产碱性磷酸酶乳杆菌的筛选鉴定、酶的纯化及特性

【背景】碱性磷酸酶(alkaline phosphatase,ALP)是生物体内参与磷酸代谢的调控酶,不同物种的ALP性质与其生理功能有关,提纯后的ALP常用作工具酶,广泛应用于基因工程中,但目前关于乳酸菌中ALP的相关研究甚少。【目的】筛选出一株产ALP且具有潜在益生作用的乳杆菌,对该酶进行分离纯化,并对其性质进行探究,为今后益生菌的开发利用和ALP的工业化生产提供新的微生物资源。【方法】采集蒙古国4个地区的酸马奶样品,通过显色反应初筛和酶活检测复筛对产酶菌株进行筛选,经形态学观察、生理生化鉴定及16S rRNA基因序列同源性比较分析进行菌种鉴定。采用超声破碎法提取ALP,经硫酸铵沉淀、DEAE-52离子交换层析、Sephadex G-200凝胶过滤层析纯化该酶,SDS-PAGE电泳法检测其纯度。【结果】从78株乳酸菌中分离筛选出一株产ALP酶活性最高的乳杆菌(编号为Z23),16S rRNA基因序列长度为1 473 bp,鉴定结果表明为鼠李糖乳杆菌。纯化后的酶比活力为180.27 U/mg,纯化倍数为48.37,酶活回收率为17.05%,该酶亚基相对分子质量为46.7 kD。菌株所产ALP的最适温度为37℃,4℃时酶活最为稳定;最适pH为9.5,在pH 9.0-10.0之间,酶活稳定性可达90%以上;Mg2+和K+对ALP有明显激活作用,Ba2+和Cu2+在低浓度时对ALP有激活作用,高浓度时有抑制作用,Ca~(2+)、Zn~(2+)和EDTA对ALP有强烈的抑制作用。以不同浓度的p-NPP为底物,测得酶的Km值为3.42 mmol/L,Vmax值为1.24 mmol/(L·min)。【结论】本研究对蒙古国地区酸马奶中的益生菌资源有了更为明确的认知,为今后碱性磷酸酶产生菌的筛选和酶的应用开辟了新途径。     

Alanine racemase from the green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Summary. Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0–2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 μmol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 °C and pH 8.4 and requires pyridoxal 5′-phosphate as a coenzyme.     

Photochemical Performance of the Acidophilic Red Alga Cyanidium sp. in a pH Gradient

The acidophilic red alga Cyanidium sp. is one of the dominant mat-forming species in the highly acidic waters of Río Tinto, Spain. The culture of Cyanidium sp., isolated from a microbial mat sample collected at Río Tinto, was exposed to 9 different pH conditions in a gradient from 0.5 to 5 for 24?h and its physiological status evaluated by variable chlorophyll a fluorescence kinetics measurements. Maximum quantum yield was determined after 30?min, 1?h, 2?h, 4?h, 6?h and 24?h of exposure after 15?min dark adaptation. The effect of pH on photochemical activity of Cyanidium sp. was observable as early as 30?min after exposure and the pattern remained stable or with only minor modifications for 24?h. The optimum pH ranged from 1.5 to 2.5. A steep decrease of the photochemical activity was observed at pH below 1 even after 30?min of exposure. Although the alga had tolerated the exposure to pH?=?1 for at least 6?h, longer (24?h) exposure resulted in reduction of the photochemical activity. At pH above 2.5, the decline was more moderate and its negative effect on photochemistry was less severe. According to the fluorescence measurements, the red alga Cyanidium sp. is well-adapted to prevailing pH at its original locality at Río Tinto, i.e. pH of 1 to 3. The short-term survival in pH?相似文献   

Effects of dibutyryl adenosine 3':5'-cyclic monophosphate and other agents on induction of alkaline phosphatase activity in monkey kidney cells

