Preliminary investigations on a polygalacturonase from Aspergillus fumigatus in Chinese Pu’er tea fermentation
- Shihui Wang1,
- Zhongshuai Lian†1,
- Liuyang Wang1,
- Xiao Yang1 and
- Yun Liu†1Email authorView ORCID ID profile
© Wang et al. 2015
Received: 26 March 2015
Accepted: 30 June 2015
Published: 21 July 2015
Polygalacturonase is one kind of pectinases which hydrolyze the alpha-1,4 glycosidic bond between galacturonic acid residue. Polygalacturonase has been widely used in the fields of food, biofuel, and textile industries, in which thermostable polygalacturonase is often demanded at high temperatures of 50–60 °C. Herein, we reported a thermostable polygalacturonase producing from Aspergillus fumigatus isolated from the pile fermentation of Pu’er tea in China.
The thermophilic polygalacturonase-producing strain was identified as A. fumigatus L45 on basis of its morphology, physicochemical properties, and 18S rDNA analysis. The crucial fermentation parameters affecting polygalacturonase activity were optimized by response surface methodology (RSM); the optimum fermentation parameters were the following: inoculums concentration of 0.07 % (v/v), fermentation time of 36 h, pH of 5.0, and temperature of 45 °C. Under the optimized conditions, the highest polygalacturonase activity of 359.1 ± 10.1 U/mL was obtained. The polygalacturonase showed good thermostability and pH stability. The enzyme was activated by metal ions Zn2+ and Mg2+, but inhibited by K+. However, Na+ and Ca2+ showed little effects on its activity. K m and V max values were estimated to be 35.0 mg/mL and 7.69 μmol/mL/min, respectively.
A polygalacturonase from A. fumigatus L45 was preliminarily investigated, the crucial fermentation parameters were optimized by RSM, and the properties of polygalacturonase was examined. The polygalacturonase showed good thermostability and pH stability, which suggested the enzyme has potential applications in the biofuel and textile industries.
KeywordsPolygalacturonase Aspergillus fumigatus Identification Response surface methodology (RSM) Enzymatic properties
Pectinases, also called pectinolytic enzymes, are a group of enzymes hydrolyzing pectin substrates in plant cell . Pectinases are mainly composed of polygalacturonase (E.C.184.108.40.206), pectin lyase (E.C.220.127.116.11), pectate lyase (E.C.18.104.22.168), and pectinesterase (E.C.22.214.171.124) . The main function of pectinases is to destroy plant tissue and cell wall structure to allow other depolymerases accession to enhance the productivity . Nowadays, pectinases have been widely used in the fields of food, oils, flavors and pigments extraction, cellulose fibers preparation, coffee and tea fermentations, and oligogalacturonides production . So, it is crucial to screen novel pectinases with remarkable characteristics from native environments [5, 6]. Pectinases are generally produced from bacteria, fungi, and plants; however, most of the commercial pectinases are from fungal sources , which optimal pH was between 3.0 and 5.5 . Aspergillus species, especially Aspergillus niger, is most commonly used for commercial pectinases production [8–10], followed by Penicillium species [11–13]. Pectinases from bacteria are mainly alkaline with the optimal pH over 7.0 and mostly used in pulp and paper processing [14–16]. Apart from Bacillus species, little information on pectinases from bacteria has been available to date.
In literature, the optimum temperature of pectinases was mostly within the ranges of 30–50 °C. For instance, pectinases from A. niger reported by Kittur et al. showed the maximal activity at 47 °C . Singh and Appu Rao  claimed that two endo-polygalacturonases from A. niger could achieve their maximal activities at 43 and 45 °C. A polygalacturonase from Saccharomyces cerevisiae isolated from marsh grape fruit pulp possessed the maximal activity at 45 °C . As well known, high temperatures of 50–60 °C are potentially useful for application in the fabric industry to separate cotton fabrics and polish enzymes, in the paper industry to remedy the retention problems in mechanical pulp bleaching, and in the food industry to clarify juice [20, 21]. Therefore, thermostable pectinases are highly demanded in these modern industries.
