Bacterial 2,3-butanediol dehydrogenases

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidized only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was partially purified (30-fold) with a specific activity of 24.5. Except NAD and NADH no other cofactors were required. Optimum pH-values for oxidation and reduction were pH 9 and pH 7, respectively. The optimum temperature was about 60°C. The molecular weight was 100000 to 107000. The K _m-values were 3.3 mM for D(-)-butanediol, 6.25 mM for meso-butanediol, 0.53 mM for acetoin, 0.2 mM for NAD, 0.1 mM for NADH, 87 mM for diacetyl, 38 mM for 1,2-propanediol; 2,3-pentanedion was not a substrate for this enzyme. The L(+)-butanediol dehydrogenase from Serratia marcescens was purified 57-fold (specific activity 22.3). Besides NAD or NADH no cofactors were required. The optimum value for oxidation was about pH 9 and for reduction pH 4.5. The optimum temperature was 32–36°C. The molecular weight was 100000 to 107000. The K _m-values were 5 mM for meso-butanediol, 10 mM for racemic butanediol, 6.45 for acetoin, 1 mM for NAD, 0.25 mM for NADH, 2.08 mM for diacetyl, 16.7 mM for 2,3-pentanedion and 11.8 mM for 1,2-propanediol.Abbreviations Bud 2,3-butanediol - DH dehydrogenase     

Production of a potentially novel anti-microbial substance by Bacillus polymyxa

A bacterial strain, SCE2, identified as Bacillus polymyxa, produced an anti-microbial substance active against yeasts, fungi and different genera of Gram-positive and-negative bacteria, in liquid medium and in plate assays. This substance appeared to be an antibiotic different from the polymyxin group, mainly because of its action against the majority of Gram-positive bacteria tested and its lack of activity against Pseudomonas aeruginosa, a species usually killed by polymyxins. Preliminary characterization showed resistance to heat (65°C, 2 h), to proteases, trypsin, lysozyme, deoxyribonuclease I, ribonuclease A, phospholipase C, ethanol, acetone, chloroform, ether and to strong alkali treatment (2 M NaOH). The molecular weight was less than 3500. The B. polymyxa strain harboured a plasmid that did not correlate with antibiotic production; after curing experiments, a derivative strain, SCE2(46), was isolated that lacked the plasmid pES1, but showed the same inhibitory spectrum as the wild-type strain.     

A gene encoding alanine racemase is involved in spore germination in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Alanine racemase is a major component of the exosporium of Bacillus cereus spores. A gene homologous to that of alanine racemase (alrA) was cloned from Bacillus thuringiensis subsp. kurstaki, and RT-PCR showed that alrA was transcribed only in the sporulating cells. Disruption of alrA did not affect the growth and sporulation of B. thuringiensis, but promoted l-alanine-induced spore germination. When the spore germination rate was measured by monitoring DPA release, complementation of the alrA disruptant reduced the rate of l-alanine-induced spore germination below that of even wild-type spores. As previously reported for spores of other Bacillus species, d-alanine was an effective and competitive inhibitor of l-alanine-induced germination of B. thuringiensis spores. d-cycloserine alone stimulated inosine-induced germination of B. thuringiensis spores in addition to increasing l-alanine-induced germination by inhibiting alanine racemase. d-Alanine also increased the rate of inosine-induced germination of wild-type spores. However, d-alanine inhibited inosine-induced germination of the alrA disruptant spores. It is possible that AlrA converted d-alanine to l-alanine, and this in turn, stimulated spore germination in B. thuringiensis. These results suggest that alrA plays a crucial role in moderating the germination rate of B. thuringiensis spores.     

Correlation between alkaline activation of Bacillus thuringiensis var. kurstaki spores and crystal production

