Detection and first characterization of a cell-wall lytic activity in Chlorella ellipsoidea C-27

Cell wall lytic activity was detected in Chlorella ellipsoidea Gernick IAM C-27 using the [¹⁴C]-labeled cell wall as substrate. The highest activity was obtained at pH 8. and the solubilized product was a polysaccharide of high molecular weight. The lytic activity appeared to be a protease and did not hydrolyse glycosidic bonds of the cell wall polysaccharides. The activity probably solubilizes the cell wall by cleaving the peptide bonds that interconnect the polysaccharides of the cell wall.     

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.     

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。     

Use of a lytic enzyme system from Cytophaga sp. in the lysis of Gram-positive bacteria

A lytic enzyme system from Cytophaga sp. has been used for lysis of the Gram-positive bacteria, Bacillus and Corynebacterium. The optimum pH and temperature for the lytic reaction were 9.2 and 50°C, respectively. The effect of substrate and enzyme concentration have also been studied. Protein release was followed and the potential of using bacteriolytic enzymes for large-scale cell lysis and release of intracellular material is discussed.     

Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.     

酶降解烟叶中细胞壁物质

烟叶中以细胞壁物质存在的碳水化合物在燃吸时产生不良影响,在一定条件下向烟叶中施加一定量的纤维素酶和果胶酶,使部分细胞壁物质降解为水溶性糖,烟质得到改善,纤维素酶和果胶酶最佳用量均为每克烟叶30u酶量（活力）,最佳作用条件为;烟叶水分25%,作用温度50℃,作用时间4h,且在真空条件下可使细胞壁物质降解更有效,可降解烟叶中细胞壁物质10%左右,烟叶的评吸质量得到明显改善。     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Cell walls of algae in the volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide ofChlamydomonas reinhardtii

A cell wall lytic enzyme (gamete wall-autolysin) and a polyclonal antiserum raised against one of the major cell wall glycopeptides ofChlamydomonas reinhardtii were used to study their cross-reactivities with the cell walls of variety of members of the Volvocales. Lytic enzyme was able to digest completely the cell walls of five species ofChlamydomonas (C. reinhardtii group), six species ofGonium and two species ofAstrephomene. The colonial structures ofGonium andAstrephomene were broken into individual cells by exposure to the enzyme and protoplasts were then formed. These organisms also showed a strong cross-reactivity with anti-cell wall glycopeptide by an indirect-immunofluorescence test. The cell walls ofChlamydomonas angulosa, Dysmorphococcus globosus, Pandorina morum, Eudorina elegans, Volvulina steinii, Pleodorina california andVolvox carteri all showed a strong cross-reactivity to the antibody, although they were insensitive to the lytic enzyme. Many other species ofChlamydomonas, Carteria crucifera, Chlorogonium elongatum, Polytoma uvella, Haematococcus lacustris, Lobomonas piriformis, Phacotus lenticularis, Pteromonas angulosa, Stephanosphera pluvialis, andPyrobotrys casinoensis had cell walls which were resistant to the enzyme and showed no or weak cross-reactivity with the antibody. Based on the results, a possible evolutionary sequence from a unicellular relative ofC. reinhardtii to the multicellular algae is discussed.     

Studies on the cell wall of Chlorella I. Quantitative changes in cell wall polysaccharides during the cell cycle of Chlorella ellipsoidea

1. 1. The cell wall of Chlorella ellipsoidea was fractionated intotwo components, alkali-soluble hemicellulose and alkali-insoluble"rigid wall". The former was composed of several neutral sugars,i.e. rhamnose, xylose, arabinose, mannose and galactose, andthe latter had glucosamine as a main constituent sugar.
2. 2.Quantitative changes in both hemicellulose and "rigid wall"contents during the cell cycle were followed using synchronouslygrown cells. The two cell wall components showed markedly differentchanges. Hemicellulose increased in proportion to the enlargementof the cell surface area in the growing phase, while the "rigidwall" remained almost constant in this phase. The "rigid wall"increased only in the reproduction phase—the time of autosporeformation.

(Received September 26, 1977; )     

Degradation of agarophytic red algal cell wall components by new crude enzyme preparations

Several new crude enzyme preparations were isolated from a marine association of the agarolytic bacterium Cytophaga diffluens and the infusorium Uronema marinum, an axenic culture of Cytophaga diffluens, some species of land micro- and macromycetes adapted to assimilate red algal biomass and from the marine mollusc Littorina littorea. Fungal and mollusc enzyme preparations were shown to have cellulase, xylanase, protease and agarase activities. Fungal agarase activity was revealed only after 3–4 passages of the culture on the medium containing algal biomass. Enzyme preparations from the association and the pure bacterial culture growing on the medium with bactoagar as the sole carbon source contained only agarase activity. The maximum specific agarase activity was found in a preparation from the marine association. The preparations obtained can be used for isolating protoplasts and single cells from red seaweed thalli. This revised version was published online in June 2006 with corrections to the Cover Date.     

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis

Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0–3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards β-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.     

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.     

