Production Of Pectinolytic Enzymes Research Articles

Isolation, Screening, and Characterization of Fungal Isolates with Pectinolytic Activity from Decaying Fruits

Filamentous fungi are potential source of pectinolytic enzyme production used in fruit processing industry can be isolated from decaying fruits as substrates. Thus, this study was conducted to isolate, screen, characterize pectinase-producing fungi from decaying fruits and assess their pectinolytic activity. Decaying fruits samples were collected from fruit stands of Iloilo City Terminal Market. Pectinolytic fungi were grown and screened for pectinolytic activity using pectinolytic activity plate assay based on the decolorization coefficient value. Results showed three genera of fungi isolated from various decaying fruits which include Aspergillus (9 isolates), Rhizopus (1 isolate), and Mucor (1 isolate). Aspergillus spp. was the most dominant fungal isolate obtained from decaying fruits of ampalaya (Momordica charantia), pear (Pyrus pyrifolia), pineapple (Ananas comosus), tomato (Lycopersicon esculentum), lemon (Citrus limon), melon (Cucumis melo), calamansi (Citrus microcarpa), and mango (Mangifera indica). The Aspergillus spp. showed significant pectinolytic activity among the fungi effectively screened based on the decolorization coefficient value obtained. Thus, common fungi with pectinolytic activities are mitosporic genera isolated from decaying fruits that can be applied in agro-biotechnological industry.

Pectobacterium parmentieri SCC 3193 Mutants with Altered Synthesis of Cell Surface Polysaccharides Are Resistant to N4-Like Lytic Bacteriophage ϕA38 (vB_Ppp_A38) but Express Decreased Virulence in Potato (Solanum tuberosum L.) Plants.

Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ϕA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ϕA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.

The chimeric GaaR-XlnR transcription factor induces pectinolytic activities in the presence of D-xylose in Aspergillus niger

Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin.Key points• Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9.• PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants.• Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.

Pectinolytic activity of aspergillus niger and Aspergillus flavus grown on grapefruit (citrus Parasidis) peel in solid state fermentation

The present study was aimed at studying pectinolytic activity of resident fungi isolated from decomposing grapefruit (Citrus parasidis) peels in solid state fermentation. Grape fruit peel was subjected to natural fermentation and the fermenting fungi were isolated, characterized and identified using standard microbiological methods. The isolated fungi were in turn used for fermentation to determine their pectinolytic activity through solid state fermentation technique. Culture parameters such as incubation period, temperature, moisture content and addition of salts supplements were optimized during the research for five days. The identified fungi were Aspergillus Niger and Aspergillus flavus. The peak of pectinolytic activity was at day three of fermentation when the highest pectinase activity of 13.32 μmol/mg/min was recorded for A. Niger and 11.32 μmol/mg/min for A. flavus. Optimum temperature and pH for pectinase activity by A. Niger and A. flavus was at 40 0C and pH 7.5 and 7.7 respectively. The use of salt supplemented substrate did not alter enzyme activity. In conclusion, the isolated fungi could be promising organisms for pectinolytic enzyme production on grape peel as substrate.
 Keywords: Grapefruit, Pectinolytic Activity, Fungi, Fermentation, Aspergillus

Pathophysiology of Fusarium oxysporum f. Sp. Dianthi in carnation (Dianthus caryophyllus L.)

The bio-chemical changes occurred in the carnation after inoculation with F. oxysporum f.sp. dianthi was studied under in vitro. Carnation plants inoculated with fourteen isolates of F.oxysporum f.sp.dianthi and monitored for their ability to production of fungal pectin-degrading enzymes viz., Pectin Methyl Esterase (PME), Polygalacturonase (PG) and Pectin Trans Eliminase (PTE) involved in development of disease symptoms. Production of pectinolytic enzymes in carnation plants were assessed from 2 days up to 8 days after inoculation at 48h intervals. The accumulation of these enzymes increased in two days after inoculation and attained a peak at six days after inoculation and slowly declined thereafter in all the inoculated plants. Among the fourteen isolates, YRPFOD2 had maximum ability to increase the activity of pectinolytic enzymes viz., Pectin Methyl Esterase (0.49 μ mole hydrogen ion / min / ml), Polygalacturonase (16.11% reduction in viscosity) and Pectin Trans Eliminase (57.59 % reduction in viscosity) after six days of inoculation in infected plants.

