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.     

Methylxanthines as inducers of pectin lyase in Penicillium griseoroseum cultured on sucrose

Pectin lyase (PL) from Penicillium griseoroseum can be induced by xanthine, theobromine, theophylline and especially by caffeine and hypoxanthine (5 mmol l⁻¹ with 0·01% yeast extract (YE)). For caffeine and hypoxanthine, PL activity was, respectively, 5·2 and 3·7 times higher than with YE alone. The simultaneous addition of caffeine or hypoxanthine (5 mmol l⁻¹) and YE (0·1%) had a synergistic effect on PL activity as compared to the addition of these substances alone (0·2% YE; 10 mmol l⁻¹ caffeine; 10 mmol l⁻¹ hypoxanthine). Increasing caffeine concentrations (0–10 mmol l⁻¹) for a constant YE content of 0·01%, resulted in an increase in PL activity and a decrease in mycelial mass. For a constant caffeine concentration (5 mmol l⁻¹) and increasing YE contents (0–0·2%), a higher PL activity and mycelial mass were detected. The addition of caffeine (10 mmol l⁻¹) at the beginning of incubation increased PL activity and decreased mycelial mass, while caffeine added after 12 and 24 h resulted in decreases in PL activity and increases in mycelial mass. The results presented here indicate that methylxanthines, especially caffeine, can induce PL in P. griseoroseum .     

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.     

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.     

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.     

Isolation and characterization of extracellular pectin lyase from Penicillium canescens

Pectin lyase A (molecular weight 38 kD by SDS-PAGE, pI 6.7) was purified to homogeneity from culture broth of the mycelial fungus Penicillium canescens using chromatographic techniques. During genomic library screening, the gene encoding pectin lyase A from P. canescens (pelA) was isolated and sequenced, and the amino acid sequence was generated by applying the multiple alignment procedure (360 residues). A theoretical model for the three dimensional structure of the protein molecule was also proposed. Different properties of pectin lyase A were investigated: substrate specificity, pH- and temperature optimum of activity, stability under different pH and temperature conditions, and the effect of Ca2+ on enzyme activity. In the course of the laboratory trials, it was demonstrated that pectin lyase A from P. canescens could be successfully applied to production and clarification of juice.     

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.     

Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization

Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a ‘smooth’ region and a ‘hairy’ region. The ‘smooth’ region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The ‘hairy’ region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40 % the viscosity of pectin with a degree of esterification ≥85 %. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8 % esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.     

Impala,a transposon from Fusarium oxysporum,is active in the genome of Penicillium griseoroseum

An autonomous impala transposon trapped in Fusarium oxysporum by insertion within the niaD gene encoding nitrate reductase was introduced in the genome of the fungus Penicillium griseoroseum, a producer of pectinase enzymes. Through a phenotypic assay, we demonstrate that this element is able to excise from the niaD gene and to reinsert at new genomic positions. As in the original host, impala inserts into a TA site and footprints left by impala excisions are generally 5 bp. The fact that impala is able to transpose in P. griseoroseum offers the opportunity to develop a gene-tagging system based on this element with the objective to detect and clone genes related in pectinase production.     

LaeA regulation of secondary metabolism modulates virulence in Penicillium expansum and is mediated by sucrose

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose‐responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175‐ and five‐fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe‐21 from Israel and Pe‐T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch‐Pe‐T01 strains to 15%–25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is‐Pe‐21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.     

黑曲霉果胶裂解酶基因毕赤酵母在Pichia pastoris GS115中的表达

利用RT-PCR技术从黑曲霉(EIM-6)中扩增得到去除信号肽的果胶裂解酶基因A,将其插入到毕赤酵母表达载体pPIC9k上,构建重组表达质粒pPIC9K-pelA,电击转化毕赤酵母GS115,得到了表达成功的工程菌株。用终浓度为1.5%的甲醇对其进行诱导,将发酵上清液浓缩后,用盐酸法测定其酶活可以达到2.3U/mL。通过对重组毕赤酵母诱导表达产物进行SDS-PAGE鉴定,发现重组毕赤酵母分泌了1个约38kD的蛋白,与该酶基因产物的理论值相符,并通过水解圈法测定验证,均说明果胶裂解酶得到正确的分泌表达.     

Purification and characterization of pectin lyase secreted by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K _m and k _cat values were found to be 1 mg/ml and 76 sec⁻¹, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992.     

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.     

Effects of sucrose on starch accumulation and rate of photosynthesis in Rosa cultured in vitro

Shootlets of Rosa multiflora L. cv. Montse were cultured in vitro with four different levels of sucrose (0, 1, 3 and 5%). Chloroplasts of shootlets grown in a medium without sucrose contained numerous, large plastoglobuli and were lacking in starch granules. The size and number of starch granules increased with the level of sucrose in the culture medium. Starch content in leaves of shootlets grown with 5% sucrose was higher (ca 1, 3%) than those grown with 3% (ca 0, 45%) and 1% sucrose (ca 0, 27%). Starch might be used by the in vitro shootlets during the acclimation period.Abbreviations BA benzyladenine - Pi orthophosphate - S sucrose - Rubisco ribulose 1,5-bisphosphate carboxylase - TEM transmission electron microscopy     

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

During glucose‐limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β‐lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady‐state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose‐limited chemostat cultures at the dilution rates (D) of both 0.03 h^?1 and 0.05 h^?1 are determined to evaluate the quantitative impact on the physiology of a high‐yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h^?1, while this is not the case at D = 0.05 h^?1. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h^?1, which are virtually absent at D = 0.03 h^?1. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose‐limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial‐scale scenario.     

Growth regulation, plastid differentiation and the development of a photosynthetic system in cultured carrot root explants as influenced by exogenous sucrose and various phytohormones

Chromoplasts, which exist in the cells of freshly isolated carrot root explants, seemed to be transformed in thylakoid containing plastids, and chlorophyll formation was initiated if the explants were cultured in a liquid medium containing inositol and IAA as a hormonal supplement. This process was intensified when kinetin was also added, but no dependence on a sucrose supply could be found.A sucrose supply of 2% in conjunction with the combination of all three hormones, however, was needed to achieve maximal thylakoid formation including stacking in individual chloroplasts and for the very extensive chloroplast multiplication in explants growing with high cell division activity. It should be noted that the number of plastids per cell is strongly increased by the sucrose supplement which leads also to starch accumulation. However, no transformation into chloroplasts occurred without the hormonal stimulus.