Tea extract as an inexpensive inducer of pectin lyase in Penicillium griseoroseum cultured on sucrose

Extracts of tea, coffee, cocoa, and yeast induced pectin lyase (PL) in Penicillium griseoroseum cultured in a mineral medium with sucrose as the carbon source. PL activity and fungal growth were similar in the treatments with 0.5% tea extract, the highest concentration tested, and 0.03% yeast extract. When tea extract was added singly to the culture medium, P. griseoroseum produced 59% and 17% of the PL activity and mycelial mass, respectively, obtained in a treatment with tea extract and sucrose. These results suggest that the production of the enzyme was not proportional to mycelial growth. No PL was produced in the medium with sucrose and without inducers. The small amounts of pectic substances present in the tea extract could not be responsible for PL induction. PL activity was detected after 12 h of growth in the medium containing sucrose and tea extract added at time zero, and after 48 h of growth when tea extract was added at times 12 and 24 h. Mycelial mass in all treatments was similar after 48 h of incubation. However, the addition of tea extract at time zero increased PL activity by 20–25%. Cyclic AMP at 5 and 10 mM in the culture medium induced 20 and 30%, respectively, of the PL activity obtained with 0.03% yeast extract, suggesting that PL induction brought about by either yeast extract or tea extract might involve the intracellular metabolism of cAMP. Received 22 October 1996/ Accepted in revised form 09 January 1997     

Pectin lyase production by Penicillium griseoroseum grown in sugar cane juice in repeated batch cultures

Penicillium griseoroseum cultured in the presence of sucrose and yeast extract produces pectin lyase (EC 4.2.2.10) (PL) in the absence of its natural inducer pectin. This fungus was cultured in a fermenter at an aeration rate of 0.5 l/min for the first 25 h and 1.0 l/min for the remainder of the culture period, and at a stirring rate of 200 rev/min for the entire culture period. Fungal spores were inoculated directly into the fermenter at a final concentration of 5 × 10⁴ spores/ml. The fungus was cultured in minimal medium supplemented with powdered dehydrated sugar cane juice, producing PL without added yeast extract. Maximum PL activity (0.067 IU/ml) was obtained after 65 h in batch culture. Pellet morphology of the mycelia made it possible to carry out three cycles of repeated batch culture. The same medium was used for renewal as for the single batch culture. The initial cycle was 53 h, after which approximately 0.103 IU/ml of PL was obtained. After this period, the medium was renewed and fermentation continued for two more cycles, which lasted approximately 20 h. Activity of PL obtained in the second cycle was approximately 0.118 IU/ml and in the third, approximately 0.109 IU/ml.     

Production of pectin lyase by Penicillium griseoroseum as a function of the inoculum and culture conditions

Optimum activity of an extracellular pectin lyase produced by Penicillium griseoroseum in submerged culture was after 120 h using 0.1% (w/v) citrus pectin as substrate. Sucrose at 0.1% (w/v) stimulated enzyme production and citrus pectin gave the highest activity of enzyme per unit growth.     

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.     

Improved pectinase production in Penicillium griseoroseum recombinant strains

Aims: To obtain recombinant strains of Penicillium griseoroseum that produce high levels of pectin lyase (PL) and polygalacturonase (PG) simultaneously. Methods and Results: A strain with high production of PL was transformed with the plasmid pAN52pgg2, containing the gene encoding PG of P. griseoroseum, under control of the gpd promoter gene from Aspergillus nidulans. Southern blot analysis demonstrated that all strain had at least one copy of pAN52pgg2 integrated into the genome. The recombinant strain P. griseoroseum T20 produced levels of PL and PG that were 266‐ and 27‐fold greater, respectively, than the wild‐type strain. Furthermore, the extracellular protein profile of recombinant T20 showed two protein bands of c. 36 and 38 kDa, associated with PL and PG, respectively. Conclusions: This recombinant strain T20 produces PL and PG using carbon sources of low costs, and an enzyme preparation that is free of cellulolytic and proteolytic activities. Significance and Impact of the Study: PL and PG play an important role in the degradation of pectin. Owing to their use in the juice and wines industries, there is a growing interest in the inexpensive production of these enzymes. This work describes an efficient system of protein expression and secretion using the fungus P. griseoroseum.     