Optimization of concurrent production of xylanolytic and pectinolytic enzymes by Bacillus safensis M35 and Bacillus altitudinis J208 using agro-industrial biomass through Response Surface Methodology

Application of crude xylanolytic and pectinolytic enzymes in diverse industrial processes make these enzymes commercially valuable and demand their production process to be cost-effective. Out of four different agrowaste biomass, wheat bran (WB) and citrus peel (CP), when amended as fermentation substrates, respectively induced the highest xylanolytic enzymes and pectinolytic enzymes from both, B. safensis M35 and B. altitudinis J208. Further, the simultaneous amendment of WB and CP yielded concurrent production of these cellulase free xylanolytic and pectinolytic enzymes. Hence, the quadratic model was developed using the Central Composite Design of Response Surface Method (CCD-RSM). The model gave the concentration values for WB and CP substrates to be amended in one single production medium for obtaining two optimized predicted response values of xylanase activity and pectinase activity units, which were further practically validated for the xylanase and pectinase production responses from the optimized production medium (OPM). These practically obtained response values from OPM were found to be in accordance with a range of 95% predicted intervals (PI) values. These observations verified the validity of the predicted quadratic model from RSM and suggested that both xylanase and pectinase enzymes can be induced concurrently from both of the bacterial strains.

COMPARACIÓN DEL POTENCIAL FERMENTATIVO Y MÉDICO DE LOS GÉNEROS Saccharomyces CON Wickerhamomyces

Comparative assessment of two different yeast genera i.e. Saccharomyces and Wickerhamomyces is carried out regarding industrial and medical applications with special focus on their role in bioethanol production. Since second generation ethanol production from lignocellulosic material has become a hot topic of research due to its noncompetitive nature with food, economically feasible conversion of lignocellulosic biomass requires an organism capable of metabolizing both the hexoses and pentoses simultaneously. Saccharomyces which is in extensive use currently for bioethanol production can effectively convert hexoses into ethanol but unable to metabolize xylose. However, Wickerhamomyces have the potential to convert both the pentoses as well as the hexoses into bioethanol. Additionally, due to its glycosidase production ability, Wickerhamomyces is tolerant to high levels of glucose and ethanol. Furthermore, the capabilities of the two genera with respect to their role in probiotics, antimicrobial agents, food, beverages, biosorption, whole cell biocatalyst, and pectinolytic enzyme production have also been compared in this review.

Production of Pectinases and Pectinolytic Enzymes: Microorganisms, Cultural Conditions and Substrates

Production of Pectinases and Pectinolytic Enzymes: Microorganisms, Cultural Conditions and Substrates

Production of Pectinolytic Enzymes by Two Bacillus spp. Strains and Their Application in Flax Degumming

This study demonstrated that Bacillus licheniformis HDYM-03 and Bacillus megaterium HDYM-09, isolated from a liquid sample of flax retting pool, were able to produce pectinolytic enzymes using polysaccharides as substrates. Bacillus megaterium HDYM-09 produced pectin lyase that exhibited the highest activity of 2116.71 ± 11.55 U/mL. Bacillus licheniformis HDYM-03 produced pectate lyase that exhibited the highest activity of 611.21 ± 14.54 U/mL. Based on these findings, we constructed four retting systems to degrade the pectin substance. The results showed that the content of galacturonic acid in the mixed system was 529.21 μg/mL, the content of reducing sugar was 98.14 mg/mL, and the weight loss ratio of cells reached 19.49%, which were significantly higher than those in other systems. The mixed system has more advantages, and the utilization rate of degumming was higher, which further ensured that the degumming can be carried out efficiently and quickly. The mixed system exhibits feasible applications in the fiber and textile industry.

Inhibition in production of cellulolytic and pectinolytic enzymes of Colletotrichum gloeosporioides isolated from dragon fruit plants in response to submicron chitosan dispersions

In vitro and in vivo antifungal potential of submicron chitosan dispersions (SCD) was studied in order to investigate the effect of SCD on cell wall degrading enzymes of Colletotrichum gloeosporioides and to control anthracnose of dragon fruit under field conditions. Four concentrations of chitosan (0.5, 1.0, 1.5 and 2.0%) were used to prepare submicron dispersions with droplet sizes of 200, 400, 600, 800 and 1000 nm. The highest inhibition in cellulolytic and pectinolytic enzymes was observed with 600 nm droplet size of 1.0% chitosan concentration. In case of in vivo studies,the area under disease progress curve (AUDPC) showed a reduction of approximately 66% of disease incidence (DI). The lower level of production of cell wall degrading enzymes in response to SCD resulted in less disease on dragon fruit plants and sustained in increasing the effective resistance against the irresistible disease of anthracnose.

Pectinolytic enzymes-solid state fermentation, assay methods and applications in fruit juice industries: a review.

