Synthesis and potential application of polygalacturonase from a Penicillium brasilianum isolate

The aim of this study was to evaluate the synthesis of pectinase from Penicillium brasilianum in shake flasks and address their potential for industrial applications. A Plackett-Burman design followed by a complete second order design were used for the screening of most important factors and to maximize the polygalacturonase activity, respectively. Maximum polygalacturonase activity was 52.8 U mL at 48 hours of bioproduction. The kinetic evaluation for substrate consumption showed that 42% total organic carbon, 52 nitrogen, 23 magnesium, and 60% potassium were consumed. The crude enzyme complex was used on commercial mango juice clarification, and, at a 0.5% concentration (v v) reduced viscosity by 10%, turbidity by 12% and clarification by 23%. Therefore, the results presented in this study could provide valuable and beneficial information for the food and enzyme industries (juice) as well as being a new landmark to microbiology by providing essential knowledge on P. brasilianum growing needs.

Usually, commercial pectinase preparations contain one or more types of microbial pectinolytic enzymes (depending on the specific use), as well as cellulases, hemicellulases, proteases, and amylases (Esawy et al., 2013). Molds such as Aspergillus niger, Coniotryrium diplodiela, Penicillum and Rhizopus species are preferred for industrial purposes since as much as 90% of the enzyme may be excreted into the culture medium (Souza, Silva, Maia, & Teixeira, 2003;Gomes et al., 2011;Pili et al., 2018).
A number of authors have studied extracellular pectinases production from Aspergillus sp. using pectic substrates, however, just a few studies using Penicillum sp. by submerged fermentation are to be found in the literature (Jayani, Saxena, & Gupta, 2005;Sandri, Fontana, & Silveira, 2015). In previous work by our research group (Stirling, 2003), a number of microorganisms capable of producing polygalacturonase were isolated from different sources. The relevance of this study lays in establishing the optimum conditions for maximum polygalacturonase production followed by kinetic evaluation of substrate consumption, pH and biomass progress from a newly isolated Penicillium brasilianum strain by submerged fermentation in a synthetic medium. Therefore, this study aimed at providing valuable and beneficial information for the food and enzyme industries, as well as being a new landmark for microbiology providing essential knowledge on P. brasilianum growing needs, which has been currently lacking in the literature.

Microorganism
The microorganism used in the present study was isolated from tea and previously identified by microcultivation technique as belonging to the Penicillium genera .
The newly isolated microorganism was identified following the molecular biology method. Fungi genomic DNA extraction was performed using liquid nitrogen for cell disruption (Tanner & Brunner, 1985) following DNA quantification using a NanoDrop 1000 model spectrophotometer (NanoDrop Technologies).
The regions ITS1, 5.8S and ITS2 of fungal rDNA were amplified using primers ITS1 and ITS4 (White, Bruns, Lee, & Taylor, 1990). The reactions were performed using a GeneAmp PCR System 9700 model thermocycler (Applied Biosystems) using the following conditions; 94º C for 5 min., followed by 33 cycles of 94ºC for 30 s, 55ºC for 30 s and 72ºC for 45 s; and a final extension of 72ºC for 10 min. The product was purified with GFX TM PCR DNA Kit and Gel Band Purification (GE Healthcare) and sequenced in an automatic ABI PRISM 3100 Genetic Analyzer sequencer (Applied Biosystems).
For the sequence consensus construction, the Phred/Phrap and Consed softwares (Altschu, Gish, Miller, Myers, & Lipman, 1990) were used and the sequence was compared with data from GenBank National Center for Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (Blast) software. The global alignment of the sequences and the phylogenetic analysis were performed using MEGA version 4.0 software. Cladistic analyses were constructed by the neighbor joining method using Jukes-Cantor for distance measurement. The confidence levels for individual branches of the resulting tree were assessed by bootstrap analysis, in which 1000 bootstrapped trees were generated from the re-sampled data.

Kinetic evaluation
The substrate consumption kinetics (total nitrogen, potassium, magnesium, and total organic carbon -TOC), cell mass, pH evolution, and PG production were followed by periodic sampling (3 -48 hours) using the maximization condition established after using the experimental design method.

