1.0 Introduction
Plant cell walls can have up to three different layers; primary cell wall, secondary cell wall and middle lamella. The middle lamella which is rich in the polysaccharide pectin forms the outermost layer of the cell wall that separates the cell from its adjacent cells. The main composition of pectin is galacturonic acid residues and the enzymes associated with this polysaccharide is known as pectinolytic enzymes. Pectinolytic enzymes can be categorized based on their approach in degrading the pectin. Currently, there are three types of pectinolytic enzymes; pectin methylesterase, polygalacturonase and pectin lyase. Polygalacturonase degrades the pectin by releasing oligomers of D-galacturonic acid through hydrolyzation of a specific type of bond between galacturonic acid residues namely the α-1, 4-glycosidic bond. There are many biotechnological applications of polygalacturonase especially in the food industry such as in extraction and clarification of fruit juices, vegetable oils, wines, fermentation of tea and coffee and as poultry feed additive. Nevertheless, the utilization of this enzyme also expands beyond the food industry such as to be used in treating waste water and removal of gum from plant fibers so they can be used in textile production such as the removal of gum (sericin) from silk. Therefore, this written work will be focusing on the isolation, purification and characterization of polygalacturonase only as summarized from Purification and Characterization of Polygalacturonase Produced by Aspergillus niger AN07 in Solid State Fermentation by Patidar, Nighojkar, Nighojkar and Kumar (2017).
2.0 Isolation of Aspergillus niger and Extraction of Polygalacturonase
Hosts are required to produce the desired enzyme polygalacturonase. In the study, soil samples from fruit processing sites in India were collected and a strain capable of to be utilized in polygalacturonase production was identified. The identification of fungal strain A. niger AN07 according to its morphology, microscopic observation and molecular descriptions was prior to screening on medium containing pectin, potato dextrose agar (PDA). The identified fungal strain was then incubated for 4 days at 30⁰C to be used for inoculation in solid state fermentation (SSF). SSF produced a significantly higher polygalacturonase formation than when fermented in submerged fermentation (SMF), hence are chosen as reported by Padma, Anuradha, Nagaraju, Kumar and Reddy (2012). SSF utilized solid substrate to cultivate the microorganisms in low moisture condition. In this study, the author narrated that a 2:1 ratio of dried papaya peel to orange peel was used as the substrate to grow the 1 ml spore suspension of polygalacturonase-producing A. niger innoculum. This utilization of agro-industrial waste was meaningful and could reduce the cost of polygalacturonase production since 30 to 40 percent of production cost was affected by the high substrate cost (Jahan, Shahid, Aman, Mujahid and Qader, 2017). Consequently, the increasing demand of pectinase can be catered while decreasing the environmental issues that were caused by the accumulation of these agro-industrial wastes.
Polygalacturonase is an intracellular product meaning that the enzyme is produced within the A. niger cells. In order to extract the enzyme, the cells were lysed physically through the subsequent process of homogenization. Firstly, chilled pestle and mortar was used to break open the cells before resorted to orbital shaker (100 rpm) and centrifuged in cold condition at the speed of 10 000 x g. Both shaking and centrifugation was held for 30 minutes each. The collected supernatant was regarded as the extracted crude polygalacturonase.
3.0 Enzyme Assay
The amount of sugar reduced in SSF was directly proportional to the degradation of pectin by polygalacturonase liberating galacturonic acids. Hence, this study utilized the Nelson and Somogyi method to measure the amount of reduced sugar by using galacturonic acid to plot a standard curve. 10 µl of the enzyme was diluted in 990 µl and 1000 µl in a solution of 0.1M acetate buffer (pH5) and 1000µl of polygalacturonic acid (0.1% w/v) and the reaction mixture was incubated for 20 minutes at 55⁰C. The liberation of one µmol of galacturonic acid per minute catalysed by one unit (U) of enzyme was regarded as the enzyme activity (unit per gram dry substrate or U/gds). In simple words, the Nelson and Somogyi method was used to measure the enzyme activity of polygalacturonase.
Meanwhile, Lowry method has been used to measure the total concentration of soluble protein by utilizing BSA standard. The principle of Lowry assay depends on two chemical reactions. In the first reaction, a complex was formed between reduced copper ions and the peptide bonds of proteins. Next, the second reaction involved the formation of copper-peptide bond complex that caused the reduction of Folin-Ciocalteu reagent resulting in the change of solution colour to blue which the maximum absorbance can be observed in the range of 650 nm to 750 nm (Johnson, 2017).