The spores of crystal-forming (Cry⁺) and non-crystal-forming (Cry^-) strains of Bacillus thuringiensis var. kurstaki and Bacillus cereus were tested for the ability to be activated by 0.1 m K₂CO₃ (pH 10). Only the spores of crystal-forming strains could be activated, and this phenotype was independent of whether crystals were present with the spores in the activation solution. The spores of a B. thuringiensis var. kurstaki strain that is temperature sensitive for protoxin accumulation could be activated by the alkaline solution when produced at the permissive temperature, whereas spores produced at the nonpermissive temperature were not activated. The results indicate that protoxin in the spore coat is responsible for the alkaline-activation phenotype and may serve an ecological function for the organism.     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, chlorine dioxide (ClO₂), and a commercial raw fruit and vegetable sanitizer (Fit powder) were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5–11.0) ClO₂ (200 mg/mL) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log cfu/mL, respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log cfu/mL resulting from treatment with 200 mg/mL chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5-log and 0.4-log cfu/mL reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO₂ (85 mg/mL), acidified (pH 3.4) ClO₂ (85 mg/mL), and a mixture of ClO₂ (85 mg/mL) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log cfu/mL, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/mL organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO₂ (400 mg/mL) for 30 min reduced populations by 4.6 and 5.2 log cfu/mL, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO₂.Published by permission of the International Association for Food Protection: Journal of Food Protection (2004) 60:1702–1708This revised version was published online in April 2005 with corrections to the text and the section heading.In section Preparation of treatment solutions the phrase 22-28°C was replaced by 22±2°C.     

Altered heat resistance in spores and vegetative cells of a mutant fromBacillus subtilis

The relationship between sporulation temperature and spore killing temperature is described.Bacillus subtilis YB886, grown and sporulated at 25°, 30°, 37°, and 45°C, produced spores having D₉₀ values of 63.5, 76.3, 89.0, and 106 min respectively. In addition, the vegetative cells of this strain also demonstrated resistance to heat killing when grown at elevated temperatures (D₅₀ of 26.6, 32.5, 39.0, and >50 min for cells grown at 25°, 30°, 37°, and 45°C). A transposon-generated mutant of strain YB886, designated as BUL786, which is missing a heat shock-induced protein (97 kDa) (Qoronfleh MW and Streips UN, BBRC, 138:526–532, 1986 and FEMS 1987), was tested for thermotolerance under similar conditions. The cells failed to respond to growth at high temperature by producing heat-resistant spores or vegetative cells. For strain BUL786 the D₉₀ of spores generated at 20°, 25°, 30°, 37°, and 45°C was 9.4, 11.3, 12.8, 14.1, and 20 min, respectively. Similarly, the D₅₀ of vegetative cells was 15, 16.8, 17.8, 19.0, and 22.3 min when the cells were grown at 20°, 25°, 30°, 37°, and 45°C. Also, sporulation of YB886 cells in the presence of cadmium chloride increased the D₉₀ values for the resulting spores (5µM CdCl₂ resulted in a D₉₀ of 160 min). Strain BUL786 failed to produce spores with any elevated D₉₀ when grown in the presence of CdCl₂.     

Persistence ofBacillus thuringiensis andBacillus cereus in soil supplemented with grass or manure

Summary Persistance of inocula ofBacillus thuringiensis spores, parasporal crystals, andBacillus cereus spores in soil supplemented with dried-grass or partly composted, dried-chicken manure (100 mg supplement per 900 mg soil,^–0.01 MPa water availability, 25°C) were monitored over a period of up to 64 days by dilution plating and bioassay with larvae ofPieris brassicae. The inoculantB. thuringiensis population increased 22 x in level in grass-supplemented soil, but declined in manure-supplemented soil to 0.22 x the original level. TheB. cereus inocula declined in both soil treatments to approximately 0.1 x the original level. Insecticidal activity of theB. thuringiensis parasporal crystal decreased exponentially in grass and manuresupplemented soils, with half-lives of approximately 9.5 and 8.5 days respectively.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

β-Chemokines Are Produced in Lungs of Mice with Mycoplasma Respiratory Disease

The abilities of Bacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere were compared; it was found that B. polymyxa colonizes the crop better. This study also showed that B. polymyxa inoculation to rice plants increased the shoot and the root growth of the crop. Efforts were made to introduce the cry1Ac gene of B. thuringiensis subsp. kurstaki into B. polymyxa so that the application of such transgenic B. polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticide and as a biofertilizer. Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain BP113, indicated efficient expression of this gene in the heterologous host. Bioassays with the first instar larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations from BP113 were toxic. Received: 12 August 1998 / Accepted: 25 September 1998     

Un nouveau candidat a la lutte biologique contre les moustiques:Bacillus thuringiensis var.Israelensis

