Purification and Properties of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

From the mycelia of Neurospora crassa (wild type No. 6068) multiple forms of a nuclease which had very close isoelectric points (pI = 9.6 (peak I), 9.4 (peak II)) were isolated by ampholine electrofocusing column chromatography (pH 8.5 ~ 10). The nuclease was about 300-fold purified from the crude extract. The two fractions of Peak I, II were indistinguishable in their enzymatic properties and were considered as manifestation of the same enzyme with minor physicochemical differences. The molecular weight was around 41,000 as estimated by the gel filtration method. The enzyme could hydrolyze both DNA and RNA in the order of heat-denatured DNA > native DNA DNA ≧ RNA. RNA competitively inhibited DNA degradation with this enzyme. The enzyme was therefore regarded as a nuclease. The pH optimum was around pH 6.5 toward native DNA, pH 6.7 toward heat-denatured DNA and pH 7.9 toward RNA. The temperature optimum was around 40°C toward these substrates and most of the activities were lost by heating at 55°C for 15 min. The enzyme required Mg²⁺ for action toward heat-denatured DNA and Mg²⁺, Mn²⁺ or Co²⁺ toward native DNA. In the presence of EDTA, the activities toward both types of DNA were lost and recovered by addition of the respective activating metallic ions. p-CMB inhibited this nuclease, but β-mercapto-ethanol and glutathione had no effect. Polyamìnes showed no activation of the nuclease for DNA degradation.     

Studies on the Autolysis of Aspergillus oryzae

Properties of nuclease O, a new intracellular enzyme which was partially purified from autolyzate of Asp. Oryzae,¹⁾ are described in this paper. The purified enzyme preferentially depolymerized RNA and heat denatured DNA, but apparently did not attack native DNA. It was activated by 0.1 mm Mg²⁺ or Mn²⁺, and inactive in the presence of EDTA. Optimum pH of the activity were 7.7 for DNA and 8.2 for RNA. By heat treatment (60°C, 10 min at pH 6) the nuclease completely lost its activity for RNA and DNA. Optimum concentration of Tris buffer for enzymatic activity was 0.15~0.2m.     

Studies on the Autolysis of Aspergillus oryzae

An intracellular nuclease inhibitor was 1270 times purified from a heat treated cell free extract of fresh mycelia of Aspergillus oryzae, by ammonium sulfate fractionation and chromatographies using DEAE-cellulose and Sephadex G-75. The purified sample of the inhibitor showed a UV absorption curve typical for protein, and it was inactivated by proteases such as chymotrypsin. The inhibitor stoichiometrically inactivated nuclease O (an intracellular nuclease of Asp. oryzae), forming an enzyme-inhibitor complex. But, it did not affect nuclease S₁, RNase T₁, RNase T₂ or pancreatic RNase. The inhibitor was insensitive to 10^?5m p-chloromercuribenzoate or 10^?4m Pb²⁺. Molecular weights estimated by the method of Andrews were 23,000 for the inhibitor, 47,000 for nuclease O, and 82,000 for the enzyme-inhibitor complex. The nuclease activity was recovered from the inactive complex by the action of chymotrypsin.

Nuclease O of Asp. oryzae was purified and crystallized from 113.5 kg of wet mycelia and 2 kl of culture filtrate, by salting out with ammonium sulfate and by chromatographies on CM-Sephadex C-50 and Sephadex G-100. The purified nuclease showed a single peak with apparent sedimentation constant 2.9S in an ultracentrifuge. The molecular weight measured by short column method was 64,000. The nuclease was completely inhibited by the specific nuclease inhibitor obtained from Asp. oryzae. The nuclease was activated by 0.1 mm Mg²⁺ and Mn²⁺, and completely inhibited by 1 mm EDTA. Optimum pH for activity was 7.6 for RNA and 7.4 for DNA. The nuclease degraded polyadenylic acid, polyuridylic acid and polycytidylic acid without forming detectable amount of mononucleotides. And, the main product from RNA was oligonucleotides. The enzyme showed no nonspecific phosphodiesterase activity.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co²⁺ and Hg²⁺, whereas Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺ and Sr²⁺ had little or no inhibitory effect. However, Mn²⁺ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t _1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca²⁺. Correspondence to: A. B. Salleh     

Nucleases in chloroplast of barley

Two barley chloroplast nuclease fractions were separated by the affinity chromatography and gel electrophoresis. Both were about 2 times more active to RNA than to native DNA and about half as active to denaturated DNA as to native DNA. Both fractions were as active to UV-irradiated (270 J m^-2) native DNA as to intact DNA but their action was inhibited by apurinic sites. The enzyme activities were inhibited by high concentrations of EDTA, NaCl, Mn²⁺, Ca²⁺, Zn²⁺ ions and by N-ethylmaleimide. They do not require Mg²⁺ ions but are stimulated or at higher concentration inhibited by their presence. Both RNase and DNase were active over a wide pH range (5.5–9), the optimum for DNase action in the presence of Mg²⁺ being 6.5, for RNA decomposing activity at pH 8.0. As no mononucleotides were detected in acid soluble form, it seems likely that DNase acts in the endonucleolytic way.     