The induction of alkaline phosphatase (ALP) by dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) was investigated in strain JTC-12 . P3 cells derived from monkey (Maccaca irus) kidney cortex. ALP activity was increased by Bt2cAMP in a dose-dependent manner, reaching a plateau at concentrations higher than 5 mM with the activity being about 4 times that of the controls. The concentration of Bt2cAMP required for half-maximal induction of ALP activity was about 0.8 mM. ALP activity was increased rapidly by Bt2cAMP for the first 5 days and then continued to increase gradually towards a plateau level. Removal of Bt2cAMP from the medium caused a rapid decrease in the activity, suggesting that the induction of ALP activity by Bt2cAMP is reversible. ALP activity was induced synergistically in the presence of 1 mM sodium butyrate together with Bt2cAMP at concentrations from 0.01 to 1 mM. It was also found that in the presence of 1 mM Bt2cAMP, sodium butyrate increased ALP activity in the same manner as Bt2cAMP did in the presence of 1 mM sodium butyrate. Although dexamethasone, a potent glucocorticoid, had no effect on ALP activity in control cells, the hormone suppressed the ALP activity induced by Bt2cAMP in a dose-dependent manner. At concentrations above 0.2 mM, two xanthine derivatives, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX), also inhibited the induction of ALP activity by 1 mM Bt2cAMP. Inhibitors of protein synthesis, cycloheximide (1.5 micrograms/ml) and pactamycin (10 micrograms/ml), as well as inhibitors of RNA synthesis, actinomycin D (2 micrograms/ml) and alpha-amanitin (50 micrograms/ml), suppressed the induction of ALP activity.     

Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells

Alkaline phosphatase (ALP) was examined in cultured human osteosarcoma cells (SAOS-2) with respect to isoenzyme form, kinetic properties toward two natural substrates, and topography and nature of attachment to the plasma membrane. ALP in SAOS-2 homogenates is the tissue-nonspecific (TNS) isoenzyme and a phosphoethanolamine (PEA) and pyridoxal 5'-phosphate (PLP) phosphatase, as demonstrated by heat and inhibition profiles and electrophoretic mobility. Kinetic studies indicate that TNSALP in SAOS-2 cells has both a low- and a high-affinity activity. The high-affinity activity (showing the greater catalytic efficiency) is active at physiologic pH toward physiologic concentrations (microM) of PEA and PLP. TNSALP was shown to be an ectoenzyme in SAOS-2 cells by our findings in intact cell suspensions, where (i) PEA and PLP degradation in the medium nearly equaled that of whole cell homogenates, (ii) greater than 85% of ALP activity was inactivated by acid treatment, and (iii) ALP activity was quantitatively released by phosphatidylinositol-specific phospholipase C. Our findings indicate that, in SAOS-2 cells, TNS (bone) ALP functions as an ectoenzyme to degrade physiologic concentrations of extracellular natural substrates at physiologic pH.     

Alkaline-resistance model of subtilisin ALP I, a novel alkaline subtilisin

The alkaline-resistance mechanism of the alkaline-stable enzymes is not yet known. To clarify the mechanism of alkaline-resistance of alkaline subtilisin, structural changes of two typical subtilisins, subtilisin ALP I (ALP I) and subtilisin Sendai (Sendai), were studied by means of physicochemical methods. Subtilisin NAT (NAT), which exhibits no alkaline resistance, was examined as a control. ALP I gradually lost its activity, accompanied by protein degradation, but, on the contrary, Sendai was stable under alkaline conditions. CD spectral measurements at neutral and alkaline pH indicated no apparent differences between ALP I and Sendai. A significant difference was observed on measurement of fluorescence emission spectra of the tryptophan residues of ALP I that were exposed on the enzyme surface. The fluorescence intensity of ALP I was greatly reduced under alkaline conditions; moreover, the reduction was reversed when alkaline-treated ALP I was neutralized. The fluorescence spectrum of Sendai remained unchanged. The enzymatic and optical activities of NAT were lost at high pH, indicating a lack of functional and structural stability in an alkaline environment. Judging from these results, the alkaline resistance is closely related to the surface structure of the enzyme molecule.     

一株产高酶活性碱性磷酸酶解淀粉芽孢杆菌的分离及其phoD碱性磷酸酶基因的克隆与表达

[背景]碱性磷酸酶作为工具酶被广泛应用于各个领域,在免疫学检测方面应用较多的是PhoA家族的碱性磷酸酶,尚无关于PhoD家族的碱性磷酸酶在免疫学检测方面的研究。[目的]筛选出一株产高酶活性PhoD家族碱性磷酸酶的细菌,并将其phoD基因进行克隆表达,研究PhoD的酶学性质,为PhoD家族的碱性磷酸酶在免疫学检测方面的应用奠定一定的基础。[方法]采取有机质丰富的土样在有机磷平板中进行细菌分离,以4-硝基苯磷酸二钠盐（4-nitrophenyl phosphate disodium salt hexahydrate,p-NPP）为底物测定有机磷平板中单菌落的酶活性,选取酶活性高的菌株作为目的菌株,克隆其phoD基因。[结果]筛选到一株产碱性磷酸酶酶活性高的菌株S2-4,通过16S rRNA基因序列同源性比较分析,鉴定该菌株为解淀粉芽孢杆菌,克隆了其phoD基因并进行诱导表达。研究了纯化后PhoD的酶学性质,PhoD的最适反应温度为70℃;最适反应pH为9.8;PhoD最适Ca²⁺浓度为3 mmol/L,Mg²⁺对PhoD的酶活性有抑制作用,K     

