Biotransformation of racemic diisophorone by <Emphasis Type="Italic">Cephalosporium aphidicola</Emphasis> and <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Racemic diisophorone (500 mg) was converted by Cephalosporium aphidicola and Neurospora crassa over 10 days at 25 °C to 8β-hydroxydiisophorone in yields of 10% (52 mg) and 20% (103 mg), respectively. The structure was established by IR, specific rotation, mass spectral, 1D and 2D-NMR studies.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 8 April 2005 and 10 May 2005     

Biodegradation of phenol and <Emphasis Type="Italic">m</Emphasis>-cresol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 under anaerobic condition

Strain Candida albicans PDY-07 was used to study the anaerobic biodegradation of phenol and m-cresol as single and dual substrates in batch cultures. The strain had a higher potential to degrade phenol than m-cresol. The cell growth kinetics of batch cultures with various initial m-cresol concentrations was investigated, and the Haldane kinetic model adequately described the dynamic behavior of cell growth on m-cresol. When cells grew on the mixture of phenol and m-cresol, substrate interactions were observed. Phenol inhibited the utilization of m-cresol; on the other hand, m-cresol also inhibited the degradation of phenol. However, the presence of low-concentration phenol enhanced m-cresol biodegradation; 100 mg/l m-cresol could be completely degraded within a shorter period of time than m-cresol alone in the presence of 150–300 mg/l phenol. The maximum m-cresol biodegradation rate was obtained at the existence of 200 mg/l phenol. Phenol was preferably utilized by the strain as a carbon and energy source. In addition, a sum kinetics model was used to describe the cell growth behavior in binary mixture of phenol and m-cresol, and the interaction parameters were determined. The model adequately predicted the growth kinetics and the interaction between the substrates.     

Exopolysaccharide production of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> BCRC 14759 and <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> BCRC 14615

In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract of both test organisms. This indicates their possible involvement in the biosynthesis of EPS.     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Composition of the lipids of <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis> and their origin from its host <Emphasis Type="Italic">Ignicoccus</Emphasis> sp. strain KIN4/I

The contents and nature of the membrane lipids of Nanoarchaeum equitans and Ignicoccus sp. strain KIN4/I, grown at 90°C, and Ignicoccus sp. strain KIN4/I, cultivated at its lowest and highest growth temperatures (75°C and 95°C) were analyzed. Both organisms contained very simple and qualitatively identical assemblages of glycerol ether lipids, showing only differences in the amounts of certain components. LC–MS analyses of the total lipid extracts revealed that archaeol and caldarchaeol were the main core lipids. The predominant polar headgroups consisted of one or more sugar residues attached either directly to the core lipid or via a phosphate group. GC–MS analyses of hydrolyzed total lipid extracts revealed that the co-culture of N. equitans and Ignicoccus sp. strain KIN4/I, as well as Ignicoccus sp. strain KIN4/I grown at 90°C, contained phytane and biphytane in a ratio of approximately 4:1. Purified N. equitans cells and Ignicoccus sp. strain KIN4/I cultivated at 75°C and 95°C had a phytane to biphytane ratio of 10:1. Sugar residues were mainly mannose and small amounts of glucose. Consistent ¹³C fractionation patterns of isoprenoid chains of N. equitans and its host indicated that the N. equitans lipids were synthesized in the host cells.     

Anaerobic biodegradation of phenol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 in the presence of 4-chlorophenol

Biodegradation of phenol and 4-chlorophenol (4-cp) using pure culture of Candida albicans PDY-07 under anaerobic condition was studied. The results showed that the strain could completely degrade up to 1,800 mg/l phenol within 68 h. The capacity of the strain to degrade phenol was higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Comparatively, low-concentration phenol from 25 to 150 mg/l supplied a carbon and energy source for Candida albicans PDY-07 in the early phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in that 50 mg/l 4-cp was degraded within less time than that without phenol. While the biodegradation of 50 mg/l 4-cp was inhibited in the presence of 200 mg/l phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and dual substrates in batch cultures. The results demonstrated that the models adequately described the dynamic behaviors of biodegradation by Candida albicans PDY-07.     

Psychrophilic <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> requires <Emphasis Type="Italic">trans</Emphasis>-monounsaturated fatty acid for growth at higher temperature

A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28°C compared to cells grown at 5°C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28°C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28°C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.     

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.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Growth of <Emphasis Type="Italic">Trametes versicolor</Emphasis> on phenol

Trametes versicolor 1 was shown to grow on phenol as its sole carbon and energy source. The culture growth and degradation ability dependence on culture medium pH value was observed. The optimal pH value of a liquid Czapek salt medium was 6.5. The investigated strain utilized completely 0.5 g/l phenol in 6 days. The dynamics of the phenol degradation process was investigated. The process was characterized by specific growth rate μ_max 0.33 h⁻¹, metabolic coefficient k = 4.4, yield coefficient Y _x/s  = 0.23 and rate of degradation Q = 0.506 h⁻¹. The intracellular activities of phenol hydroxylase (0.333 U/mg protein) and cis,cis-muconate lactonizing enzyme (0.41 U/mg protein) were demonstrated for the first time in this fungus. In an attempt to estimate the occurrence of gene sequences in T. versicolor 1 related to phenol degradation pathway a dot blot analysis with total DNA isolated from this strain was performed. Two synthetic oligonucleotides were used as hybridizing probes. One of the probes was homologous to the 5′end of phyA gene coding for phenol hydroxylase in Trichosporon cutaneum ATCC 46490. The other probe was created on the basis of cis,cis-muconate lactonizing enzyme coding gene in T. cutaneum ATCC 58094. The results of these investigations showed that T. versicolor 1 may carry genes similar to those of Trichosporon cutaneum capable to degrade phenol.     

