地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。     

Thermostable and alkalophilic maltogenic amylase of Bacillus thermoalkalophilus ET2 in monomer-dimer equilibrium

A gene encoding a thermostable and alkalophilic maltogenic amylase (BTMA) was cloned from the thermophilic bacterium Bacillus thermoalkalophilus ET2. BTMA was composed of 588 amino acids with a predicted molecular mass of 68.8 kDa. The enzyme had an optimal temperature and pH of 70°C and 8, respectively, the highest among maltogenic amylases reported so far. The Tm of BTMA at pH 8 was 76.7°C with an enthalpy of 113.6 kJ mol^-1. Both hydrolysis and transglycosylation activities for various carbohydrates were evident. β-Cyclodextrin (β-CD) and soluble starch were hydrolyzed mainly to maltose, and pullulan to panose. Acarbose, a strong amylase inhibitor, was hydrolyzed by BTMA to glucose and acarviosine-glucose. The K _m and k _cat values of BTMA for β-CD hydrolysis were 0.128 mM and 165.8 s^-1 mM, respectively. The overall catalytic efficiency (k _cat/K _m) of the enzyme was highest toward β-CD. BTMA was present in a monomer-dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer (pH 8.0). This equilibrium could be affected by KCl and enzyme concentrations. The multi-substrate specificity of the enzyme was modulated by the structural differences between monomeric and dimeric forms. Starch was hydrolyzed more readily when monomeric BTMA was prevalent, while the opposite was observed for β-CD.     

Enhanced fructose levels in field grown potato tubers expressing a thermostable glucose isomerase

A thermostable glucose isomerase was expressed in Solanum tuberosum Desirée using the tuber-specific granular-bound starch synthase promoter. The fructose content was substantially increased in microtubers, greenhouse grown tubers as well as tubers produced in field trials as compared with the controls. Furthermore, the tuber yield of field grown potatoes was enhanced by 30% in the transgenic lines (from 1.04 kg/plant in the wild type to 1.36 kg/plant in the transgenic lines).     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Identification of a 3.2 kb 5'-flanking region of the murine keratocan gene that directs beta-galactosidase expression in the adult corneal stroma of transgenic mice

The mouse keratocan gene (Ktcn) expression tracks the corneal morphogenesis during eye development and becomes restricted to keratocytes of the adult, implicating a cornea-specific gene regulation of the mouse Ktcn [J. Biol. Chem., 273 (1998) 22 584–22 588]. To examine the functionality of the mouse Ktcn promoter, we have cloned and sequenced a 3.2 kb genomic DNA fragment 5′ of the mouse Ktcn gene, which was used to prepare a reporter gene construct that contained the 3.2 kb 5′ flanking sequence, exon 1 and 0.4 kb of intron 1 of Ktcn, and β-geo hybrid reporter gene. The β-galactosidase (βGal) activity was assayed in tissues of two of five transgenic mouse lines obtained via microinjection. In adult transgenic mice, βGal activity was detected only in cornea, not in other tissues (e.g. lens, retina, sclera, lung, heart, liver, diaphragm, kidney, and brain). During ocular development, the spatial–temporal expression patterns of the βGal recapitulated that of endogenous Ktcn in transgenic mice. Using XGal staining, strong βGal activity was first detected in periocular tissues of E13.5 embryos, and restricted to corneal keratocytes at E14.5 and thereafter. Interestingly, in addition to cornea, βGal activity was transiently found in some non-ocular tissues, i.e. ears, snout, and limbs of embryos of E13.5 and E14.5 but was no longer detected in those tissues of E16.5 embryos. The transient expression of endogenous keratocan in non-ocular tissues during embryonic development was confirmed by in situ hybridization. Taken together, our results suggest that the 3.2 kb Ktcn promoter contains sufficient cis-regulatory elements to drive heterologous minigene expression in cells expressing keratocan. The identification of keratocyte-specific expression of βGal reporter gene in the adult transgenic mice is an important first step in characterizing the Ktcn promoter in order to use it to drive a foreign gene expression in corneal stroma.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Esterase isozyme polymorphism, specific and nonspecific esterase, syngenic lines development and natural occurrence of a thermostable esterase in the tropical silkworm Bombyx mori L