Résumé Le nouveau sérotype H₁₄ individualisé dans le groupe desBacillus thuringiensis Berliner, sous le nom de variétéisraelensis est un pathogène puissant et essentiel des larves de moustiques. Son pouvoir larvicide n'a aucun équivalent chez les autres sérotypes connus deB. thuringiensis et appara?t très compétitif avec celui deB. sphaericus. A dose forte,B. thuringiensis var.israelensis tue les larves d'Aedes aegypti L. en 20 à 30 mn et celles d'Anopheles stephensi (Liston), en 100 à 110 mn, les DL 50 en 24 h étant de l'ordre de 2,4.10⁴ spores/ml pourAedes et de 9,8.10⁴ spores/ml pourAnopheles. La toxicité deB. thuringiensis var.israelensis pour les larves de moustiques est liée à une endotoxine protéique présente dans les cristaux, de nature et de mode d'action comparables à ceux des endotoxines des autres souches deB. thuringiensis pathogènes pour les lépidoptères. L'étude histopathologique surAedes aegypti montre que l'effet primaire correspond à une désintégration de l'épithélium intestinal par gonflement, distorsion puis éclatement des cellules. L'absence d'action deB. thuringiensis var.israelensis sur les lépidoptères étudiés:Anagasta kuehniella, Z.,Plutella maculipennis Curtis etProdenia litura F., ainsi que son innocuité ?per os? pour les mammifères tendent à prouver une certaine spécificité pour les diptères. Toutes ces qualités, jointes à la parfaite connaissance du groupethuringiensis découlant de sa longue utilisation en forêts et en cultures, devraient faire de la variétéisraelensis un candidat préférentiel pour la lutte biologique contre les moustiques.

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Summary The new serotype 14 which has been discovered in theBacillus thuringiensis Berliner group and named varietyisraelensis is a major pathogen for mosquito larvae. Its larvicidal power has been found without any equivalence in comparison with the other knownB. thuringiensis serotypes, and very competitive with the larvicidal activity ofBacillus sphaericus. At high doses,B. thuringiensis var.israelensis kills theAedes aegypti L. larvae in 20 to 30 mn, and theAnopheles stephensi (Liston) larvae in 100 to 110 mn. The DL₅₀ in 24 h are about 2,4.10⁴ spores/ml forA. aegypti and 9,8.10⁴ spores/ml forA. stephensi. The toxicity ofB. thuringiensis var.israelensis for mosquito larvae is linked with a proteic endotoxin in its crystals, the nature and mode of action of which look like these ones of the otherB. thuringiensis strains, pathogens for lepidoptera larvae. The histopathological study onA. aegypti has shown that the primary action consists in the loss of integrity of the gut epithelium, as a result of the swelling, distortion and finally bursting of the cells. The lack of activity ofB. thuringiensis var.israelensis on the tested Lepidoptera:Anagasta kuehniella Z.,Plutella maculipennis Curtis andProdenia litura F. and its innocuity “per os” for mammals lead to suggest some specificity for Diptera. All these qualities, which are enhanced by the detailed knowledge ofB. thuringiensis group coming from its long practical use on large scale in forests and cultures, ought to put theisraelensis variety as a preferential candidate for the biological control of mosquitoes.

     

Thermal Resistance ofBacillus cereus spores in custard preparations

Summary The thermal resistance ofBacillus cereus spores expressed in terms ofD value (an expression of time in minutes required for the heat destruction of one log cycle of spores) in custard preparations decreased from 90°C to 100°C. With an increase in pH of custard preparations from 6.2 to 7.2, theD value ofB. cereus spores increased from 3.1 to 3.7 min at 90°C, from 2.5 to 3.0 min at 95°C and from 1.7 to 2.2 min at 100°C. However, theZ value (the change in temperature necessary to cause a tenfold change in theD value) remained unaffected by the change in pH. A decrease in the number of heatedB. cereus spores was observed with the increase in the concentration of the antimicrobial agent sodium benzoate and the antioxidants butylated hydroxyanisole and butylated hydroxytoluene.

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Résistance thermique des spores deBacillus cereus dans les préparations de crème

Résumé La résistance thermique de spores deBacilluscereus, exprimée par la grandeur D (l'expression du temps en minutes requis pour la destuction thermique d'une décade logarithmique de spores) dans les préparations de crème, décroît de 90 °C à 100 °C. Avec une augmentation de pH des préparations de crème de 6.2 à 7.2, la valeur D pour les spores deB. cereus augmente de 3.1 à 3.7 min. à 90 °C, de 2.5 à 3.0 min. à 95 °C et de 1.7 à 2.2 min. à 100 °C. Toutefois, la grandeur Z (le changement de température requis pour engendrer un changement d'un facteur 10 dans la valeur D) n'est pas affectée par ce changement de pH. Une diminution du nombre de spores deB. cereus chauffé est observée avec l'augmentation de la concentration du benzoate de soude, un agent antimicrobien, ainsi que de l'anisole hydroxy-butylé et du toluène hydroxybutylé, deux agents antioxidants.