A plethora of solid substrates, cultivation conditions and enzyme assay methods have been used for efficient production and estimation of polygalacturonase and pectin methylesterase enzymes. Recent developments in industrial biotechnology offer several opportunities for the utilization of low cost agro-industrial waste in Solid State Fermentation (SSF) for the pectinolytic enzyme production using fungi. Fruit waste mainly citrus fruit waste alone and along with other agro-industrial waste has been explored in SSF for enzyme production. Agro-industrial waste, due to the economic advantage of low procuring cost has been employed in SSF bioreactors for pectinolytic enzyme production. Acidic pectinases produced by fungi are utilized especially in food industries for clarification of fruit juices. This review focuses on the recent developments in SSF processes utilizing agro-industrial residues for polygalacturonase and pectin methylesterase production, their various assay methods and applications in fruit juice industries.

Oxygen Availability Influences Expression of Dickeya solani Genes Associated With Virulence in Potato (Solanum tuberosum L.) and Chicory (Cichorium intybus L.).

Dickeya solani is a Gram-negative necrotrophic, plant pathogenic bacterium able to cause symptoms in a variety of plant species worldwide. As a facultative anaerobe, D. solani is able to infect hosts under a broad range of oxygen concentrations found in plant environments. However, little is known about oxygen-dependent gene expression in Dickeya spp. that might contribute to its success as a pathogen. Using a Tn5 transposon, harboring a promoterless gusA reporter gene, 146 mutants of D. solani IPO2222 were identified that exhibited oxygen-regulated expression of the gene into which the insertion had occurred. Of these mutants 114 exhibited higher expression under normal oxygen conditions than hypoxic conditions while 32 were more highly expressed under hypoxic conditions. The plant host colonization potential and pathogenicity as well as phenotypes likely to contribute to the ecological fitness of D. solani, including growth rate, carbon and nitrogen source utilization, production of pectinolytic enzymes, proteases, cellulases and siderophores, swimming and swarming motility and the ability to form biofilm were assessed for 37 strains exhibiting the greatest oxygen-dependent change in gene expression. Eight mutants expressed decreased ability to cause disease symptoms when inoculated into potato tubers or chicory leaves and three of these also exhibited delayed colonization of potato plants and exhibited tissue specific differences in gene expression in these various host tissues. The genes interrupted in these eight mutants encoded proteins involved in fundamental bacterial metabolism, virulence, bacteriocin and proline transport, while three encoded hypothetical or unknown proteins. The implications of environmental oxygen concentration on the ability of D. solani to cause disease symptoms in potato are discussed.

Harnessing soybean hulls for improved polygalacturonase production by Aspergillus sojae through fine‐tuning of ambient pH

AbstractBACKGROUNDSoybean hulls result from the processing of the bean for producing oil and protein products. This by‐product generated massively in America has virtually no commercial value, so substantial effort is being applied to its exploitation for generating value‐added goods. This work evaluates soybean hulls as inducer of the production of pectinolytic enzymes, through optimization studies regarding polygalacturonase production by Aspergillus sojae in submerged cultures.RESULTSA 2‐fold improvement in polygalacturonase yield was found by varying the initial pH of the culture in a very narrow acid pH range (2.40–2.80). The optimized fermentation process was successfully transferred to stirred‐tank bioreactors in terms of volumetric productivity, and final polygalacturonase yields were 42 U mL‐1 and 1.39 U g‐1 soybean hulls, which are among the highest reported with this by‐product. Morphological characterization of A. sojae during cultivation showed that the fungus mainly developed in dispersed mycelia at initial pH of 2.40–2.80 while, conversely, fungal pellets predominated in cultures performed at initial pH of 5.40.CONCLUSIONHigh enzyme titers are possibly connected to the formation of dispersed mycelia, as well as to acid‐induced expression of the respective gene/s. It is foreseen that this data will be helpful regarding the production of fungal pectinases or other acid‐induced enzymes. © 2017 Society of Chemical Industry

Combinatorial control of gene expression in Aspergillus niger grown on sugar beet pectin

Aspergillus niger produces an arsenal of extracellular enzymes that allow synergistic degradation of plant biomass found in its environment. Pectin is a heteropolymer abundantly present in the primary cell wall of plants. The complex structure of pectin requires multiple enzymes to act together. Production of pectinolytic enzymes in A. niger is highly regulated, which allows flexible and efficient capture of nutrients. So far, three transcriptional activators have been linked to regulation of pectin degradation in A. niger. The L-rhamnose-responsive regulator RhaR controls the production of enzymes that degrade rhamnogalacturonan-I. The L-arabinose-responsive regulator AraR controls the production of enzymes that decompose the arabinan and arabinogalactan side chains of rhamnogalacturonan-II. The D-galacturonic acid-responsive regulator GaaR controls the production of enzymes that act on the polygalacturonic acid backbone of pectin. This project aims to better understand how RhaR, AraR and GaaR co-regulate pectin degradation. For that reason, we constructed single, double and triple disruptant strains of these regulators and analyzed their growth phenotype and pectinolytic gene expression in A. niger grown on sugar beet pectin.