Nuclease Stn α from <Emphasis Type="Italic">Streptomyces thermonitrificans</Emphasis>: Characterization of the Associated Adenylic Acid Preferential Ribonuclease Activity

Nuclease Stn α from Streptomyces thermonitrificans hydrolyses DNA and RNA at the rate of approximately 10:l. The optimum pH and temperature for RNA hydrolysis were 7.0 and 45°C. The RNase activity of nuclease Stn α had neither an obligate requirement of metal ions nor was it activated in the presence of metal ions. The enzyme was inhibited by Zn²⁺, Mg²⁺, Co²⁺, and Ca²⁺; inorganic phosphate; pyrophosphate; NaCl; KCl; and metal chelators. It was stable at high concentrations of urea but susceptible to low concentrations of Sodium dodecyl sulfate and guanidine hydrochloride. The rates by which nuclease Stn α hydrolysed polyribonucleotides occurs in the order of poly A >> RNA >> poly U > poly G > poly C. The enzyme cleaved RNA to 3′ mononucleotides with preferential liberation of 3′AMP, indicating it to be an adenylic acid preferential endonuclease.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF THE ORGANIC SOLVENT-TOLERANT LIPASE PRODUCED BY Pseudomonas fluorescens RB02-3 ISOLATED FROM MILK

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50°C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd²⁺, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.     

An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence: purification and characterization

In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca²⁺, which cannot be replaced by Mg²⁺ or Mn²⁺. The features of the enzyme and its relation to plant DNA metabolism are discussed. Received: 26 March 1998 / Accepted: 4 June 1998     

Peroxidase from Bacillus sp. VUS and its role in the decolorization of textile dyes

Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li²⁺, Co²⁺, K²⁺, Zn²⁺, and Cu²⁺ where as it showed less activity in the presence of Ca²⁺ and Mn²⁺. Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while sodium azide (NaN₃) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage of the dye.     

A thermoalkaliphilic lipase of <Emphasis Type="Italic">Geobacillus</Emphasis> sp. T1

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70°C and pH 9, respectively. It was stable up to 65°C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na⁺, Ca²⁺, Mn²⁺, K⁺ and Mg²⁺ , but inhibited by Cu²⁺, Fe³⁺ and Zn²⁺. Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10–C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T _m for T1 lipase was around 72.2°C, as revealed by denatured protein analysis of CD spectra.     

Properties of a Zn2+-glycerophosphocholine cholinephosphodiesterase from bovine brain membranes

A Zn²⁺-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 μmole/min·mg protein from bovine brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis, the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight of approximately 54 kDa. The Km value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 μM and pH 10.5, respectively. The phosphodiesterase was inhibited by Cu²⁺, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co²⁺ or Zn²⁺, but not Mn²⁺ or Mg²⁺. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn²⁺ or Zn²⁺, but not Co²⁺ or Mg². In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase may be stabilized in-vivo.     

Purification and characterization of the α-glucosidase produced by thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI 756

An α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0–9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na⁺, Ba²⁺, Co²⁺, Ni²⁺, Mg²⁺, Mn²⁺, Al³⁺, Zn²⁺, Ca²⁺ this enzyme maintained 90–105% of its maximum activity and was inhibited by Cr³⁺, Ag⁺, and Hg²⁺. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The K_m measured for the α-glucosidase was 0.07 μM, with a V_max of 318.0 μmol/min/mg.     

Purification and characterization of extracellular tannin acyl hydrolase from <Emphasis Type="Italic">Aspergillus heteromorphus</Emphasis> MTCC 8818

A tannase (E.C. 3.1.1.20) producing fungal strain was isolated from soil and identified as Aspergillus heteromorphus MTCC 8818. Maximum tannase production was achieved on Czapek Dox minimal medium containing 1% tannic acid at a pH of 4.5 and 30°C after 48 h incubation. The crude enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography. Diethylaminoethyl-cellulose column chromatography led to an overall purification of 39.74-fold with a yield of 19.29%. Optimum temperature and pH for tannase activity were 50°C and 5.5 respectively. Metal ions such as Ca²⁺, Fe²⁺, Cu¹⁺, and Cu²⁺ increased tannase activity, whereas Hg²⁺, Na¹⁺, K¹⁺, Zn²⁺, Ag¹⁺, Mg²⁺, and Cd²⁺ acted as enzyme inhibitors. Various organic solvents such as isopropanol, isoamyl alcohol, benzene, methanol, ethanol, toluene, and glycerol also inhibited enzyme activity. Among the surfactants and chelators studied, Tween 20, Tween 80, Triton X-100, EDTA, and 1, 10-o-phenanthrolein inhibited tannase activity, whereas sodium lauryl sulfate enhanced tannase activity at 1% (w/v).     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.     

