Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.     

Species-specificity of the lytic activity of the cell wall in the genus Chlorella

Cell wall lytic activity was compared among strains IAM C-27, C-87, SAG 211-1c, -1d, -9a, -8b, -8c, -8l, -11f, -8k, -11g, and -11h/9 of the genus Chlorella . The optimal pH was alkaline in strains with glucosamine as the characteristic group of the rigid wall, and acidic in strains characterised by glucan groups. The lytic enzymes of strains in the former type of algae lyzed the cell wall mainly to soluble high molecular oligosaccharides. The lytic activity of the Chlorella cell wall thus appears species- and strain-speicific.     

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and -1,4-mannanase activity coeluted in gel filtration and were separated from -d-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization 6. The soluble degradation products of the cell wall consisted of 92–96% mannose and 4–8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.     

Growth Delay and Intracellular Changes in Chlorella ellipsoidea C-27 as a Result of Deuteration

The effects of deuterium (D) on Chlorella ellipsoidea C-27 wereinvestigated. Cells grown in a medium prepared with deuteriumoxide (D₂O) showed pronounced delays in cell growth and division;the length of a cell cycle in medium with 100 mol% D₂O was morethan 5 times longer than that in medium prepared in H₂O Thedelay caused by D₂O was not overcome by either indoleaceticacid or kinetin. The biological and ultrastractural characteristicsof deuterated .Chlorella (D-Chlorella) cells were examined.The responses of D-Chlorella to cell wall-digesting enzymesdid not differ from those of normal (H-Chlorella) cells. D-Chlorellacells were enlarged, and cellular components, such as proteins,nucleic acids, lipids and ATP, were present in larger quantitiesthan those in H-cells. The chloroplast of D-Chlorella was enlarged,but the levels of component photosynthetic pigments were significantlyreduced. By contrast, mitochondria of D-Chlorella were smallerthan those of H-cells. These changes in levels of cellular componentsand in the sizes of organelles seem to be unique to deuteration. (Received May 13, 1992; Accepted July 28, 1992)     

Characteristics of the Photosynthetic Metabolism of Carbon in Deuterated Chlorella ellipsoidea C-27

The photosynthetic metabolism of carbon in fully deuteratedcells of Chlorella ellipsoidea C-27 (D-Chlorella), obtainedby culture in medium prepared with 100 mol% D₂O, was characterizedby examining the activities of several enzymes and the levelsof metabolic regulators in a comparison with those of ordinarycells (H-Chlorella). The cellular content of starch in D-Chlorellawas more than twice that in H-Chlorella, whereas those of sucroseand glucose were significantly lower in D-Chlorella. Deuterationof Chlorella caused marked alterations in the activities ofenzymes involved in starch metabolism. There was a significantdecrease in the activity of phosphorylase, a catabolic enzyme,and a significant increase in the activity of starch synthase,an anabolic enzyme. These alterations are probably responsiblefor the increase in the amount of starch in cells. By contrast,no marked changes were observed in the activities of enzymesand the levels of metabolic inhibitors that are involved inthe synthesis of sucrose. It seems likely, therefore, that thedecrease in the amount of sucrose in D-Chlorella was causedmainly by a deficiency in sources of carbon in the cytoplasm,as a consequence of an increase in levels of starch in chloroplasts. (Received May 13, 1992; Accepted December 1, 1992)     

Molecular cloning and characterization of a cDNA encoding nitrate reductase from Chlorella ellipsoidea (Chlorophyta)

Nitrate reductase (NR) genes have beencloned from higher plants, fungi and algae.Based on seven of the amino acid residuesmost strongly conserved between Chlorella vulgaries and Chlamydomonasreinhardtii NR gene, a degenerate primerwas designed. This degenerate primer wasused to amplify the corresponding homologyin Chlorella ellipsoidea. A 3304 bpfull-length cDNA was cloned by rapidamplification of cDNA ends (RACE). Thededuced amino acid sequence of this cDNAhas a high degree of similarity withpreviously identified members of the NRgene. This suggests that the amplified cDNAencodes a functional NR. Northern blotexpression analysis suggests that this geneis strongly induced by nitrate, but isrepressed by ammonium. The nucleotidesequence data reported in this paper willappear in the DDBJ/EMBL/GeneBank databasesunder accession number AY275834.     