Production of pectin lyase by Penicillium griseoroseum cultured on sucrose and yeast extract for degumming of natural fibres

Sucrose, a non-pectic carbon source, with yeast extract (YE) added was able to support the production of pectin lyase (PL) by Penicillium griseoroseum Dierckx. However, in the absence of YE, the fungus did not produce PL but grew and caused a marked reduction in culture medium pH. Furthermore, in the absence of YE, only a culture medium with a high buffering capacity permitted the production of PL in the presence of pectin. On the other hand, in the presence of 0.06% YE and of 0.1% pectin, the fungus produced maximum growth and specific PL activity during a 48-h period of culture, with a small variation in medium pH. In the absence of sucrose, YE concentrations from 0 to 0.6% did not support enzyme production, indicating synergism between sucrose and YE for production of the enzyme.     

Production of lyases byPhoma exigua associated with seed-rot ofVigna radiata

Phoma exigua associated with seed-rot ofVigna radiata produced lyases which varied with the media tested. The production of lyases was higher in pectin-supplemented media.Vigna seed meal medium was not suitable for induction of lyase production. The pectin lyase and pectate lyase was maximum after 11 d of incubation by which time the pH was shifted to alkaline side. Temperature of 25 °C and pH 9 was found to be optimum for the activity of pectin lyase and pectate lyase. Fungicides (antracol and panoctine), phenols (pyrocatechol and gallic acid) and growth substances (gibberellic acid and yeast extract) adversely affected the enzyme secretion.     

Production of pectinolytic enzymes pectinase and pectin lyase by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SAV-21 in solid state fermentation

Pectin-degrading enzymes (pectinase and pectin lyase) were produced in solid state fermentation by Bacillus subtilis SAV-21 isolated from fruit and vegetable market waste soil of Yamuna Nagar, Haryana, India, and identified by 16S rDNA sequencing. Under optimized conditions, maximum production of pectinase (3315 U/gds) and pectin lyase (10.5 U/gds) was recorded in the presence of a combination of orange peel and coconut fiber (4:1), with a moisture content of 60% at 35 °C and pH 4.0 after 4 days and 8 days of incubation, respectively. Pectinase yield was enhanced upon supplementation with galactose and yeast extract, whereas pectin lyase production was unaffected by adding carbon and nitrogen source to the basal medium. Thus, B. subtilis SAV-21 can be exploited for cost-effective production of pectinase and pectin lyase using agro-residues.     

Green mould of oranges caused by Penicillium digitatutn Sacc.; effect of additives on spore germination and infection

Water extracts of rind, essential oil and juice from oranges, also citrus pectin and citric acid promoted the formation of lesions when spores of Penicillium digitatum were placed in wounds 1·0 mm deep in flavedo of oranges; fructose, glucose and sucrose had little effect. Rind extracts were less effective in wounds 0·5 mm deep but orange juice and pectin still increased infection. None of the substances allowed the parasite to infect fruit through unwounded surfaces. Germination of spores in water increased as spore concentration decreased but was poor even at low concentrations. Almost all spores germinated in aqueous extracts of flavedo, albedo or whole rind, or in wounds on the surface of fruit. Fructose, glucose, sucrose and xylose were less effective but still caused over two-thirds of spores to germinate but only in the presence of phosphate buffer. Without buffer, germination was little different from that in water. Arabinose and galactose stimulated germination to a lesser extent but with the same phosphate effect. Carboxymethylcellulose and pectin did not affect germination. A variety of substances containing nitrogen increased germination but to different degrees, decreasing in the order, casamino acids, yeast extract, ammonium salts, nitrate. Thiamin and to a lesser extent biotin were also effective. Volatile substances from rind infected with P. digitatum stimulated spore germination and growth of germ tubes. The significance of these results is discussed in relation to infection.     

Carbon,nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ～7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.     