Thermal properties of NADP:ferredoxin oxidoreductase and ferredoxin isolated from a thermophilic blue-green alga

NADP:ferredoxin oxidoreductase (EC. 1.6.7.1.) isolated from a thermophilic blue-green alga, Synechococcus sp., was stable at temperatures up to 65°C. The diaphorase and cytochrome c reductase activities of the enzyme were low at 25°C but increased with elevated temperature to reach a maximum at about 60°C. The pH-profile of the diaphorase activity showed a peak at pH 9.0 at 55°C, whereas the activity was largely independent of pH at 25°C. High concentrations of NaCl suppressed activity at both high and low temperatures. In the cytochrome c reductase activity catalyzed by the enzyme, ferredoxin served as an electron carrier in a temperature-insensitive manner over a wide range of temperature. The results support the view that the optimum and the upper limiting temperatures for photosynthesis in this alga are related to thermal properties of proteins.     

The Effect of Some External Factors on Nitrogenase Activity in the Free-Living and Endophytic Nostoc of the Liverwort Blasia pusilla

The effects of pH, light intensity, temperature, oxygen and dehydration on nitrogenase activity in the free-living and endophytic Nostoc sp. of Blasia are described. The endophyte exhibits greater nitrogenase activity at lower pH's than the free-living alga. Maximal activity in the free-living alga is attained at much lower light intensities than those required by the endophyte. Both algae have low activities below 10°C, the free-living alga showing high activity above 12°C and the endophyte above 17°C. Increasing pO₂ levels cause a decrease in activity in both algae. The free-living alga is more tolerant to dehydration than the endophyte, although both algae are protected by mucilage. The results are discussed and possible benefits resulting from the symbiosis, with respect to nitrogen fixation rates, considered.     

链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２株脂肪酶产生菌中,链霉菌Ｚ９４－２产脂肪酶活力为５９６ｕ／ｍＬ,其最适培养基（ｇ／Ｌ）为：糊精１０、黄豆饼粉３０、尿素１０、Ｋ２ＨＰＯ４０．５、ＭｇＳＯ４０．５、ＮａＣｌ１和ＡＥＯ９０．５,产酶的最适条件为：初始ｐＨ９．５～１０．０,在２６℃培养４８ｈ。用ＰＶＡ橄榄油乳化系统测定该酶的最适ｐＨ９．８,最适温度３７℃,在ｐＨ８．６～１０．２于５℃存放２４ｈ,酶活力不变。０．１４ｍｏｌ／Ｌ的氯     

Alkaline phosphatase (tissue-nonspecific isoenzyme) is a phosphoethanolamine and pyridoxal-5''-phosphate ectophosphatase: normal and hypophosphatasia fibroblast study.

To clarify its physiologic role, alkaline phosphatase (ALP) was examined in normal skin fibroblasts and was shown to be the tissue-nonspecific (TNS) isoenzyme type (as evidenced by heat and inhibition profiles) and to be active toward millimolar concentrations of the putative natural substrates phosphoethanolamine (PEA) and pyridoxal-5'-phosphate (PLP). Fibroblast ALP has a low-affinity activity, with a distinctly alkaline pH optimum (9.3), toward 4-methylumbelliferyl phosphate (4-MUP), PEA, and PLP but a more physiologic pH optimum (8.3) toward physiologic concentrations (micromolar) of PEA and PLP. Normal fibroblast ALP is linked to the outside of the plasma membrane, since in intact cell monolayers (1) dephosphorylation rates of the membrane-impermeable substrates PEA and PLP in the medium at physiologic pH were similar to those observed with disrupted cell monolayers, (2) brief exposure to acidic medium resulted in greater than 90% inactivation of the total ALP activity, and (3) digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) released about 80% of the ALP activity. Hypophosphatasia fibroblasts were markedly deficient (2%-5% control values) in alkaline and physiologic ALP activity when 4-MUP, PLP, and PEA were used as substrate. The majority of the detectable ALP activity, however, appeared to be properly lipid anchored in ecto-orientation. Thus, our findings of genetic deficiency of PEA- and PLP-phosphatase activity in hypophosphatasia fibroblasts, as well as our biochemical findings, indicate that TNS-ALP acts physiologically as a lipid-anchored PEA and PLP ectophosphatase.     