利用椭圆小球藻硝酸还原酶缺失突变体为生物反应器表达兔防御素NP-1蛋白

利用转基因小球藻为生物反应器生产兔防御素NP-1蛋白具有重要的应用价值。本研究利用椭圆小球藻(Chlorella ellipsoidea)硝酸还原酶(nitrate reductase)缺失突变体为受体, 构建了包含NPTII基因和硝酸还原酶基因两个筛选标记的兔防御素蛋白表达载体, 采用电激法将目的基因转入椭圆小球藻硝酸还原酶缺失突变体nrm-4, 获得了正确表达防御素蛋白的转基因藻, 从而表明通过硝酸还原酶作为筛选标记基因并结合硝酸还原酶缺失突变体可作为较好的小球藻生物反应器生产模式。     

Stable Integration and Functional Expression of Flounder Growth Hormone Gene in Transformed Microalga, Chlorella ellipsoidea

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 μg of fGH protein expression per one liter culture containing 1 × 10⁸ cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding. Received March 26, 2001; accepted July 10, 2001.     

Phenoloxidase but not lytic activity reflects resistance against Pasteuria ramosa in Daphnia magna

The field of ecological immunology strongly relies on indicators of immunocompetence. Two major indicators in invertebrates, the activity of phenoloxidase (PO) and lytic activity have recently been questioned in studies showing that, across a natural range of baseline levels, these indicators did not predict resistance against a manipulated challenge with natural parasites. We confirmed this finding by showing that baseline levels of PO and lytic activity in the host Daphnia magna were not related to spore load of the parasite Pasteuria ramosa. Yet, PO levels in infected hosts did predict spore load, indicating PO activity can be useful as an indicator of immunocompetence in this model parasite–host system.     

绿色木霉对小球藻细胞壁的酶解作用

【目的】探讨绿色木霉分泌液能否分解小球藻细胞壁。【方法】用海藻酸钠和氯化钙固定绿色木霉,游离绿色木霉和固定化绿色木霉分别培养一段时间,离心培养液,用分光光度计法检测上清液中纤维素酶活性。在上清液中加入浓缩的小球藻悬浮液,用显微镜计数细胞壁破碎的小球藻。【结果】绿色木霉能同时分泌内切葡聚糖酶、外切葡聚糖酶及β-1,4葡萄糖苷酶3种纤维素酶,其中外切葡聚糖酶活性最高。固定化绿色木霉反复使用5次后,分泌的纤维素酶活性能保持到初次的67.4%。市售纤维素酶、游离绿色木霉、固定化绿色木霉初次及第5次分解小球藻细胞壁的效率分别为47.3%、86.5%、81.5%、52.1%。【结论】市售纤维素酶、游离绿色木霉、固定化绿色木霉都能分解小球藻细胞壁,其中固定化绿色木霉因可重复使用,具有潜在的应用前景。     

Mannuronan C-5 epimerase activity in protoplasts of Laminaria digitata

Biosynthesis of alginate in algae may be studied by following the cell wall regeneration of brown seaweed protoplasts in culture. The enzyme mannuronan C-5 epimerase will control the composition of the alginate being synthetized.Freshly isolated protoplasts from the thallus of young Laminaria digitata plants showed only low expression of this enzyme. However, after prolonged periods in culture, this activity increased 15-fold. The synthesis of C-5 epimerase by the protoplasts is probably essential for the formation of a new cell wall.After cellular disruption by osmotic shock and centrifugation, most of the epimerase activity resided in the pellet fraction. This may indicate that the enzyme is membrane associated.     