4.0 Enzyme Purification
Protein purification steps are important to be able to characterize the function, structure and interactions with the protein of interest. The proteins may be of similar properties, hence the challenge is to be able to separate one protein from another. Many purification steps can be utilized to separate the proteins based on physical and chemical properties such as solubility, hydrophobicity, binding interaction, isoelectric point, size and charged surface residues (Berg, Tymoczko, & Stryer, 2002). These purification steps can be classified into three main stages; preliminary concentration step, intermediate step and polishing step. In this study, ammonium sulfate precipitation and desalting by using Sephadex G-25 column were utilized in the attempt to purify the protein preliminarily. In the next intermediate step, anion exchange chromatography on DEAE-cellulose was performed and in the final polishing step, the enzyme was purified by using gel filtration by using Sephadex G-200.
4.1 Preliminary concentration steps
Subsequent addition of ammonium salt was used by the study to achieve three levels of salt concentration by referring to the ammonium sulfate nomogram and measured volume of supernatant prior to centrifugation at 10 000 x for 30 minutes between each saturation. The three saturations are 0 to 30% saturation, 30 to 60% saturation and 60 to 90% saturation. 75 mM sodium phosphate buffer calibrated to pH 7 was used to resuspend the pellets collected after each centrifugation. Then, the dissolved pellets were tested for polygalacturonase activity. The known 60 to 90% fraction centrifuged enzyme was passed through Sephadex G-25 column for removal of salt from the solution.
4.2 Intermediate step
In this step, anion exchange chromatography was performed by loading the desalted solution of enzyme onto a DEAE cellulose column. The column was equilibrated with a pH 7 sodium phosphate buffer and the same buffer was used to prepare a 200 ml sodium chloride with linear gradient concentration between 0 to 1 M to be used for enzyme elution. Since anion exchange chromatography is considered as an absorption chromatography, the enzyme will bind to the stationary phase (in this case the DEAE resin). Therefore, before starting the gradient, two bed volumes of buffer were used to collect the unbound proteins that eluted out. These unbound proteins showed no activity of polygalacturonase while at 0.4M NaCl concentration, the enzyme was eluted out and showed a major peak of polygalacturonase activity. Reverse dialysis by using a nitrocellulose membrane against 200 g solid sucrose was then utilized to pool and concentrate the fractions (~20 ml) that have polygalacturonase activity. The elution profile of polygalacturonase enzyme from anion exchange chromatography performed as shown as follows;
Figure 1: Polygalacturonase elution profile from DEAE-cellulose anion exchange chromatography. Elution of enzyme was performed in 75 mM phosphate buffer (pH 7) using a 0 to 1 M NaCl linear gradient (Patidar, et al., 2017).
4.3 Polishing Step
After incubation overnight in the reverse dialysis step, the enzyme volume has been reduced from approximately 20 ml to only 2 ml. Sephadex G-200 column was then used in gel filtration chromatography to further purify the concentrated enzyme. When observing the enzyme activity, a single peak of enzyme activity was observed in this chromatography (Figure 2).
Figure 2: Elution profile of polygalacturonase from gel filtration.
Table 1: Purification table of polygalacturonase from A. niger (Patidar et al., 2017).
The summary of all the purification steps of polygalacturonase including its observed enzyme units, total protein (mg), specific activity, fold purification and yield is tabulated in Table 1. Based on the table, the fold purification of this enzyme extracted from A.niger AN07 is 24.8 which is very high when compared to previous reports of polygalacturonase isolated from other fungi. For example, the isolation of polygalacturonase from different strain of A. niger CFR 305 utilizing activated charcoal as the substrate only has the fold purification of 6.5 (Murthy & Naidu, 2011). Since the fold purification is calculated by dividing the specific activity of purified enzyme to the specific activity of crude enzyme, high fold purification indicates the high specific activity of polygalacturonase purified in this study than the previous study.
5.0 Enzyme Characterization
Many methods are used to characterize the enzyme polygalacturonase isolated from A.niger AN07. Among the methods listed in this study are native PAGE and zymogram study, SDS-PAGE, manipulation of substrate concentrations, manipulation of pH, manipulation of temperature, manipulation of incubation time to test for enzyme stability and thin layer chromatography. The native-PAGE method was employed to determine the homogeneity of the purified enzyme by replicating the electrophoresis twice through loading of purified enzyme in a well of each half of the gel. Two different staining methods were used; staining by cetyl trimethylammonium bromide (CTAB) in the first half of gel and staining by Coomassie brilliant blue in the second half. The staining by using CTAB revealed a transparent band on an opaque background indicating polygalacturonase activity while blue protein bands were observed by the later stain. Both bands however still showed enzyme molecular mass estimation to be 64.5 (±1.6) kDa which is similar to the molecular mass figured out through SDS-PAGE study though these two methods (native-PAGE and SDS-PAGE) employed separation based on two different factors. Native-PAGE essentially separates proteins based on their mass to charge ratio while SDS-PAGE only separated protein based on size (Nowakowski, Wobig and Petering, 2014).