Antioxidant activity of mycosporine-like amino acids isolated from three red macroalgae and one marine lichen

Several standard in vitro assays were performed in order to determine the potential antioxidant capabilities of purified aqueous extracts of the mycosporine-like amino acids (MAAs), porphyra-334 plus shinorine (P-334 + SH), isolated from the red alga Porphyra rosengurttii, asterina-330 plus palythine (AS-330 + PNE), from the red alga Gelidium corneum, shinorine (SH), from the red alga Ahnfeltiopsis devoniensis, and mycosporine -glycine (M-Gly), isolated from the marine lichen Lichina pygmaea. The scavenging potential of hydrosoluble radicals (ABTS⁺ decolorization method), the antioxidant activity in lipid medium (β-carotene/ linoleate bleaching method) and the scavenging capacity of superoxide radicals (pyrogallol autooxidation assay) were evaluated. In terms of scavenging of hydrosoluble radicals, the antioxidant activity of all MAAs studied was dose-dependent and it increased with the alkalinity of the medium (pH 6 to 8.5). M-Gly presented the highest activity in all pH tested; at pH 8.5 its IC₅₀ was 8-fold that of L-ascorbic acid (L-ASC) followed by AS-330 + PNE while P-334 + SH and SH showed scarce activity of scavenging of hydrosoluble free radicals. AS-330 + PNE showed high activity for inhibition of β-carotene oxidation relative to vitamin E and superoxide radical scavenging whilst the activity of P-334 +SH and SH were moderate. According to these results, the potential of MAAs in photoprotection can be considered high due to a double function: (1) UV chemical screening with high efficiency for UVB and UVA regions of the solar spectrum, and (2) their antioxidant capacity.     

Analysis of liver/bone/kidney alkaline phosphatase mRNA, DNA, and enzymatic activity in cultured skin fibroblasts from 14 unrelated patients with severe hypophosphatasia.

Hypophosphatasia is a heritable disorder characterized by defective bone mineralization and a deficiency of liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity in serum and tissues. Severe forms of the disease, which are generally lethal in infancy, are inherited in an autosomal recessive fashion. The gene defects that produce hypophosphatasia are poorly understood, but many are likely to occur at the L/B/K ALP locus. To investigate these gene defects, we analyzed L/B/K ALP DNA, RNA, and enzyme activity in cultured dermal fibroblasts from 14 patients with perinatal or infantile hypophosphatasia and from 12 normal individuals. Southern blot analyses of the L/B/K ALP genes from patients and controls revealed identical restriction patterns. Control fibroblast ALP activity correlated with the corresponding L/B/K ALP mRNA levels estimated by blot hybridization analysis and densitometry (r = .94, P less than .0001). In contrast, fibroblasts from the hypophosphatasia patients were deficient in ALP enzyme activity but expressed apparently full-sized L/B/K ALP mRNA at normal levels. Bone specimens from one of the patients were examined and found to be deficient in histochemical ALP but contained immunologic cross-reactive material detected by anti-human liver ALP antiserum. Our results demonstrate that the deficiency of ALP activity in fibroblasts from 14 patients with severe hypophosphatasia is not due to decreased steady-state levels of the corresponding mRNA. The presence of enzymatically inactive L/B/K ALP protein in one of these patients is consistent with a point mutation or small in-frame deletion in the coding region of L/B/K ALP gene.     

Alkaline phosphatase as an indicator of true ejaculation in the rhinoceros

The objective was to determine if seminal alkaline phosphatase (ALP) can serve as an indicator of true ejaculation in the rhinoceros. Concentrations of ALP activity were determined in seminal fractions collected from African black rhinos (Diceros bicornis), an African white rhino (Ceratotherium simum), and an Indian rhino (Rhinoceros unicornis) during electroejaculation. In addition, seminal fractions collected during penile massage of a Sumatran rhino (Dicerorhinus sumatrensis) were assessed. Correlations between ALP activity and sperm concentration, fraction pH, and fraction osmolality were evaluated in the Indian rhino and black rhino. Concentrations of ALP activity in rhino ejaculate fractions ranged from < 5 to 11,780 U/L and were positively correlated (P < 0.05) with sperm concentration for both Indian rhino (r = 0.995) and black rhino (r = 0.697), but did not exhibit a strong correlation with either pH or osmolality (P > 0.05). Data were insufficient for establishing meaningful correlation coefficients in the Sumatran rhino and white rhino, but preliminary results were in accordance with findings in the Indian rhino and black rhino. We concluded that ALP was present in rhinoceros semen, likely originated from the epididymides and/or testes, and could serve as a useful tool for assessing the production of ejaculatory versus pre-ejaculatory fluid in the rhinoceros.     