Effect of pectin on pectinases in autolysis of Botrytis cinerea

Pectic activity in autolyzed cultures of Botrytis cinerea in a medium with and without pectin was similar, but in the medium with pectin maximal activities occurred in younger cultures. The pectic activities found were polygalacturonase, polymethylgalacturonase, endo activity (pectin as substrate) and pectin lyase. The molecular weights of polygalacturonase, polymethylgalacturonase and endo activity (pectin as substrate) were 36000, 33000 and 30200 daltons respectively, and the molecular weight of pectin lyase was 18200 daltons. By gel electrophoresis four different pectic activities were detected, three in the top of the gel and one in the bottom. Two enzymes were characterized, the polygalacturonase activity (first band in the top) inhibited by Ca⁺⁺ and the pectin lyase activity (in the bottom) which was not inhibited by Ca⁺⁺. These enzymes are not induced by the presence of pectin in the medium during degradation of Botrytis cinerea.     

Isolation of a homocysteine gamma-lyase-producing bacterium and study of its enzyme production conditions

Aim: To investigate the possibility of finding a new homocysteine (Hcy) γ‐lyase with the desired properties for Hcy measurement in bacteria. Methods and Results: Through a process of enrichment, the Hcy γ‐lyase‐producing bacterium strain N2‐1 was isolated from soil. Based upon its morphological, physiological, and biochemical characteristics, as well as its 16S rDNA sequence and phylogenetic tree analysis, this isolate belongs to the genus Serratia. The effects of pH, aeration, inducers, carbon (C) and nitrogen (N) sources on enzyme production were studied. Methionine, yeast extract, and glucose were selected as the optimal inducer, C and N sources, respectively. Maximum production of Hcy γ‐lyase was obtained when the isolate was cultured at 30°C at pH 6·5 for about 36 h in the optimum medium. Results also showed that this Hcy γ‐lyase has relatively high specificity towards Hcy. Conclusions: Because of its high specificity for Hcy, this bacterial Hcy γ‐lyase has the potential application in Hcy determination. Significance and Impact of the Study: In addition to isolating a bacterium that produces Hcy γ‐lyase suitable for Hcy determination, this study also indicates that the bacterium could be a source for production of Hcy γ‐lyase for clinical applications.     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.     

Culture conditions for the production of pectinolytic enzymes by the white-rot fungus Trametes trogii on a laboratory scale

Different cultural parameters that regulate pectinolytic enzyme production in vitro by Trametes trogii were studied. When grown in a medium containing pectin, T. trogii produced extracellular polymethylgalacturonase, polygalacturonase and pectin lyase but no pectate lyase activity. No significant differences in the maximum enzyme activities measured were observed with the addition of xylan, carboxymethylcellulose or both to the medium containing pectin. The addition of glucose to that medium considerably decreases all the activities studied, and in a medium with glucose as the sole carbon source no galacturonase activity could be measured, and pectin lyase activity was at its minimum. The low synthesis of pectin lyase in cultures containing glucose suggests that this enzyme is constitutive in contrast to the polygalacturonases that were not detected. The increase in pectin concentration stimulated growth and enzyme production. The highest specific activities were attained with the greatest concentration tested (15 g/l). Casamino acids were the best nitrogen source for enzyme production. Maximum growth was measured at pH 3.3; pH values of around 4.5 stimulated enzyme production, but high pectinase activities were also detected in media with more alkaline initial pH values (6.2 for galacturonases and 6.6 for lyases), probably owing to the specific induction of particular isoforms. In the range of 23 to 28°C, good results were obtained in growth as well as in enzyme production. The addition of Tween 80 promoted growth and gave the highest yield of polymethylgalacturonase and pectin lyase (0.37 and 36.2 E.U./ml, respectively). The highest polygalacturonase activity (1.1 E.U/ml) was achieved with polyethylene glycol. Tween 20 and Triton X-100 inhibited growth and pectinase production.     

Partial purification and properties of pectin lyase from Penicillium expansum

A pectin lyase, poly(methoxygalacturonide) lyase, EC 4.2.2.10, from a culture filtrate of Penicillium expansum was partially purified 33-fold with 7.3% yield. The enzyme was monomeric with a molecular mass of 36.5 kDa. The enzyme did not contain pectate lyase activity and degraded citrus and apple pectin best at pH 7.0 and 40 to 45°C. The K _m for citrus pectin was 9 mg ml^-1.     