Esterase isozyme polymorphism was documented for digestive juice and haemolymph of the tropical multivoltine silkworm, Bombyx mori L., breed CB5 (GP) and its syngenic lines (CB5Lm^e-1, CB5Lm-2 and CB5Lm-5) using - and β-naphthylacetate separately as nonspecific substrates (Ogita, Z., Kasai, T., 1965. Genetico-biochemical analysis of specific esterases in Musca domestica. Jpn. J. Genet. 40, 173–184). Polymorphism existed in the isozyme pattern of -esterase with two or three bands in digestive juice and three to five bands in haemolymph. No polymorphism was observed in β-esterase isozyme pattern having four bands in digestive juice and two bands in haemolymph. During the course of esterase isozyme studies, the presence of some specific -esterase bands (Est-1, 4 and 5) in haemolymph and β-esterase bands (Est-1, 2 and 3) in digestive juice were observed. But both - and β-esterase bands Est-3 and 4 in digestive juice and Est-2 and 3 in haemolymph were found to be nonspecific. Nonspecific β-esterase band (Est-3) in haemolymph of CB5 (GP) and its syngenic lines withstood a temperature up to 80±1°C for 10 min. No thermostable band was observed in the isozyme zymogram of -esterase in digestive juice and haemolymph or β-esterase in digestive juice. Overall, this study discusses the presence of esterase heterogeneity in the CB5 (GP) genepool, syngenic lines development, occurrence of specific - and β-esterase bands in digestive juice and haemolymph and thermostable β-esterase band Est-3 in haemolymph in tropical silkworm Bombyx mori L.     

Effect of lactic acid fermentation of wheat and barley whole meal flour on carbohydrate composition and digestibility in mink (Mustela vison)

Wheat and barley whole meal flours (WMFs) were subjected to treatment by fermentation, autoclaving, and fermentation followed by autoclaving. The WMFs were analysed for chemical composition, formulated into wet diets (282 g kg⁻¹) and fed to adult mink (Mustela vison) for determination of total tract digestibility of total starch, total carbohydrate, crude protein and fat. Fermentation of WMF/water mixtures inoculated with a Lactobacillus sp. (strain AD₂) was performed at 30°C for 16 h. Autoclaving was carried out for 60 min at 120°C. Fermentation increased colony-forming units (CFUs) to about 10⁸ g⁻¹ and lowered pH to 3.7–3.8 in both WMFs. All carbohydrate parameters were affected by type of cereal, and were, except for total starch, affected by treatment. Levels of total dietary fibre and β-glucans decreased by fermentation in both WMFs. The decrease in total β-glucans from 33.5 to 18.4 g kg⁻¹ in barley WMF, was mainly restricted to the soluble fraction. Glucose levels in barley WMF increased simultaneously from 0.6 to 12.3 g kg⁻¹. The main effects of autoclaving were increased levels of total dietary fibre, maltose, and increased hydration capacity. With fermentation prior to autoclaving, increases in levels of the fibre fractions and maltose were prevented while hydration capacity prevailed as an effect of autoclaving. Compared with fermentation alone, the combined treatment increased damaged starch levels and hydration capacity. Digestibilities of total carbohydrate, crude protein and fat were significantly higher for wheat than for barley. Fermentation had no effect on digestibility of total starch or total carbohydrate of wheat, but increased digestibility of total starch of barley significantly from 0.742 to 0.880, and of total carbohydrate from 0.457 to 0.616. Autoclaving had no significant effect on digestibility of total starch and total carbohydrate of wheat. Digestibility of total starch and total carbohydrate in barley increased significantly after autoclaving. Total starch and total carbohydrate digestibility of both wheat and barley were significantly enhanced by combined fermentation and autoclaving compared with fermentation alone. Compared with autoclaving alone, combined fermentation and autoclaving promoted no significant improvement of total starch and total carbohydrate digestibility in wheat, whereas total carbohydrate digestibility in barley increased from 0.605 to 0.672. Fat digestibility was slightly improved by both fermentation and autoclaving. Autoclaving of cereals reduced significantly the faecal dry matter contents of mink. This effect could be counteracted by preceding fermentation. In conclusion, lactic acid fermentation of wheat and especially barley provided chemical changes of benefit for carbohydrate digestion in the mink.     