Algal biomass: A sustainable, economical and renewable approach for microbial production of pectinolytic enzymes using submerged and solid state fermentation techniques

In the past decade, algal waste has been used as useful natural resource for production of enormous range of products that have wide economical and commercial importance. Pectinases are group of enzymes that have wide commercial applications. Hence, current study was designed to utilize algal biomass for the production of pectinases using submerged (SmF) and solid state fermentation (SSF) techniques. Different algal sources including brown (Dictyopteris polypodioides, Sargassum wightii and Dictyopteris divaricata) and green algae (Ulva lactuca and Codium tomentosum) were used and U. lactuca was found to be the most suitable substrate. Several bacterial and fungal strains were screened and among them Bacillus licheniformis KIBGE-IB4 was selected based on maximum pectinase production. SmF and SSF were studied utilizing U. lactuca as a substrate and results revealed that enzyme production was favoured by SmF (2457 ± 3.31 U mg−1) as compared to SSF (1432 ± 1.46 U mg−1). Parametric optimization of pectinase production indicated that B. licheniformis KIBGE-IB4 requires 10.0 g L–1 U. lactuca as a biomass in the medium with a pH 7.0 when incubated at 37 °C for 24 hours. Likewise, production of pectinase using algal resource was also compared with that of the conventional agricultural biomass and it was observed that when U. lactuca was used, the selected bacterial isolate produced a higher yield of enzyme than sugarcane bagasse and rice husk. Hence, it is anticipated that algal biomass can be efficiently utilized as an environmental friendly bioresource for the production of industrially important hydrolytic enzymes.

Temperature‐responsive genetic loci in pectinolytic plant pathogenic Dickeya solani

Fifty‐four Dickeya solani thermoregulated genes were identified using Tn5 transposon mutagenesis with an inducible gusA reporter system; 45 genes were up‐regulated at 37 °C, whereas nine were up‐regulated at 18 °C. The relative level of gene up‐regulation ranged from 2–1200 and 5–650 U/mg total proteins at 18 and 37 °C, respectively. Among the temperature‐regulated loci, genes coding for proteins involved in fundamental bacterial metabolism, membrane‐related proteins and pathogenicity‐corresponding factors and several hypothetical unknown proteins were found. The mutants were tested for their pathogenicity in planta and for features known to be important for D. solani virulence viz. production of pectinolytic enzymes, cellulases, proteases, siderophores and auxins as well as for motility and the ability to form a biofilm. Eight Tn5 mutants, four up‐regulated at high and four up‐regulated at low temperature, expressed visible phenotypes including the decreased ability to cause symptoms on potato tubers and chicory leaves, impairment in phospholipase production and/or deficiency in biofilm formation. The implications of environmental temperature on the ability of D. solani to cause disease symptoms in potato are discussed.

Attenuation and quantitation of virulence gene expression in quorum-quenched Dickeya chrysanthemi.

N-Acyl-homoserine lactones (AHLs)-dependent quorum sensing (QS) system(s) is recruited by the soft rot bacterium Dickeya chrysanthemi for coordinating its social activities such as secretion of plant cell wall-degrading enzymes, while the main signal molecule and quantity dependence of virulence to QS in this bacterium have not been clarified. To do this end, the involvement of AHLs in African violet leaves and potato tuber maceration; swarming motility; pectate lyase and polygalacturonase enzymes production and in planta expression of virulence genes including pelE, pehX and pemA by electroporating two quorum-quenching vectors. The expression of two types of AHL-lactonase expressing vector caused dramatic decrease in swarming motility, production of pectinolytic enzymes and macerating of plant tissues. The maximum ability of quenching of QS in repression of D. chrysanthemi virulence was assessed quantitatively by q-RT-PCR, as expression of pelE, pehX and pemA genes were decreased 90.5-92.18% in quenched cells. We also showed that virulence and pathogenicity of this bacterium was under the control of DHL-dependent QS system and that the existence of second DHL operating system is probable for this bacterium. Thus, this signal molecule would be the key point for future research to design DHL-specific lactonase enzymes using bioinformatics methods.