Proteolytic activity of a yeast cell wall lytic Arthrobacter species

AIMS: To investigate properties of the proteolytic activity of a yeast cell wall lytic soil bacterium identified as an Arthrobacter species. METHODS AND RESULTS: The organism was grown at pH 7.5 and 30 degrees C in shake flasks on media with different complex subtrates. Highest proteolytic activity assayed with azocaseine was detected in media with wheat gluten. In addition, l-leucine, l-alanine exopeptidase activity and esterase activity were found. The proteolytic activity showed stability up to pH 12, with a maximum at pH 11. The temperature optimum was at 55 degrees C, but there was a loss in enzyme activity of 50% within 2 h. The proteolytic activity was inhibited by 3,4-dichloroisocumarin, whereas there was little or no effect with EDTA, pepstatin A or E64. CONCLUSIONS: The proteolytic activity is highly alkaline stable. The formation of the enzyme can be induced by media with high protein content.     

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.     

海水小球藻抗菌蛋白的分离纯化及性质研究

海水小球藻(Chlorella pacifica)提取液经硫酸铵沉淀、DEAE-52离子交换层析和SephadexG-200凝胶过滤层析后分离纯化出1种抗菌蛋白.经SDS-PAGE测定,两个亚基的相对分子量分别为61 kD和70 kD;该抗菌蛋白对热稳定,氨基酸组成分析表明含17种氨基酸,其中谷氨酸的含量最高,其次为甘氨酸与天冬氨酸,胱氨酸的含量最低.在抗菌活性中,纯化的蛋白质对产黄青霉(Penicillium chrysogenum)和中华根霉(Rhizopus chinensis)有较强的抑制作用,对金黄色葡萄球菌(staphy lococcus aureus)和肠炎病病原菌(Ameromonas punctata)也有抑制作用,其抗真菌活性比抗细菌强.     

Studies on the cell wall of Chlorella II. Mode of increase of glucosamine in the cell wall during the synchronous growth of Chlorella ellipsoidea

The dry weight and glucosamine content of the alkali-insoluble"rigid wall" of Chlorella ellipsoidea were measured in synchronouslygrowing and dividing cells. The content of glucosamine, the major component of the rigidwall, was measured by the Elson-Morgan and the ninhydrin reactions.The results revealed, in agreement with previous observations,that the amount of the rigid wall, measured in terms of wholedry weight or glucosamine content, remains almost constant duringthe growing phase and increases only in the reproduction phase. (Received February 19, 1979; )     

Wall-protein antibodies as inhibitors of growth and of autolytic reactions of isolated cell wall

Antiserum raised against protein prepared from corn (Zea mays L. hybrid B73 X Mo17) seedling cell wall exhibits antigrowth activity and inhibits autolytic reactions of isolated cell wall. The growth of corn coleoptile segments pretreated in wall-protein antiserum was inhibited 35% compared to tissue which had been exposed to normal serum. Since the cuticle constituted a barrier to the entry of serum protein the coleoptile surface was abraded prior to exposure to the serum. Growth inhibition observed in nonabraded sections (8 to 10%) was apparently the result of the entry of serum protein via the cut ends of the tissue. Subsections recut from the central region of antiserum-treated, nonabraded coleoptiles responded normally in growth experiments. Antiserum brought in contact with isolated cell wall prior to its use with intact coleoptiles was less effective as a growth inhibitor, which suggested that the antibodies which impede growth responses bind to cell wall. Wall-protein antiserum also strongly inhibited autolytic reactions of isolated cell wall. Wall treated in normal serum or buffer liberated sugars at a rate of 8.6 μg- (mg wall dry-weight)^?1·h^?1 while antiserum-treated wall autolyzed at a rate of 2.6 μg·mg^?1·h^?1 representing a 70% reduction in autolytic potential. After 24 h yields from antiserum-treated wall were 50% lower than yields obtained from control wall. The antiserum had no effect on the molecular-weight distribution of the autolytically-liberated products, and direct assays of wall-bound and solubilized endo- and exo-β-d -glucanases involved in autolytic reactions demonstrated no significant impairment of their catalytic activity. However, the addition to antiserum-treated wall of a wall-protein preparation containing autolytic enzymes resulted in a significant enhancement (132%) in autolytic potential. The evidence suggests that enzyme mobility may be critical in the autolytic hydrolytsis of plant cell wall.     

Behaviour of the cell walls of Aspergillus niger during the autolytic phase of growth

Abstract Autolysis of Aspergillus niger cultures has been studied. The stability of the cell wall fraction was followed throughout a 57-day period of autolysis. The results indicated that both a persistence of existing walls and an extra wall deposition due to continued synthesis of walls or wall components during the first 12 days of autolysis seem to take place.     

Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation

Abstract From autolysed cultures of Mucor rouxii , two chitosanases, A and B, were purified to electrophoretic homogeneity. Apparent M _r values of 76 000 and 58 000 and p I values of 4.9 and 4.7 were determined for A and B, respectively. Both chitosanases showed a high specificity for chitosan and chitosan derivatives. They had optimum activities at pH 5.0 and at temperatures of 55°C and 50°C for A and B, respectively. Enzyme A was inhibited by acetate ions and enzyme B by high substrate concentration. Both enzymes showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of dimer, trimer and higher molecular mass oligomers of glucosamine. Glucosamine oligosaccharides were poorly hydrolysed by these enzymes. Both enzymes extensively degraded the chitosan extracted from M. rouxii cell walls.     

Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).