Changes in the Carbonic Anhydrase Activity and the Rate of Photosynthetic O2 Evolution during the Cell Cycle of Chlorella ellipsoidea C-27

Rhythmical changes in carbonic anhydrase activity(CA) and inphotosynthesis were observed during the cell cycle of Chlorellaellipsoidea C-27 synchronized at various concentrations of dissolvedCO₂ (dCO₂ with a regime of 16 h of light and 8 h of darkness.At a constant low concentration of dCO₂ (11 {diaeresis}M), intracellularCA activity showed obvious fluctuations with a peak at 8 h afterthe initiation of illumination, while extracellular CA activity,located on the cell surface, showed only minor fluctuationsalthough the activity was as high as the maximum activity ofintracellular CA. In contrast, obvious changes in the activitiesof intra- and extracellular CA activities were not observedat a high concentration of dCO₂ (520 {diaeresis}M). The ratioof photosynthetic activity at limiting versus saturating concentrationsof dCO₂, which is indicative of the affinity of cells for CO₂,showed clear rhythmical changes during the cell cycle and theratio was higher in low-CO₂ cells than in high-CO₂ cells. Thechanges in the ratio seemed to reflect the changes in CA activity. When the cells that had been synchronized under high CO₂ conditionswere transferred to low CO₂ conditions at any given stage inthe cell cycle, CA activity was induced in every case but thecapacity for induction of CA was greater in young cells thanin mature cells. This result suggests that the capacity of cellsto induce CA over the course of the cell cycle is closely relatedto endogenous aging of the cell. (Received August 29, 1988; Accepted December 28, 1988)     

The acquisition and accumulation of inorganic carbon by the unicellular green alga Chlorella ellipsoidea

Abstract. The uptake and accumulation of inorganic carbon has been investigated in Chlorella ellipsoidea cells grown at acid or alkaline pH. Carbonic anhydrase (CA) was detected in ceil extracts but not in intact cells and CA activity in acid-grown cells was considerably less than that in alkali-grown cells. Both cell types demonstrates low K_1/2 (CO₂) values in the range pH 7.0–8.0 and these were unaffected by O₂ concentration. The CO₂ compensation concentrations of acid- and alkali-grown cells suspended in aqueous media were not significantly different in the range of pH 6.0–8.0, but at pH 5.0, the CO₂ compensation concentrations of acid-grown cells (57.4cm³ m⁻³) were lower than those of alkali-grown cells (79.2cm³ m⁻³). The rate of photo-synthetic O₂ evolution in the range pH 7.5–8.0 exceeded the calculated rate of CO₂ supply two- to three-fold, in both acid- and alkali-grown cells, indicating that HCO₃⁻ was taken up by the cells. Accumulation of inorganic carbon was measured at pH 7.5 by silicone-oil centri-fugation, and the concentration of unfixed inorganic carbon was found to be 5.1 mol m⁻³ in acid-grown and 6.4mol m⁻³ in alkali-grown cells. These concentrations were 4.6- and 5.9-fold greater than in the external medium. These results indicate that photorespiration is suppressed in both acid- and alkali-grown cells by an intracellular accumulation of inorganic carbon due, in part, to an active uptake of bicarbonate.     