Affinity chromatography of mustard β-amylase on starch columns

An affinity chromatography method for purification of β-amylase from cytoledons of whit mustard seedlings (Sinapsi alba L.) is described. β-Amylase is bound to starch column, while other contaminating proteins are eluted with the binding buffer. The bound β-amylase is eluted by including dextrin (1%, w/v) in binding buffer. This method yielded a homogenous preparation of β-amylase enzyme, which migrated as a single polypetide band in SDS electrophoresis.     

X-ray scattering study on potato (Solanum tuberosum L.) cultivars during winter storage

The nanometer range structure of potato (Solanum tuberosum L.) tubers was examined by wide-angle, small-angle and ultra small-angle X-ray scattering methods. The crystallinity of starch, the lattice constants of the hexagonal lattice of amylopectin, the average crystallite size in the direction [100], the lamellar distance and the thickness of lamella stacks were determined from the data. A new achievement presented in this paper is that reasonable results for these parameters of potato starch were obtained by carrying out experiments on slices and mashes of raw potato tubers. The effects of sample preparation were also investigated by doing experiments on air-dried and re-hydrated potato samples, and on isolated potato starch as well. Changes in the structure of three different cultivars grown in Finland (S. tuberosum cv. Satu, Saturna and Lady Rosetta) were studied monthly from August to May. The physiological ageing caused changes in the crystallinity and in the crystal structure. The mean values (±SD) were determined from the data measured between September and January (30 samples). The lattice constants a=18.4±0.06 and c=10.4±0.04 Å, the crystallinity of starch 24±2% and the crystallite size 118±10 Å were obtained. The lamellar distance was 97±3 Å and the thickness of lamella stacks 513±6 Å. The structural parameters did not vary significantly between Satu, Saturna and Lady Rosetta. For comparison, two cultivars grown in the Netherlands were studied in December. The Dutch cultivars showed the same structural parameters as the Finnish cultivars.     

Influence of elevated CO2 and ozone concentrations on late blight resistance and growth of potato plants

Potato plants (Solanum tuberosum L. cv. Indira) with high susceptibility to the late blight pathogen Phytophthora infestans were exposed for 4 weeks to two different CO₂ concentrations (400/700 ppm) combined with ambient and double ambient ozone concentrations (first experiment) and with 1/5 ambient and ambient ozone concentrations (second experiment) in climate chambers. Leaves of the potato plants were then inoculated with Phytophthora infestans zoospores. Plants from the “high CO₂” variant showed a significantly increased resistance to the pathogen, verified by visual evaluation and quantitative real-time PCR, whereas plants treated with double ambient ozone were slightly more susceptible. An increase in the constitutive activities of the PR-proteins β-1,3-glucanase and osmotin in leaves of plants exposed to 700 ppm CO₂ correlated with the increase in resistance at this CO₂-concentration. Biomass parameters were barely affected by the elevated CO₂-concentration but decreased with increasing ozone concentrations. Biochemical analyses revealed that the content of starch as well as the content of soluble sugars in leaves were highest at the double ambient ozone/700 ppm CO₂ variants pointing to an ozone-induced inhibition of assimilate allocation from leaves to tubers. Leaf C/N-ratio increased at elevated CO₂-concentrations due to a decrease in N-content. The effect of the ozone- and CO₂-induced biochemical changes on the resistance response of potato towards Phytophthora infestans is discussed.     

Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus

The amylases produced by a Bacillus stearothermophilus were purified through a series of four steps. Two separable enzyme fractions having starch hydrolysing activity were eluted from a DEAE-cellulose column by NaCl gradient elution. The homogeneity of the purified enzymes was checked on polyacrylamide gel electrophoresis. The product formation studies indicated that fraction I was an -amylase whereas fraction II was a β-amylase. The molecular weights were determined to be 48 000 and 57 000 and the carbohydrate moiety was found to be 13.2 and 0.8% for - and β-amylase, respectively. The protein digest of these enzymes indicated a total number of 15 amino acids with aspartic and glutamic acid showing the highest value. The purified amylase showed maximal activity at 80°C and pH 6.9. Fe³⁺, Cd²⁺, Pb²⁺, Hg²⁺, Ni²⁺ and Ag¹⁺ were potent inhibitors whereas Zn²⁺, Mg²⁺, Mn²⁺ and Al³⁺ were mild inhibitors. Ca²⁺, Ba²⁺, Sr²⁺ and K⁺ stimulated amylase activity in the order of Ca²⁺ > Ba²⁺ > Sr²⁺ > K⁺. PCMB, EDTA and sodium iodoacetate were inhibitory whereas glutathione (GSH) and cysteine afforded protection of enzyme activity. EDTA showed dose-dependent noncompetitive inhibition of both - as well as β-amylase activities. EDTA inhibition was reversed by the addition of Ca²⁺ and PCMB inhibition by the addition of glutathione (reduced). The K_m for - and β-amylases were found to be 1.05 and 1.25 mg starch per ml, respectively.     

Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1

Transgenic potato ( Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene ( SnRK1 ) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%−167% compared with that in the wild-type. Glucose levels were decreased, at 17%−56% of the levels of the wild-type, and the starch content showed an increase of 23%−30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, α-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%–60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.     

The action pattern and physiological role of Tenebrio larval amylase

The larval midgut amylase of Tenebrio molitor L. may be classified as a typical -amylase on the basis of its action pattern on amylopectin, glycogen, and β-amylase limit dextrin. When the development is on the preferred diet of wheat-bran, the amylatic activity of the larvae is impaired by an -amylase inhibitor present in wheat. The inhibitor has been found to be stable to digestion by the larvae. Wheat β-amylase plays the major role in the digestion of starch by larvae. The implications of these relations are discussed.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

Rapid method for the affinity purification of thermostable α-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Summary A rapid and efficient method the exploiting affinity of α-amylase for its substrate starch is described. α-amylase from Bacillus licheniformis was purified to homogeneity by ammonium sulphate precipitation and affinity chromatography with 230-fold purification. The α-amylase adsorption to various starches was examined in order to screen its ability for highest binding to starch. The α-amylase was bound to starch more tenaciously, hence various eluants like maltose, soluble starch and high salts could not elute the bound α-amylase. However, the bound α-amylase was instantly eluted using 2% (w/v) dextrin. The purified enzyme showed a single polypeptide on SDS-PAGE, with a molecular weight of 58 kD. Western blot analysis confirmed the specificity of antibody raised against purified α-amylase.     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.     

Influence of sucrose on the rheology and granule size of cross-linked waxy maize starch dispersions heated at two temperatures

Cross-linked waxy maize (CWM) starch dispersions (STDs) of concentration 50 g kg⁻¹ were heated in sucrose solutions containing 0–600 g kg⁻¹ (g sucrose/kg dispersion) at 85 °C at low shear and in intermittently agitated cans at 110 °C. The STDs heated in 0–300 g kg⁻¹ sucrose exhibited antithixotropic behavior, while those heated in 400–600 g kg⁻¹ sucrose exhibited thixotropic behavior. The mean starch granule diameter of the starch dispersions did not show strong dependence on sucrose concentration. The dispersions, especially those with high sucrose concentrations and heated at 110 °C, exhibited G′ versus frequency (ω) profiles of gels. The STDs exhibited first normal stress differences that increased in magnitude with the concentration of sucrose. Values of the first normal stress coefficient of canned dispersions calculated from dynamic rheological data plotted against ω and experimental values plotted against shear rate of some of the STDs overlapped.