     

Rapid viability assessment of spores of several fungi by an ionic intensified fluorescein diacetate method

Fluorescein diacetate (FDA) was applied to the viability assessment of spores of Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, and Penicillium citrinum. The fluorescence of individual cells was quantitated with a charge coupled device (CCD) detector. When staining was carried out in a phosphate buffer solution (10 mM, pH 7.0), weak or no fluorescence was emitted from viable spores of A. niger and R. stolonifer, which made it difficult to distinguish between viable (nontreated) and nonviable (heat treated at 90°C for 30 min) spores. The addition of NaCl, KCl, or MgCl₂ to the staining solution caused an increase in the fluorescence intensity of A. niger viable spores, from which nonviable spores could be distinguished. The same effect of NaCl was observed in staining the spores of other species.     

Production and characterization of polygalacturonase fromGeotrichum candidum

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The K_m and V_max values for polygalacturonic acid were 4.2 mg/ml and 0.19mm galacturonic acid/min, respectively. The enzyme was not substrate inhibited.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Bacillus thuringiensis growth,sporulation and δ-endotoxin production in oxygen limited and non-limited cultures

The production of crystals and spores ofBacillus thuringiensis var.israelensis was studied under different aeration conditions. The results with 4 l batch cultures showed that for O₂ non-limited, cultures cell yield, toxin production and spore count were constant for all oxygen transfer rates (OTR). Under O₂ limitation, °-endotoxin concentrations and spore counts were lower than those obtained in non-limited cultures. In addition, -endotoxin yields diminished under O₂ limitation, suggesting that the toxin synthesis mechanism could have been affected.     

Le titrage biologique des cristaux deBacillus thuringiensis Berliner par réduction de consommation au laboratoire de La Minière

Summary The principles, technics, advantages, limits and extension possibilities of the biological titration of crystals are briefly discussed. The figure 1 illustrates the independance of consummation reduction regarding the heat-stable toxin and the spores. The figure 2 shows the results of seven biological titrations of threeB. thuringiensis products.

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Texte présenté lors de la démonstration du Symposium sur le titrage des préparations deBacillus thuringiensis Berliner, Paris 5–7 mars 1964.     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

Regulation of mesophyll photosynthesis in intact wheat leaves by cytoplasmic phosphate concentrations

The effect of D-(+)-mannose, inorganic phosphate (Pi) and mannose-6-phosphate on net mesophyll CO₂ assimilation rate (A) and stomatal conductance (g_s) of wheat (Triticum aestivum L.) leaves was studied. The compounds were supplied through the transpiration stream of detached leaves from plants grown in sand in growth cabinets or glasshouses, with different concentrations of Pi (0.25, 1.0 and 4.0 mM) supplied during growth. In all cases, 10 mM D-(+)mannose caused 40–60% reduction of A within 30 min, though the time courses differed for flag leaves and the sixth leaf on the mainstem of glasshouse- and cabinet-grown plants. D-(+)Mannose had a similar effect on A in leaves having a fourfold range in total phosphate content. Effects of D-(+)mannose in reducing g_s were always slower than on A. When the CO₂ concentration in the leaf chamber was adjusted to maintain intercellular CO₂ concentration (C_i) constant as A declined after mannose supply, g_s still declined indicating that stomatal closure was not caused by changing C_i. Supplying mannose-6-phosphate at 10 and 1 mM and Pi at 5 and 10 mM concentrations caused rapid reductions in g_s and also direct reductions in A. The observed effects of mannose and Pi on assimilation are consistent with the proposed regulatory role of cytoplasmic Pi in determining mesophyll carbon assimilation that has been derived previously using leaf discs, protoplasts and chloroplasts.Abbreviations and symbols A net mesophyll CO₂-assimilation rate - C_a, C_i external (assimilation-chamber) and intercellular CO₂ concentration, respectively - g_s stomatal conductance - Man6P mannose-6-phosphate - Pi orthophosphate     

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 °C, but N,N-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)₂] were selectively produced from chitin. At 45 °C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 °C (GlcNAc)₂ was the major product (86%) with a yield of 35% within 5 d.Revisions requested 29 September 2004; Revisions received 1 November 2004     

Protoplasts from the cyanobacterium,Spirulina platensis

Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.