Figure 3: (A) Native PAGE analysis. Lane 1, Lane 2, Lane 3 and Lane 4 were identified as molecular weight markers, crude enzyme, enzyme purified by ion exchange chromatography and purified enzyme respectively and (B) Purified enzyme zymogram (Patidar et al., 2017).
Figure 4: SDS-PAGE of purified enzyme. Lane 1, Lane 2 and Lane 3 were identified as Bio-Rad molecular weight markers, crude enzyme and purified enzyme respectively (Patidar et al., 2017)
Next, the polygalacturonic acid substrate concentration (prepared in 0.1 M sodium buffer acetate buffer) ranging from 1 to 10 mg/l were used in assay to estimate the Km and Vmax of values through Lineweaver-Burk plot plotted. Based on the low Km value of 2.6 mg/l and high Vmax value of 181.8 µmol/ml/min, it was revealed that the enzyme polygalacturonase has a high affinity towards the substrate.
Based on the manipulation of pH and temperature in this study, the optimum pH of polygalacturonase was found to be at pH 5.0 while the optimum temperature was at 55⁰C. The stability of enzyme was of wide pH range (pH 4 – pH 7). This was determined because following pre-incubation in pH 4 – pH 7 for 1 hour, 100 percent of enzyme activity was retained. Apart from having wide pH range, polygalacturonase from this strain is also stable at high temperature of 55⁰C when exposed to for 1 hour but the enzyme activity decreased progressively when further increase in temperature was applied.
Figure 5: (A) Polygalacturonase activity affected by pH. Maximum activity observed is at pH 5. (B) Polygalacturonase affected by pH stability showing 100 percent stability at pH 4 to pH 7 (Patidar, et al., 2017).
Figure 6: (A) Polygalacturonase activity affected by temperature (optimum activity at 55⁰C) (B) Polygalacturonase activity affected by thermal stability (pH stability up to 55⁰C over duration of 1 h) (Patidar et al., 2017).
Polygalacturonase can be subdivided into two; exo-polygalacturonase and endo-polygalacturonase. These two subdivisions of enzyme essentially differ in their means of degrading pectin through hydrolyzation. Endo-polygalacturonase hydrolyzes the pectin network randomly and produce oligosaccharide as its product. By using exo-polygalacturonase, non-reducing end of polymer was hydrolysed forming monosaccharide as the product. Thin layer chromatography was used in this study for assessment of product released by the enzyme action on polygalacturonic acid. The degradation of polygalacturonic acid into oligosaccharides was shown by the results of thin layer chromatography (Figure 7). This result placed confidence to this study that the enzyme isolated was the endo-polygalacturonase.
Figure 7: Pattern of product formation catalyzed by endo-polygalacturonase on silica gel by employing thin layer chromatography at 55⁰C based on different time of incubation. Lane 1 to Lane 6 was control (only polygalacturonic acid), 0 min, 10 min, 20 min, 40 min and conrol 2 (only galacturonic acid) respectively.
6.0 Conclusion
In conclusion, the best polygalacturonase has been continually produced from many bacteria and fungi in order to address the high cost of production and low yield of enzyme. In this study, the cost of production can be significantly decreased by the utilization of agro-industrial waste such as papaya and orange peels as the substrates instead of the utilization of more expensive substrate such as activated charcoal that has been mentioned in previous literature. The polygalacturonase enzyme isolated from A. niger strain AN07 also showed a high yield with 52.6 percent recovery with high specific activity of enzyme indicated by a whooping 24.8 fold purification through purification done with anion exchange chromatography and gel filtration chromatography. Preliminarily, the enzyme was purified through ammonium sulfate precipitation and desalting by using Sephadex G-25 column. The enzyme weighed 64.5 kDa as determined by native-PAGE and SDS-PAGE. The manipulation of substrate enabled the determination of Km (2.6 mg/L) and Vmax (181.8 µlmol/ml/min) that characterized the high affinity of polygalacturonase to the polygalacturonic acid substrate. Conducting enzyme assay at different range of temperature and pH over a period of time (1 hour) allowed the determination of optimum pH and temperature that were 5 and 55⁰C respectively and determination of enzyme stability that was stable between pH range of 4 to 7 and high temperature of 55⁰C. Finally, the identification of product formed through degradation of polygalacturonic acid can be confirmed through thin layer chromatography which in this case polygalacturonase from A.niger strain AN07 was identified as endo-polygalacturonase due to oligosaccharides formed as visualized on silica gel.
2017-11-12-1510520320