Simultaneous Synthesis of Pectin Lyase and Carotovoricin Induced by Mitomycin C,Nalidixic Acid or Ultraviolet Light Irradiation in Erwinia carotovora

When Erwinia carotovora Er, a bacteriocinogenic strain, was induced after irradiation by ultraviolet (UV) light or inhibitors of DNA synthesis, such as mitomycin C or nalidixic acid, pectin lyase and bacteriocin (designated carotovoricin) activity appeared in the culture fluid. The optimal dose of each of these agents for producing the enzyme or bacteriocin was identical, and the time courses for both were essentially the same. Therefore, we assumed that the synthesis of the enzyme and bacteriocin was regulated by the same mechanism, in which a repressor inactivated by UV light, mitomycin C or nalidixic acid was involved. The other three bacteriocinogenic strains of E. carotovora also formed pectin lyase, in addition to carotovoricin in the presence of mitomycin C, indicating that simultaneous syntheses of pectin lyase and carotovoricin were widespread phenomenon in bacteriocinogenic strains of E. carotovora.     

Production,purification and characterization of pectinase from a Bacillus sp. DT7

A soil isolate, Bacillus sp. DT7 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as pectin lyase (EC 4.2.2.10). By optimizing growth conditions, Bacillus sp. DT7 produced higher amount of pectin lyase (53 units/ml) than that has been reported in the literature. Using gel filtration and ion exchange chromatography, this enzyme was purified and found to have a molecular mass of 106 kDa. The purified enzyme exhibited maximal activity at a temperature of 60 C and pH 8.0. The presence of 100 mM concentrations of CaCl₂ and mercaptoethanol significantly enhanced pectinase activity of the purified enzyme. This pectinase has tremendous applications in textile industry, plant tissue maceration and fruit juice wastewater treatments.     

Statistical medium optimization and DO-STAT fed-batch fermentation for enhanced production of tyrosine phenol lyase in recombinant Escherichia coli

Abstract

Tyrosine phenol lyase (TPL) is a robust biocatalyst for the production of L-dihydroxyphenylalanine (L-DOPA). The improvement of TPL production is conducive to the industrial potential. In this study, the optimization of culture medium of recombinant Escherichia coli harboring TPL from Fusobacterium nucleatum (Fn-TPL) was carried out. Sucrose and combination of yeast extract and peptone were selected as carbon and nitrogen source, respectively. Their optimal concentrations were determined by Box-Behnken design and the synergistic effect between yeast extract and peptone was found to be significant, with p-value < 0.05. The DO-STAT fed-batch fermentation under optimized culture condition was established and the oxygen level was fixed at 20%. Both the biomass and Fn-TPL activity were significantly increased, which were 35.6 g dcw/L and 12292 U/L, respectively. The results obtained significantly promote the industrial production of L-DOPA production.     

The Influence of Exogenous Nutrients on the Abundance of Yeasts on the Phylloplane of Turfgrass

Four experiments were conducted to assess the effect of foliar applications of various nutrient solutions on the phylloplane yeast community of tall fescue (Festuca arundinacea Schreb.). In the first three experiments, increasing concentrations of sucrose (2–16%), yeast extract (0.5–2.5%), and sucrose plus yeast extract (2.5–18.5% total) were applied and the yeast colony forming units (cfu) enumerated 14 h later by dilution plating. Significant positive linear relationships were observed between the number of yeast cfu and applications of both yeast extract and sucrose plus yeast extract. Foliar applications of sucrose alone had no significant effect on yeast community abundance, indicating that phylloplane yeasts of turfgrass are not limited by the amount or availability of carbohydrates. In the fourth experiment, five different solutions were applied to tall fescue to investigate the response of the yeast community to organic and inorganic nitrogen sources. Tryptone or yeast extract, both with considerable amino acid composition, significantly increased the yeast population, while yeast nitrogen base (with or without amino acids) and ammonium sulfate had no affect on yeast abundance. These results suggest that organic nitrogen stimulate yeast community growth and development on the phylloplane of tall fescue, while carbohydrates, inorganic nitrogen, and non-nitrogenous nutrients have little positive effect.