Retting and degumming of natural fibers by pectinolytic enzymes produced from Bacillus tequilensis SV11-UV37 using solid state fermentation.

The present study demonstrated the simultaneous production and optimization of pectinolytic enzymes (pectate lyase and polygalacturonase) under SSF from Bacillus tequilensis SV11-UV37 using wheat bran as a substrate, which is commercially viable and cost-effective. Optimization by one variable-at-a-time-approach showed a maximum yield of pectate lyase (1371.25 U/gds) and polygalacturonase (85.45 U/gds) with wheat bran using 80 % (v/w) moisture, 0.7 mm particle size, 20 % (v/w) inoculum, 1 % (w/w) pectin at 37 °C, pH 6 and 72 h of incubation. In addition, optimization using central composite design achieved 1.6-fold improvement in both pectate lyase (1828.13 U/gds) and polygalacturonase (105.55 U/gds) yield at optimum levels of pectin (3 %, w/w), inoculum size (20 %, v/w) and moisture level (80 %, v/w). Further, Retting studies concluded that the enzyme mixture was efficient in separating the whole fiber from kenaf and part (>75 %) from sunn hemp. In degumming of sunn hemp fibers, amount of galacturonic acid released and percentage weight loss was higher in successive alkali and enzymatic treatment than their independent treatments. The scanning electron microscopic analysis also confirmed that alkali followed by enzymatic treatment effectively removed non-cellulosic gummy material from the fiber; hence, this enzyme mixture may find feasible applications in the fiber and textile industry.

IMPACT OF BACTERIAL QUORUM SENSING SYSTEM ON CHANGES OF ORGANOLEPTIC MARKERS OF STORAGE CABBAGE

The regulation of phenotypes of vegetable-associated bacteria can be mediated through the production of acylated homoserine lactones (AHLs). This knowledge is essential for successful control of bacterial diseases of vegetable. The purpose of these investigations was to define the AHL patterns of gram-negative bacteria presented in storage cabbage by LC/MS technique. The phenomenon of regulating the pectinolytic activity and the exopolysaccharide (EPS) production by AHLs that are associated with microbial spoilage of cabbage was also examined. Among 100 strains isolated from storage cabbage, 47 isolates produced AHLs. The results of the 16S rDNA sequence analysis indicated that selected microflora was highly closely related to Pantoea agglomerans, Rahnella aquatilis, Pseudomonas fluorescens, Pseudomonas syringae, and Pseudomonas cedrina (approximately 98%-99% confidence). The chemical nature of AHLs produced by selected microflora differ from species to species. The pattern of AHLs of Ps. cedrina consisting of C8-HSL, 3-oxo-C10-HSL, and 3-hydroxy-C14-HSL, has not been previously reported. The present study demonstrates that bacterial spoilage of storage cabbage is influenced by quorum sensing. Application of furanone C-30 that acts as quorum sensing inhibitor, caused the significant reduction in the production of EPS and pectinolytic enzymes by examined bacteria.

Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae

Strain improvement is a powerful tool in commercial development of microbial fermentation processes. Strains of Aspergillus sojae which were previously identified as polygalacturonase producers were subjected to the cost-effective mutagenesis and selection method, the so-called random screening. Physical (ultraviolet irradiation at 254 nm) and chemical mutagens (N-methyl-N'-nitro-N-nitrosoguanidine) were used in the development and implementation of a classical mutation and selection strategy for the improved production of pectic acid-degrading enzymes. Three mutation cycles of both mutagenic treatments and also the combination of them were performed to generate mutants descending from A. sojae ATCC 20235 and mutants of A. sojae CBS 100928. Pectinolytic enzyme production of the mutants was compared to their wild types in submerged and solid-state fermentation. Comparing both strains, higher pectinase activity was obtained by A. sojae ATCC 20235 and mutants thereof. The highest polygalacturonase activity (1,087.2 ± 151.9 U/g) in solid-state culture was obtained by mutant M3, which was 1.7 times increased in comparison to the wild strain, A. sojae ATCC 20235. Additional, further mutation of mutant M3 for two more cycles of treatment by UV irradiation generated mutant DH56 with the highest polygalacturonase activity (98.8 ± 8.7 U/mL) in submerged culture. This corresponded to 2.4-fold enhanced polygalacturonase production in comparison to the wild strain. The results of this study indicated the development of a classical mutation and selection strategy as a promising tool to improve pectinolytic enzyme production by both fungal strains.