Among the aforementioned four pectinases, polygalacturonase is the most commonly used in food industries. So many researchers have been focusing on the isolation and characterization of thermostable polygalacturonases. For instance, a polygalacturonase from thermophilic fungus Thermoascus aurantiacus had a high thermostability at 60 °C . The polygalacturonase from Penicillium SPC-F 20 isolated from citrus fruit was found to show the highest activity at 60 °C . Maller et al.  revealed that the polygalacturonase from Aspergillus niveus showed maximum activity at 55 °C and pH 4.0, and the enzyme was stable at 60 °C for 90 min. Martins et al.  found that the polygalacturonase from T. aurantiacus achieved its highest activity at 60–65 °C and pH 4.5–5.5 and was stable at 50 °C for 1 h.
In this work, a novel thermostable polygalacturonase produced from the microorganism in the pile fermentation of Pu’er tea in Yunnan province, China, is reported for the first time. The microorganism was then identified on basis of its morphology, physicochemical properties, and 18S rDNA analysis. After that, the crucial fermentation parameters affecting polygalacturonase activity were optimized by response surface methodology (RSM). Finally, the enzymatic characterization of the thermostable polygalacturonase was investigated. It is hoped that the findings in this work could facilitate the application of polygalacturonase in biofuel and textile industries.
Pu’er tea was gifted from Yunnan Pu’er Tea (Group) Co. (Yunnan province, China). The reason of screening the thermophilic strain from Pu’er tea fermentation pile was the fact that the temperature of the pile fermentation of Pu’er tea could always reach more than 60 °C. The strain was isolated, purified, and identified in our lab and kept in −80 °C fridge. Pectin from citrus peel (galacturonic acid ≥74.0 % (dried basis)) with an item number of P9135 was brought from Sigma-Aldrich (Beijing, China). D-(+)-galacturonic acid was also purchased from Sigma-Aldrich (Beijing, China). DNeasy plant Mini Kit with a production number of 69104 was bought from Qiagen Co. (Beijing, China). TaKaRa Ex Taq DRR001A, 10× Ex Taq Buffer DRR001A, DL2000 DNA Marker SN125-1, dNTPs, and primers were from Takara Co., Ltd (Dalian, China). All other reagents were of analytical grade and bought from local market in Beijing, China.
Compositions of Luria-Bertani (LB) medium are the following: peptone 1 g, yeast extract 0.5 g, NaCl 1 g, agar 1.5 g, and deionized water 100 mL. Compositions of yeast extract peptone dextrose (YEPD) medium are the following: peptone 2 g, yeast extract 1 g, glucose 2 g, agar 1.5 g, and deionized water 100 mL. Compositions of Congo red medium are the following: pectin 0.1 g, sucrose 2 g, MgSO4 0.05 g, KCl 0.05 g, Fe2(SO4)3 1 mg, K2HPO4 0.1 g, NaNO3 0.3 g, agar 1.5 g, Congo red 0.02 g, and deionized water 100 mL. Compositions of submerge medium are the following: KCl 0.05 g, MgSO4 0.1 g, NaNO3 0.1 g, KH2PO4 0.1 g, FeSO4 0.01 g, yeast extract 0.1 g, peptone 0.1 g, sucrose 1.0 g, and deionized water 100 mL. The pH of the medium was adjusted to 5.0 with 1.0 N HCl.
Isolation and identification of thermophilic strain
Isolation of thermophilic strain
Sample preparation of 20 kg of Pu’er tea was fermented for 43 days as optimized previously. The pile of the tea was artificially divided into upper (1.0–10.0 cm), middle (10.0–30.0 cm), and lower (>30.0 cm) layers. The middle layer, with the temperature of 55–65 °C and pH of 4.5–6, was most suitable for the screening of thermophilic strains. So, in this layer, four monitoring points were set for sample collection. Every 3 days, 10 g of tea samples were obtained from each monitoring point at eight o’clock in the morning. Each tea sample was added with 90 mL normal saline and homogenized for 3 min. The homogenate was used for primary screening.
Primary screening: The homogenate was divided into two parts. Each part was respectively incubated in LB and YEPD media at 45 °C for 2 days. After cultivation and crossed purification, the strains were stored in the tube culture at 4 °C. The crossed purification on the solid medium was processed until the strains with the same morphology were examined by microscope. Then the strains were numbered and incubated on Congo red medium using pectin as the solo carbon source. The diameters of the pectin degradation halos (D p) and the potency index (defined as the ratio of D p to the diameters of the colonies (D c)), D p/D c) were calculated and used as the standard to screen the pectinase-producing strain.