Characterization of chitinase from Streptomyces luridiscabiei U05 and its antagonist potential against fungal plant pathogens

Streptomyces luridiscabiei U05 was isolated from wheat rhizosphere. It produced chitinase, which showed in vitro antifungal properties. The crude enzyme inhibited the growth of Alternaria alternata, Fusarium oxysporum, F. solani, Botrytis cinerea, F. culmorum and Penicillium verrucosum. The chitinase enzyme of the molecular weight of 45 kDa was purified using affinity chromatography of chitin. Streptomyces luridiscabiei U05 produced different chitinolytic enzymes. The highest enzyme activity was observed with the use of 4‐MU‐(GlcNAc)_, which points to the presence of an β‐N‐acetylhexosaminidase. The optimum activity was obtained at 35–40°C and pH 7–8. The enzyme showed thermostability at 35–40°C during 240 min of preincubation and lost its activity at 50°C and 60°C in 60 min. The chitinase activity from S. luridiscabei U05 was strongly inhibited by Hg²⁺ and Pb²⁺ ions, and sodium dodecyl sulphate (SDS). The Ca²⁺, Cu²⁺ and Mg²⁺ ions stimulated the activity of the enzyme.     

Purification and characterization of endoxylanase Xln-1 from <Emphasis Type="Italic">Aspergillus niger</Emphasis> B03

An extracellular endoxylanase was isolated from the xylanolytic complex of Aspergillus niger B03. The enzyme was purified to a homogenous form using consecutive ultrafiltration and anion exchange chromatography. The endoxylanase was a monomer protein with a molecular weight of 33,000 Da determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 34,000 Da determined by gel filtration. The optimal pH and temperature values for the enzyme action were 6.0 and 60°C, respectively. Endoxylanase was stable at 40°C, pH 7.0 for 210 min. The thermal stability of the enzyme was significantly increased in the presence of glycerol and sorbitol. The enzyme activity was inhibited by Cu²⁺, Fe²⁺, Fe³⁺, and Ag¹⁺, and it was activated by Mn²⁺. The substrate specificity and kinetic parameters of the enzyme were determined with different types of xylans. Endoxylanase displayed maximum activity in the case of oat spelt xylan, with an apparent K _m value of 8.19 mg/ml. The substrate specificity and the product profile of the enzyme suggested it to be an endoxylanase.     

Purification and characterization of β-agarase from seaweed decomposing bacterium <Emphasis Type="Italic">Microbulbifer</Emphasis> sp. Strain CMC-5

Microbulbifer strain CMC-5 was isolated from decomposing seaweeds, and was found to degrade agar, alginate, carboxymethyl cellulose, carrageenan, xylan, and chitin. The extracellular agarase enzyme from strain CMC-5 was purified 103-fold by ultrafiltration, ion-exchange chromatography, using diethylaminoethyl sepharose FF, and gel filtration, using sephacryl S-300HR, with a yield of 6.7%. Zymogram and protein staining of the purified agarase on a SDS-polyacrylamide gel revealed a single band, with an apparent molecular weight of 59 kDa. The purified enzyme was endo-type β-agarase, as it was able to hydrolyze the β-1, 4 glycosidic linkages of agarose, releasing neoagarotetraose and neoagarohexaose as the end products. The optimum pH and temperature of agarase were 7 and 50°C, respectively. Thermal stability studies indicated that the agarase retained 62% of its activity after incubating at 50°C for 30 min. Treatment with EDTA reduced the agarase activity by 54%. The agarase activity was stimulated by the presence of Ca²⁺ and Mg²⁺ ions; whereas, Zn²⁺, Hg²⁺, Cu²⁺, Fe²⁺, and Co²⁺ abolished the activity. Further, the presence of NaCl at concentrations lower than 100 mM caused a decrease in the agarase activity; whereas, the activity was enhanced up to a concentration of 500 mM.     

Strong extracellular nuclease activity displayed by barley (Hordeum vulgare L.) uninucleate microspores

 Electroporation is becoming an increasingly important technique for plant transformation. Nevertheless, no positive results were achieved in barley when uninucleate microspores were used as target cells. Since it was previously demonstrated that electric shocks create pores in the microspore cell wall, experiments were designed to verify the presence of nucleases in the electroporation mix. Aliquots of all the solutions used for microspore extraction, purification and transformation were collected and analysed using supercoiled pBI 221 as a substrate; a nuclease activity was detected in all samples. Though microspore rinsing removed most nucleolytic activity in the supernatants, DNA preservation in the electroporation buffer was difficult to achieve, because microspores appeared capable of synthesising and releasing endonucleases at any time. Microspore chilling at 0°C was fairly effective in reducing nuclease secretion in the mix, whereas 1%PEG or 10 mM EDTA maintained most of the DNA in a supercoiled or circular relaxed form. EDTA effects were counterbalanced by Mg²⁺, but not Ca²⁺ or Zn²⁺, and enhanced by Mn²⁺. Barley microspore nucleases actively degraded different DNAs as well as TMV RNA, and apparently had a molecular weight above 30 kDa. Nuclease inactivation with EDTA did not alter microspore viability and allowed a transient expression of the uidA gene in electroporated barley microspores. Received: 13 January 1997 / Accepted: 28 February 1997