Crude enzymatic extracts partial characterization
The temperature stability of enzymatic extract was determined by enzyme incubation at a fixed pHinitial (5.5) and different temperatures: 25, 35, 45, and 55ºC. The pH stability was achieved by incubating the extract obtained at 40ºC at pHs 4.0, 5.0, 7.0, and 9.0. The samples were withdrawn at regular time intervals.

Crude enzymatic extract application in juice clarification
A commercial mango juice (Del Valle brand) was treated by the crude enzymatic extract obtained at, previously established, maximized bioproduction conditions. 0.01, 0.05, 0.1, and 0.5% enzyme concentrations (v v -1 ) at 40ºC, 100 rpm for 60 min. were used by evaluating viscosity, turbidity, and juice clarification percentage.

Analytical methodology
Polygalacturonase (PG) was determined by measuring the reducing groups release using the acid dinitrosalisilic (DNS) method, initially mentioned by Miller (1959). One PG unit was defined as the amount of enzyme that releases 1 moL D-galacturonic acid per minute of reaction (U= moL min. -1 ). Pectin methylesterase (PME) was determined following a method described by Hultin, Sun, & Bulger (1966), with modifications. One PME unit was established as the amount of enzyme capable to catalyze the demethylation of pectin corresponding to the consumption of 1 µmol NaOH min. -1 mL -1 under assay conditions. Pectin lyase (PMGL) was determined using method described by Pitt (1988), with modifications. One enzyme activity unit was established as the amount of enzyme that changes 0.01 absorbance at 550 nm under assay conditions. Initial and final pH values of the culture medium were determined using a digital pH meter (Digimed DMPH-2). Cell mass was quantified by drying at 105ºC (Fanem SE-320) until reaching a constant mass.
Total organic carbon (TOC) was determined by oxidation using catalytic combustion at 680ºC and infrared detection (Shimadzu TOC-VCSH model).
The total nitrogen content in the medium was determined by the Kjedahl method (VELP DK-20 and UDK-126 D) according to the procedure described by Association of Official Analytical Chemists (AOAC, 2000). Macronutrients (Mg and K) were determined by flame atomic absorption spectrometry -FAAS (Varian Spectra AA-55), according to method described by AOAC (2000).
The viscosity reduction of mango juice after enzymatic treatment with crude extract was evaluated using a Falling-ball viscometer (Abbas, Abdulkarim, Saleh, & Ebrahimian, 2010). Mango juice clarification after enzymatic treatment with crude extract was determined based on color intensity (Chatterjee, Chatterjee, Chatterjee, & Guha, 2004), and it was expressed as clarification (%), taking into account the control juice color intensity (without enzymatic treatment) and the enzyme-treated juice. Turbidity reduction (%) was calculated based on the control juice absorbance (Chatterjee et al., 2004).

Statistical analysis
The results were treated using the Statistic 5.0 software (Statsoft, Tulsa, OK, USA). All analyses were performed considering a 95% confidence level (p < 0.05).

Microorganism identification
According to the method described in the previous section, the newly isolated microorganism was identified as Penicillium brasilianum with a 100% identification rate as a result of BLAST. This result was enhanced by a dendrogram (not shown) generated by the neighbor joining method, using the Jukes-Cantor model as distance measurement and 1,000 bootstrap replicates, where the fungal isolate was compared to NCBI sequences.

Pectinases bio-production
The Plackett-Burman design results are seen in Table 1. It was noted that the pectin concentration presented a positive significant effect (p < 0.05) under the polygalacturonase activity, within the studied range ( Figure 1). L-asparagine, potassium phosphate, and iron (II) variables presented a significant negative effect (p < 0.05). Therefore, these variables on level -1 were excluded from the fermentation process as the concentration was null. However, the magnesium sulphate and yeast extract variables showed a significant negative effect (p < 0.05) on level -1, the concentration used was zero and these variables was excluded from the process.
After analysing the first design, a second one was performed. Table 2 presents the coded and real values for the complete 2³ factorial design and the responses in terms of polygalacturonase activity and pH. The maximum enzyme activity was obtained from assay 1.