Secondary screening: After primary screening test, seven microbes with higher potency indexes and D p values were further tested by submerged fermentation at 45 °C. At regular intervals (12, 24, and 36 h), aliquots of fermentation broth were withdrawn and centrifuged at 12,000 rpm and 4 °C. The supernatant was served as the crude pectinase. The pectinase activity was assayed from the aspect of polygalacturonase activity, i.e., the enzyme activity was determined by measuring the release of reducing groups using the method of 3,5-dinitrosalicylic acid (DNS) . 0.2 mL of crude enzyme solution was mixed with 0.8 mL of 1 % (w/v) pectin and incubated at 50 °C for 10 min following by adding 1 mL DNS reagent. The mixture was heated in boiling water bath for 5 min. After cooling, the solution was diluted to 10 mL and centrifuged at 6000 rpm for 10 min. The supernatant was collected to determine enzyme activity by measuring absorbance at 540 nm. One “polygalacturonase unit” was defined as the amount of the enzyme in 1 mL fermentation broth that hydrolyzed 1 μg reducing sugar (herein, it is galacturonic acid) per minute under standard assay conditions.
Morphology and culture characters of the strain
The morphological and cultural characteristics of the strain were observed by naked eyes after incubated on LB, YEPD, and Congo red media for 24 h. The spores of the strain were examined by SU1510 Hitachi scanning electron microscope (SEM) (Hitachi City, Japan). Before observation, the strain cell was first fixed by glutaraldehyde (2.5 %, pH 7.4) and osmic acid solution (1 %) and then dehydrated by ethanol followed by being dried in a vacuum freeze dryer for 12 h. The specimen was sputter-coated with gold in an ion coater for 2 min.
Physicochemical properties of the strain
A total of 15 tests were conducted for physiological characterization experiments, including 9 types of carbon sources and 6 types of nitrogen sources utilization in submerged fermentation medium. The tested carbon sources were sucrose, pectin, fructose, glucose, maltose, lactose, galactose, and soluble starch. The tested nitrogen sources were beef extract, peptone, NaNO3, (NH4)2SO4, urea, and yeast extract. The culture stability of the strain growth on submerged fermentation medium was also examined at 45 °C for 48 h.
18S rDNA identification and phylogenetic analysis of the strain
The methods of 3,5-dinitrosalicylic acid (DNA) isolation, 18S rDNA amplification, and phylogenetic analysis of the strain were in accordance with the methods described by Lin and Liu . The nucleotide sequence coding for the 18S rDNA gene of strain (607 bp) was deposited in the GenBank database with accession number of KJ818296. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5.0 software.
Optimization of fermentation parameters using RSM
where Y represented polygalacturonase activity (dependent variable); β 0 was the constant coefficient; β i , β ii , and β ij were the coefficients for the linear, quadratic, and interaction terms, respectively; and X i and X j represented the independent variables. Subscript i and j were the numbers 1 to 3.
Coefficient of determination (R 2) and analysis of variance (ANOVA) were used to evaluate the fit of the second-order polynomial equation (Eq. 1). The contour plots and three-dimensional response surfaces were attained by holding one of the independent variances constant while changing the level of other variables.
Enzymatic characterization of the polygalacturonase
The purification of polygalacturonase
The polygalacturonase-producing strain A. fumigatus was incubated in submerged medium for 36 h. The fermentation broth was centrifuged at 12,000 rpm and 4 °C for 10 min. The supernatant was filtered with a polyethersulfone membrane (0.22 μm) followed by being dialyzed with a dialysis bag (molecular weight cut off, 1 kDa) at 4 °C for 24 h. Then the solution was transferred into another dialysis bag with a molecular weight cut off of 25 kDa to be concentrated by 10 % (m/v) polyethylene glycol (PEG) at 4 °C for 24 h. Mixture solutions of trichloroacetic acid/acetone (v/v, 1:4) were put into the concentrated enzyme solution to precipitate the enzyme at −20 °C for 12 h. The volume ratio of enzyme solution to mixture organic solvents was 1:4. After precipitation, the enzyme solution was centrifuged at 12,000 rpm for 10 min. The protein pellet was obtained and washed by 100 % acetone for three times and dried as the crude enzyme for the following experimental use. The enzyme protein content was monitored by Bradford protein assay method using Bradford reagent from BioRad at a wavelength of 595 nm with a standard calibration curve of BSA. The purity and molecular weight of polygalacturonase were measured by SDS-PAGE .