一株裂解性奶牛乳房炎源大肠杆菌噬菌体的分离鉴定及生物学特性分析

【目的】从新疆石河子地区奶牛粪样中分离裂解性大肠杆菌噬菌体(Escherichia coli phage),对其进行纯化及生物学特性分析。【方法】利用双层平板法从奶牛粪样中分离、纯化噬菌体,将纯化后的噬菌体浓缩液用醋酸双氧铀负染后通过透射电子显微镜观察其形态特征。对该噬菌体进行全基因组测序和遗传进化分析,同时测定噬菌体的宿主谱、最佳感染复数、一步生长曲线、热稳定性及酸碱稳定性。【结果】分离并纯化出一株裂解性噬菌体vB_EcoM_XJ2,噬菌斑圆形不透明,直径0.7 mm–1.2 mm;电镜显示其头部呈正多面体对称,有可伸缩性尾部;核酸类型为双链DNA,基因组大小为75.617 kb,G+C%含量为42.09%;其核酸序列与大肠杆菌噬菌体NJ01和vB_EcoP_SU10相似性高达94%。生物学特性研究显示该噬菌体能裂解多株临床分离的大肠杆菌;能耐受60°C左右高温,在pH 5.0–11.0范围内效价稳定;最佳感染复数为0.1,潜伏期为15 min,暴发期为95 min,裂解量约为10.6 PFU/cell。【结论】vB_EcoM_XJ2是一株在不同温度、不同酸碱性环境中有较强适应能力的裂解性肌尾科大肠杆菌噬菌体。     

Purification and characterization of a novel haemagglutinin from Chlorella pyrenoidosa

We previously identified a strong haemagglutination activity in the freshwater unicellular green alga, Chlorella pyrenoidosa. Here, we sought to purify and characterize the haemagglutinin associated with this activity. Ammonium sulfate precipitation, gel filtration on sephacryl S-200 and DEAE-Sepharose ion-exchange chromatography were used to purify the haemagglutinin, which was designated CPH (Chlorella pyrenoidosa haemagglutinin). The molecular weight of CPH was estimated as 58 kDa by SDS-PAGE and 60 kDa by gel filtration of the native protein, indicating that this haemagglutinin exists as a monomer. The haemagglutinin activity of CPH was inhibited by glycoproteins, especially yeast mannan, but not by monosaccharides or disaccharides, indicating that CPH is carbohydrate-specific. In addition to the composition of CPH shown to be rich in glycine and acidic amino acids, heamagglutinating activity of CPH was insensitive to variations in pH or the presence of divalent cations, and atomic force microscopy revealed that the protein is rod-shaped. These results indicate that the characteristics of CPH are consistent with its identification as a haemagglutinin, and suggest that CPH may be a viable candidate for applications in a variety of biomedical fields.     

Composition and positional distribution of fatty acids in polar lipids from Chlorella ellipsoidea differing in chilling susceptibility and frost hardiness

The composition and positional distribution of fatty acids in the polar lipids from 4 strains of Chlorella differing in chilling susceptibility and frost hardiness were analyzed by enzymatic hydrolysis and gas-liquid chromatography. Analysis of the polar lipids from chilling-sensitive, chilling-resistant and chilling-sensitive revertant strains of Chlorella ellipsoidea IAM C-102 showed that the sum of palmitic and trans -3-hexadecenoic acid in phosphatidylglycerol (PG) is about 60% for the sensitive strains and 53% for the resistant strain. The sum of dipalmitoyl and 1-palmitoyl-2-( trans -3-hexadecenoyl) PG as estimated from the positional distribution of their fatty acids, is about 10% in the case of each of the three strains. The contents of unsaturated fatty acids in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were higher in the resistant than in the sensitive strain. This suggests that unsaturation of fatty acids in not only PG but also PC and PE is involved in chilling sensitivity of Chlorella . On the other hand, lipid changes during the development of frost hardiness of C. ellipsoidea IAM C-27, a frost hardy strain, were examined. The results showed that fatty acids in most lipid classes are unsaturated in the hardening process but their degree of unsaturation is not greatly different from that of the chilling-resistant strain, suggesting that not only unsaturation of fatty acids in lipids but also other factors are necessary for the development of frost hardiness.     

Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus

As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a "living fossil" for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head(59.2 nm in length, 31.9 nm in width) and a tail(43.2 nm in length). Bacteriophage VMY22 was classified as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at p H 5.0–9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.     

Detection of surface forces by the cell-wall mechanosensor Wsc1 in yeast

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Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)