Kinetic evaluation
For substrates consumption (Figure 3b) it was observed a TOC decrease in the first 6 hours of fermentation (from 152.6 to 134 mg L -1 ). After this period the concentration remained constant until reaching 24 hours. A TOC concentration reduction after this time could be observed and it was noted that the polygalacturonase activity was at its maximum at 48 hours with the TOC concentration of 88 mg L -1 (42% TOC consumption).
The potassium content gradually decreased in the first 27 hours of fermentation (from 453 to 385 mg L -1 ). After this period, a 78% reduction at 60 hours could be observed. Magnesium consumption presented similar behavior as it reduced by 10 in the first 30 hours. The highest polygalacturonase activity was achieved at 23% magnesium consumption after 48 hours evaluation.
In this work, pH values of all assays were monitored and it was noted that the behavior was not associated with the polygalacturonase production. A slight pH reduction could be observed (from 5.5 to 4.5) in all assays, as seen in Table 1 and 2. According to Cordeiro & Martins (2009), this pH reduction could be due to the glucoronic acid release into the medium caused by pecnolytic enzymes action. Such enzymes are produced by the microorganisms during the first hours of fermentation.

Crude enzymatic extracts partial characterization
The stability of the crude enzymatic extracts obtained by P. brasilianum fermentations in relation to temperature (Figure 4) was verified in the 25 to 55ºC range at a fixed 5.5 pH value.
The pH stability ( Figure 5) was assessed in the 4.0 to 9 range using 100 µmoL L -1 sodium phosphate buffer at 37ºC. The PG produced from P. brasilianum presented higher stability at pH 4.0 to 5.0 and Acta Scientiarum. Technology, v. 42, e48042, 2020 55ºC. Tari, Dogan, & Gogus (2008), when investigating the effect of pH on stability, found that polygalacturonase from A. soybean was quite stable at pH 5.0 and retained 60 and 70% of its activity at pHs 3.0 and 7.0, respectively. Kant, Vohra, & Gupta (2013) studied the stability of purified polygalacturonase from A. niger MTCC 3323 and found enzymatic stability from pH 4.0 to 5.5 for 1 hours. Gomes et al. (2011) evaluated the stability of PG produced from A. niger using a complex medium (32 g L -1 pectin, 2 g L -1 L-asparagine, 0.06 g L -1 potassium phosphate and 1.0 g L -1 iron sulfate) and observed that at pH 5.0 60% of the PG initial activity during 150 hours of storage was maintained. Silva et al. (2007) evaluated pectinolytic enzymes from Penicillium viridicatum RFC3 and obtained the maximum activity at pH 6.0 and 60ºC. Table 3 shows a turbidity reduction and a clarification increase of commercial mango juice treated with the crude enzymatic extract. It shows a 12% reduction in turbidity and a 23% increase in clarification, obtained using a 0.5% enzymatic concentration (v v -1 ) at 40ºC, 100 rpm for 60 min.  Acta Scientiarum. Technology, v. 42, e48042, 2020 Table 3. Viscosity, turbidity, and clarification reduction of commercial mango juice treated with pectinolytic crude enzymatic extract.
This effect was also observed by Chatterjee et al. (2004), and might is possibly be related to the presence of other pectinases in crude enzymatic extracts, such as pectin methylesterase (PME) with 6.0 U mL -1 activity and pectin lyase (PMGL) with 6.61 U mL -1 activity. Thus, the results could have been influenced by these and other enzymes whose activities have not been quantified in the study. Clemente and Pastore (1998) and Vámos-Vigyázó (1981), using a commercial enzyme (Pectinex), which showed cellulases activity, verified a better performance of the clarification process for peach juice compared to the isolated enzymes. Poletto, Renosto, Baldasso, Zeni, and Silveira (2015), in the clarification of blackberry juices, verified a reduction in viscosity and turbidity of 40 and 50%, respectively. According to Echavarría, Torras, Pagán, and Ibarz (2011), the reduction of these parameters is paramount to ensure juice stability during storage.

Conclusion
The crude enzyme-complex as well as the pectinolytic activity of polygalacturonase (52.8 U mL -1 ) showed pectin methylesterase (6 U mL -1 ) and pectin lyase (6.6 U mL -1 ) activities. The crude enzymatic complex with a 0.5% concentration (v v -1 ) used for commercial mango juice clarification reduced the viscosity by 10%, turbidity by 12% and clarification by 23%. In this way, the results presented.