Effects of pH, temperature, and metal ions on polygalacturonase activity
Besides, the pH stability and thermostability of polygalacturonase were also evaluated. For pH stability, the enzyme was incubated at a certain pH (pH 2–8) for 24 h before activity measurement. In view of thermostability, the enzyme was incubated at a certain temperature (20–80 °C) for 2 h before activity measurement.
Determination of K m and V max values of enzyme kinetics
Therefore, the values of K m and V max can be obtained from the intercept and slope of the 1/v vs. 1/[S] plot . Herein, the substrate (pectin) concentrations used were in the range of 0.2 to 1.8 % (w/v). Experiments were conducted at pH 5 and 45 °C.
Results and discussion
Isolation and identification of thermophilic strains
Isolation of thermophilic polygalacturonase-producing strains
Primary selection of polygalacturonase-producing isolates on Congo red medium
Potency index (D p/D c)
Number of isolates
D p/D c > 3.5
3.5 > D p/D c > 2.0
2.0 > D p/D c
D p/D c = 0
Identification of the strain L45
Fermentation parameters optimization by RSM
RSM design and experimental results
Inoculums concentration (%)
Polygalacturonase activity (U/mL)
The main effective analysis of variance (ANOVA) of RSM data
Sum of squares
Lack of fit
In order to verify the reliability of the derived model (Eq. 5), experiments were carried out in triplicate under optimized conditions. The results showed that the practical polygalacturonase activity of 359.1 ± 10.1 U/mL was in highly agreement with the predicted value of 361.3 U/mL. As a result, the validity of the regression model was satisfactory.
In literature, many studies referred to the optimal fermentation parameters for the production of polygalacturonase. For instance, a polygalacturonase produced from A. fumigatus Fres. MTCC 4163 showed maximum activity of 1270 U/g when fermented at 50 °C and pH 4.0–5.0 for 2–3 days . A polygalacturonase activity was the highest (ca. 2 U/mL) when the strain T. aurantiacus was cultivated at 45 °C and pH 5.5 for 5 days in submerged fermentation with pectin as the substrate . Penicillium SPC-F 20 produced the maximum polygalacturonase activity of ca. 1.8 U/mL when the fungus was cultivated at 30 °C and pH 5.0 for 3 days with pectin as the substrate . A. niger DMF 27 produced the maximum polygalacturonase activity (3.8 U/mL for endo-polygalacturonase and 11.1 U/mL for exo-polygalacturonase) when fermented at 30 °C and pH 5.0 for 72 h using pectin as the substrate . A. foetidus var. pallidus Ege-K-730 produced the highest polygalacturonase activity of 122 U/mL when incubated at pH 2.3 for 72 h . By comparison, these results with ours (maximum activity of 359.1 U/mL at 45 °C and pH 5 for 36 h), it is obviously seen that the optimal fermentation temperature and pH in this study were moderate, while the fermentation time was much shorter than that in literature. Besides, since the activity could be greatly affected by different source of substrates, the polygalacturonase activities in literature could just be roughly compared. In summary, A. fumigatus L45 would be a promising fungus to produce thermostable polygalacturonase in the future.
Enzymatic properties of the polygalacturonase
pH effect and pH stability
Comparisons of enzymology properties of polygalacturonase reported in the literatures
Optimal temperature (°C)
K m (mg/mL)
Mostly inactive at 65 °C,
Aspergillus fumigatus strain 4
Stable between 3.0 and 9.0 and at 4–6 °C;
activity reduce by 24 % at 26–30 °C
T m = 43 °C
T m = 46 °C
Penicillium viridicatum Rfc3
Stable in neutral pH range and at 40 ºC for 1 h
Thermoascus aurantiacus 179-5
Stable in the acidic to neutral pH range at 60 °C for 1 h
Penicillium SPC-F 20
A residual activity of 48 % after 2 h of incubation at 60 °C
Rhizopus microsporus var. rhizopodiformis
Activity decreased by 28 and 39 at 60 and 65 °C, respectively, for 90-min incubation
Stable in pH 3.0–4.5 at 55 °C for 1 h
Stable up to 50 °C for 120 min at pH 4.0–5.0;
decreased rapidly above 60 °C and above pH 5.0
Penicillium oxalicum SX6
Stable at pH 3.5–6.0 and 40 °C
Stable at pH 2.0–7.0 and 60 °C for 2 h
In our work
Temperature effect and thermostability
Experiments were conducted to investigate the effect of temperature on polygalacturonase activity and its thermostability, and the results were shown in Fig. 7b. It was revealed that the optimum temperature for polygalacturonase activity was 60 °C. Even at 80 °C, the polygalacturonase could still keep 63 % catalytic activity. The thermostability of the polygalacturonase showed that the residual activities of 100, 80, and 54 % were obtained at 50, 60, and 80 °C for 2 h incubation, respectively.