Influence of temperature on the properties of the xylanolytic enzymes of the thermotolerant fungus<Emphasis Type="Italic"> Aspergillus phoenicis</Emphasis>

This study reports on the effects of growth temperature on the secretion and some properties of the xylanase and -xylosidase activities produced by a thermotolerant Aspergillus phoenicis. Marked differences were observed when the organism was grown on xylan-supplemented medium at 25 °C or 42 °C. Production of xylanolytic enzymes reached maximum levels after 72 h of growth at 42 °C; and levels were three- to five-fold higher than at 25 °C. Secretion of xylanase and -xylosidase was also strongly stimulated at the higher temperature. The optimal temperature was 85 °C for extracellular and 90 °C for intracellular -xylosidase activity, independent of the growth temperature. The optimum temperature for extracellular xylanase increased from 50 °C to 55 °C when the fungus was cultivated at 42 °C. At the higher temperature, the xylanolytic enzymes produced by A. phoenicis showed increased thermostability, with changes in the profiles of pH optima. The chromatographic profiles were distinct when samples obtained from cultures grown at different temperatures were eluted from DEAE–cellulose and Biogel P-60 columns.     

Negative Evidence of <Emphasis Type="Italic">Wolbachia</Emphasis> in the Predaceous Mite <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis>

The cytoplasmically inherited bacterium Wolbachia is widespread in arthropod species and has been repeatedly detected in the predaceous mite Phytoseiulus persimilis. Our original goal was to assess the prevalence of Wolbachia infection in P. persimilis and the potential fitness consequences for this host. To accomplish that goal, seven P. persimilis strains were obtained from Europe, Africa and the USA and reared on the phytophagous mite Tetranychus urticae as prey. After preliminary results showed that the T. urticae used was infected with Wolbachia, the minimum starvation time of the predators to prevent false positive results from undigested prey was determined. We tested DNA samples by PCR (polymerase chain reaction) after starving the predators or feeding them Wolbachia-free T. urticae for various periods. Those experiments showed that Wolbachia could not be detected after 16 h at 25 °C and 48 h at 20 °C. To verify the results of the PCR analyses, we furthermore conducted crossing experiments with antibiotic-treated and untreated individuals. No indications of Wolbachia effects were recorded. Additionally, we screened live eggs of four of the seven strains reared in our laboratory and alcohol samples of 10 other P. persimilis strains for the occurrence of Wolbachia by PCR, none of which tested positive. Synthesis of our study and previous reports suggests that infection of P. persimilis with Wolbachia is extremely rare and of minor importance. We discuss the significance of our findings for future studies on the presence of Wolbachia in predaceous arthropods.     

Diverse<Emphasis Type="Italic"> Mesorhizobium plurifarium</Emphasis> populations native to Mexican soils

Forty-six Mesorhizobium strains associated with the leguminous plants Leucaena leucocephala and Sesbania herbacea in an uncultivated Mexican field were characterized using a polyphasic approach. The strains were identified as Mesorhizobium plurifarium based upon the close relationships with the reference strains for this species in PCR-based restriction fragment length polymorphism analyses, sequencing of 16S rRNA genes, multilocus enzyme electrophoresis, and DNA-DNA hybridization. Although the strains isolated from both plants formed the same group in multilocus enzyme electrophoresis and cross-nodulations were observed in the laboratory, different electrophoretic types were obtained from the two plants grown in natural soils, indicating the existence of a preferable association between the plants and the rhizobia. The M. plurifarium strains from Mexico and the reference strains from Africa and Brazil formed different phenotypic clusters in a numerical taxonomy. The Mexican strains did not grow at 37 °C and were sensitive to salty-alkaline conditions, while the reference strains from Africa and Brazil grew at 42 °C and were more resistant to salty-alkaline conditions. These results demonstrate that both the plants and environmental factors affected the evolution of rhizobia and that the Mexican strains had adapted to the neutral soils and the cool climate where they were isolated.     

Biodegradation of phenol and 4-chlorophenol by the yeast <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Biodegradation of phenol and 4-chlorophenol (4-cp) using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2,000 mg l⁻¹ phenol alone and 350 mg l⁻¹ 4-cp alone within 66 and 55 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Phenol beyond 800 mg l⁻¹ could not be degraded in the presence of 350 mg l⁻¹ 4-cp. Comparatively, low-concentration phenol from 100 to 600 mg l⁻¹ supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in the fact that 4-cp biodegradation velocity was higher than that without phenol. And the capacity of C. tropicalis to degrade 4-cp was increased up to 420 mg l⁻¹ with the presence of 100–160 mg l⁻¹ phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

Overexpression of the <Emphasis Type="Italic">plg</Emphasis>1 gene encoding pectin lyase in <Emphasis Type="Italic">Penicillium griseoroseum</Emphasis>

The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml⁻¹ respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.