogel P-300 or Ultragel AcA-34. These three isozymes had almost the same K_m's and relative values of V_m's for several substrates, suggesting that they had the same common active site.PII-D and PII-T are more strongly embedded in the membrane than PII-M, because the proportion of PII-D and PII-T was much increased when the remaining glucosidase in the precipitate after the first solubilization was reextracted by DOC. A large peak of α-glucosidase isozyme P-IV which preferentially hydrolyze sucrose eluted just after P-II (soluble P-II) when the supernatant fraction of the labellar homogenate was chromatographed on DEAE-Sephadex A-50. P-IV was scarcely present in the precipitate fraction.Soluble P-II had the same mol. wt as PII-M and had similar properties to PII-M except for the ratio of V_m's.A large proportion of PII-D was contained in the well washed labellar integuments, a preparation rich in labellar chemosensilla. It suggests that most of the insoluble α-glucosidase contained in the dendrite in labellar chemosensilla is PII-D. PII-D (and PII-T) are possible sites of the pyranose receptor molecule because their properties and localization agree well with those of the receptor.     

Investigations on the interactions of DiAmsar with serum albumins: Insights from spectroscopic and molecular docking techniques

Diamine‐sarcophagine (DiAmsar) binding to human serum albumin (HSA) and bovine serum albumin (BSA) was investigated under simulative physiological conditions. Fluorescence spectra in combination with Fourier transform infrared (FT‐IR), UV‐visible (UV–vis) spectroscopy, cyclic voltammetry (CV), and molecular docking method were used in the present work. Experimental results revealed that DiAmsar had an ability to quench the HSA and BSA intrinsic fluorescence through a static quenching mechanism. The Stern–Volmer quenching rate constant (K_sv) was calculated as 0.372 × 10³ M^‐1 and 0.640 × 10³ M^‐1 for HSA and BSA, respectively. Moreover, binding constants (K_a), number of binding sites (n) at different temperatures, binding distance (r), and thermodynamic parameters (?H°, ?S°, and ?G°) between DiAmsar and HSA (or BSA) were calculated. DiAmsar exhibited good binding propensity to HSA and BSA with relatively high binding constant values. The positive ?H° and ?S° values indicated that the hydrophobic interaction is main force in the binding of the DiAmsar to HSA (or BSA). Furthermore, molecular docking results revealed the possible binding site and the microenvironment around the bond. Copyright © 2014 John Wiley & Sons, Ltd.     

Two temperature-sensitive photosystem II mutants of pea

Two nuclear gene mutants of pea, chlorotica-887 and chlorina-5756, are temperature-sensitive in the development of photosystem II activity. Low temperature flourescence emission spectra of leaves show that the peak at 697 nm from the reaction center of photosystem II is present when the mutants have been grown at 18°C, but absent when they have been grown at 30°C. For leaves of chlorina-5756 grown at 18°C the relative size of the peak at 697 nm is reduced compared to that of leaves of the wild type or chlorotica-887 grown at this temperature. Flourescence induction curves of leaves from wild type plants and chlorotica-887 grown at 18°C possess two steps, while those of leaves from chlorina-5756 grown at 18°C or 30°C and chlorotica-887 grown at 30°C show at fast rise to the maximal level of fluorescence. Measurements on chloroplasts isolated from the mutants indicated that the photosystem I activity per g leaf material is comparable for plants grown at 18°C and plants grown at 30°C. In contrast, no photosystem II activity was detected when the mutants had been grown at 30°C. It is suggested that these mutants are affected in a component required for the assembly of functional photosystem II complexes.     

Evaluation of Tetrahydropalmatine Enantiomers on the Activity of Five Cytochrome P450 Isozymes in Rats Using a Liquid Chromatography / Mass Spectrometric Method and a Cocktail Approach

The aim was to evaluate the effects of tetrahydropalmatine (THP) enantiomers on the activity of five cytochrome P450 (CYP450) isozymes in vivo. A liquid chromatography / mass spectrometric (LC‐MS) method was developed for simultaneous determination of five specific probe substrates including metoprolol (2D6), caffeine (1A2), dapsone (3A4), chlorzoxazone (2E1), and tolbutamide (2C9) in rat plasma. Analytes were separated with the mobile phase consisting of 0.1% acetic acid aqueous solution and acetonitrile in a gradient elution. The mass spectrometric detection via selected ion monitoring (SIM) was operated in both positive ion mode (for metoprolol m/z 268, caffeine m/z 195, and dapsone m/z 249) and negative ion mode (for chlorzoxazone m/z 168 and tolbutamide m/z 269) in the same run. Linear correlation was obtained (r² > 0.99) over the concentration range of 0.050–25.0 µg/mL for caffeine and dapsone, 0.025–10.0 µg/mL for metoprolol, 0.050–50.0 µg/mL for chlorzoxazone, and 0.25–100.0 µg/mL for tolbutamide. Intra‐ and interday precision were less than 12.09%. The matrix effect ranged from 87.50% to 109.25% and the absolute recoveries were greater than 70%. The method was successfully applied to evaluate the effect of THP enantiomers on the activity of CYP450 isozymes by a cocktail approach. The pharmacokinetic results of five probe drugs indicated that there were stereoselective differences between the two THP enantiomers, i.e., d‐THP had the potential to inhibit the activities of CYP2D6 and CYP1A2 isozymes, while l‐THP inhibited CYP1A2 isozyme and induced CYP3A4 and CYP2C9 isozymes. Chirality 27:551–556, 2015. © 2015 Wiley Periodicals, Inc.     

Induced Heat Sensitivity of Wheat Roots and Protecting Effect of Ethanol and Kinetin

Wheat seedlings were subjected to heat shock for 2 min at 45°C. The seedlings were then incubated at 25°C or higher temperatures (usually 35°C). At 25°C the root tips survived the heat shock, but not at temperatures above 34°C, unless they had been pretreated with ethanol or kinetin, After 1 h in ethanol and after more than 15 h in kinetin the root meristem survived a high incubation temperature after the heat shock. Immediately after heat treatment the glyceride content in treated root tips was higher than in untreated roots. The same was observed after heat treatment of root tips pretreated in ethanol and kinetin. The content of ether extractable lipids was not changed by the heat shock.