In literature, a polygalacturonase from A. fumigatus strain 4 showed the highest activity at 65 °C. However, the enzyme was only stable at 4–6 °C. When stored at 26–30 °C, its activity was reduced by 24 % . Another pectinase from A. fumigatus showed maximal activity at 40 °C and pH 5.5 and was stable up to 40 °C . A polygalacturonase produced by Penicillium strain presented a residual activity of 48 % after 2 h incubation at 60 °C . A polygalacturonase producing from T. aurantiacus showed a residual activity of 13 % after 1-h incubation at 60 °C . Other related data were shown in Table 4. Through comparison of characters of polygalacturonase reported in literature, it was confirmed that the polygalacturonase from A. fumigatus L45 showed good thermostability.
Effects of metal ions on enzyme activity
The effects of different metal ions on the polygalacturonase activity were evaluated, and the results were shown in Fig. 7c. It can be seen that metal ions Na+ and Ca2+ showed almost noninfluence on the polygalacturonase activity, K+ inhibited the polygalacturonase activity by ca. 25 %, Mg2+ enhanced the polygalacturonase activity by 25 %, and Zn2+ could increase the polygalacturonase activity to 1.80-fold. The activity of a polygalacturonase from A. niger was inhibited by K+, Zn2+, and Ca2+ to 55.83, 43.11, and 73.02 %, respectively, while promoted by Mg2+ by 11.55-fold . The activity of another polygalacturonase from thermophilic fungus T. aurantiacus was inhibited 59, 77, and 100 % by Zn2+, Mn2+, and Hg2+, respectively .
Kinetic parameters (K m and V max)
The enzyme kinetic parameters K m and V max from Michaelis-Menten equation were determined through the Lineweaver-Burk plot (seen in Fig. 7d). V max represents the maximum rate the system can achieved at saturating substrate concentrations. K m is the substrate concentration at which the reaction rate is half of V max; therefore, K m is negatively related to the affinity of the enzyme to the substrate. The K m and V max values of polygalacturonase were 35.0 ± 2.8 mg/mL (or 3.5 ± 0.28 %) and 7.69 ± 0.59 μmol/mL/min, respectively, in this work. Further, we obtained the K cat value of 1.38 min − 1 following the equation of K cat = V max/enzyme concentration. So K cat/K m was 0.04 mg/mL/min.
Kant et al.  demonstrated that the K m and V max values of a purified polygalacturonase from A. niger MTCC 3323 were 0.083 mg/mL and 18.21 μmol/mL/min, respectively, using polygalacturonic acid as a substrate. Rashad et al.  confirmed that K m and V max values of a purified polygalacturonase from Pleurotus ostreatus were 1.33 mg/mL and 28.6 μmol/mL/min, respectively, using pectin as a substrate. Other K m values from references were presented in Table 4. The high K m and lower V max values of our polygalacturonase mainly stemmed from the fact that the enzyme was just primarily purified and had a relatively low purity.
A thermophilic polygalacturonase-producing strain A. fumigatus L45 was screened from the pile fermentation of Pu’er tea in China. The fermentation parameters of the strain were optimized by RSM, and the result showed that the polygalacturonase achieved a maximum activity of 359.1 ± 10.1 U/mL under the optimized conditions (inoculum concentration of 0.07 % (v/v), temperature of 45 °C, and pH 5.0). The optimal temperature and pH for polygalacturonase were 60 °C and pH 5.0. The enzyme maintained 100, 80, and 54 % of its activity after incubated at 50, 60, and 80 °C for 2 h, respectively. The good thermostability and pH stability suggested that the enzyme has potential applications in the biofuel and textile industries. The polygalacturonase gene cloning, protein structural biology, and applications are ongoing in our lab.
analysis of variance
- D c :
the diameter of the colony
- D p :
the diameters of the pectin degradation halos
- R 2 :
coefficient of determination
response surface methodology
scanning electron microscope
yeast extract peptone dextrose
- ΒB design:
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