Small intestinal alkaline phosphatase activity and changes induced by cysteamine administration in fecal excretions of male Wistar rats

Alkaline phosphatase (ALP) activity in fecal excretions was measured in male Wistar rats. Total daily ALP activity in fecal extracts was 133.1 +/- 21.2 mumoles/min per rat weighing approximately 150 g. We found that 63.7% of the fecal ALP activity was inhibited by 30 mM L-phenylalanine (L-Phe), a specific inhibitor for intestinal ALP. As body weight increased from 150 g to 300 g, total daily ALP activity in fecal extracts decreased rapidly to 33.7 +/- 6.08 mumoles/min/rat. However, the percentage of L-Phe-sensitive ALP to total enzyme activity was less variable (40-65%) in the growing rats. Cysteamine-HCl, an ulcerogenic drug, was injected subcutaneously to adult rats (300 g b. w) at a dosage of 400 mg/kg. b. w. Daily excretion of L-Phe-sensitive ALP in fecal extracts decreased to one-third 2 days after injection. Afterwards, a steep and transient increase in the enzyme activity was detected in fecal extracts between days 4 and 7 after injection. ALP activity in fecal excretions may be a clinical indicator of duodenal mucosal damage.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

Inorganic Carbon Accumulation and Photosynthesis in a Blue-green Alga as a Function of External pH

The blue-green alga Coccochloris peniocystis photosynthesizes optimally over the pH range of 7.0 to 10.0, but the O₂-evolution rate is inhibited below pH 7.0 and ceases below pH 5.25. Measurement of the inorganic carbon pool in this alga in the light, using the silicone-fluid filtration technique demonstrated that the rate of accumulation of dissolved inorganic carbon remained relatively constant over a wide pH range. At external dissolved inorganic carbon concentrations of 0.56 to 0.89 millimolar the internal concentration after 30 seconds illumination was greater than 3.5 millimolar over the entire pH range. Intracellular pH measured in the light using [¹⁴C]5,5-dimethyloxazolidine-2,4-dione and [¹⁴C]methylamine dropped from pH 7.6 at an external pH of 7.0 to pH 6.6 at an external pH of 5.25. Above an external pH of 7.0 the intracellular pH rose gradually to pH 7.9 at an external pH 10.0. Ribulose-1,5-bisphosphate carboxylase activity of cell-free algal extracts exhibited optimal activity at pH 7.5 to 7.8 but was inactive below pH 6.5. It is suggested that the inability of Coccochloris to maintain its intracellular pH when in an acidic environment restricts its photosynthetic capacity by a direct pH effect on the principal CO₂ fixing enzyme.     

有机溶剂对缢蛏碱性磷酸酯酶分子构象和催化活力的影响

本文初步研究了三种有机溶剂甲醇、二甲基甲酰胺、二氧六环对缢蛏碱性磷酸酯酶(ALP)分子构象和催化活力的影响。发现在有机溶剂作用下,ALP的荧光发射光谱、紫外差光谱、园二色光谱都发生了显著的变化,而酶经甲醇、二甲基甲酰胺预处理后酶活力提高,经二氧六环预处理后活力下降,但当测活体系中有三种有机溶剂存在时,酶的活力却被大大抑制。     

A sensitive enzyme-catalytic nanogold-resonance scattering spectral assay for alkaline phosphate

In pH 8.9 Tris-HCl buffer solutions, alkaline phosphatase (ALP) catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP) substrate to form ascorbic acid. Then H(3)PO(4) was added to stop the enzymatic reaction and HAuCl(4) was used to react with ascorbic acid to generate gold nanoparticles that exhibited a resonance scattering (RS) peak at 600 nm. Under the selected conditions, when the activity of ALP increased, the formed ascorbic acid and gold nanoparticles also increased. Thus, the RS intensity at 600 nm enhanced linearly. The linear range was 0.06-22 U/L, with a detection limit of 0.03 U/L. The ALP in serum was analyzed, and the results were in agreement with those of the fluorescence method.