MATERIALS AND METHODS

Study area: All the experiments in the studies were performed during December, 2021-March, 2022 in the Food Microbiology Laboratory, the Food Chemistry Laboratory, Department of Food Technology and Central Laboratory, Universitas Padjadjaran, Bandung City, Indonesia.

Microorganism preparation: Isolate T. versicolor (L.) Lloyd was obtained from the Indonesian Culture Collection (InaCC) LIPI, Indonesia. Trametes versicolor was maintained at Potato Dextrose Agar (PDA) and stored at 4°C. The culture was grown in a modified submerged fermentation system to produce crude laccase.

Qualitative assay of laccase: Laccase assay was determined using guaiacol. The test was performed by culturing the fungus Trametes versicolor (L.) Lloyd’s in a mixture of PDA agar plates with 0.02% guaiacol. The mycelium was placed at 5 points on the Petri dish. Samples were incubated at 30°C for 3 days. A reddish brown or orange colour around the colony is a positive sign of the laccase enzyme in guaiacol agar¹⁴.

Laccase desalting using column chromatography: Crude laccase enzyme with an amount of 3 mL was poured into the column chromatography contained with Sephadex G-25 matrix¹⁵. The crude laccase enzyme was then eluted using 0.5 M acetate buffer pH 5. The pooled fraction was then tested for protein content and enzyme activity.

Laccase purification using column chromatography: The 3 mL enzyme fraction from the desalting process was poured into the column chromatography contained with Sephadex G-100 matrix¹⁶. The enzyme was eluted using 0.5 M acetate buffer pH 5. The pooled fraction was then tested for protein content, enzyme activity and pH characterization.

Laccase activity assay: Laccase activity was determined spectrophotometrically¹⁷. The mixture of 60 μL acetate buffer pH 5 (0.5 M), 20 μL 2.2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) (1 mM) and 50 μL crude laccase enzyme in microplate was incubated at 30°C for 30 min. The absorbance was measured at 420 nm. Enzyme unit was defined as the laccase activity that oxidized 1 μmol of ABTS per minute.

Protein content determination: Protein content was determined by the Bradford method¹⁸. The bovine serum albumin (BSA) standard curve was performed by performing different concentrations with a mixture of 5 μL sterile aqua dest and 100 μL Bradford reagent as blank. The BSA standard solution concentration variations were 1, 3, 5, 7 and 9 ppm, 5 μL each. About 100 μL of Bradford’s reagent was added to each microplate containing the BSA standard solution, then homogenized for 10 sec and incubated for 10 min. Absorbance was measured by a spectrophotometer at 595 nm. The protein standard curve was generated by plotting the BSA concentration (x) against the absorbance of the standard solution (y) to obtain the linear regression equation:

The 5 μL enzyme fraction was mixed with 100 μL Bradford’s reagent in the microplate. The mixture was then homogenized for 10 sec and incubated for 10 min. The absorbance was measured with a spectrophotometer at 595 nm. The sample protein content was calculated by entering the absorbance value of the sample (y) into the linear regression equation:

so, that the concentration or protein content of the sample (x) can be determined.

Laccase pH characterization: About 40 μL of enzyme purified by column chromatography Sephadex G-100 was mixed with 60 μL of acetate buffer pH 3, 4, 5, 6 and 7, respectively, which had been added with 20 μL ABTS 1 mM. The mixture with a total volume of 120 μL was then homogenized. The absorbance of the mixture was measured at 420 nm for 0 and 30 min at 30°C¹⁹.

RESULTS

Qualitative assay of laccase: A positive indicator of laccase activity in PDA substrate-guaiacol supplemented was demonstrated by the appearance of a reddish brown or orange colour around the colony. The reddish brown colour appearing on PDA was the result of the oxidation of guaiacol by the enzyme laccase. This result indicated that Trametes versicolor (L.) Lloyd can secrete laccase.

Laccase desalting using column chromatography: The crude laccase enzyme sample was then desalted by column chromatography. The gel substrate used is Sephadex G-25 which separates the enzymes from the smaller molecules. The total number of enzyme fractions pooled was 24. The results were shown in Fig. 1, there were 5 parts with the highest protein content, which were parts 8, 9, 14, 15 and 16. The highest absorbance peaks were represented by parts 8 and 14, indicating the existence of laccase in the mentioned sections, with absorbance values of 0.448 and 0.515, respectively. The 5 fractions were then pooled and purified by Sephadex G-100.

Laccase purification using column chromatography: The desalting enzymes obtained from the desalting step were then purified by column chromatography using the Sephadex G-100 substrate.

Fig. 1:

Chromatogram of protein from desalting using Sephadex G-25 matrix

Fig. 2:

Chromatogram of protein from purification using Sephadex G-100 matrix

Fig. 3:

Effect of pH on laccase activity

Sephadex G-100 was used to separate a specific laccase enzyme from another molecule based on the size of the laccase molecule and the pores of the substrate. The total amount of synthetic enzyme was 20 fractions. The results of the laccase purification were shown in Fig. 2, the highest peaks of absorbance were in fractions 14 and 15 with the absorbance value of 0.453 and 0.432, respectively. The high absorbance value in the collected fraction was assumed to be an indicator of the presence of the purified laccase enzyme.

Quantitative laccase assay: Laccase enzyme samples from each purification step were assayed quantitatively by measuring the enzyme’s protein content, enzyme activity and specific activity. The result of the tests can be seen in Table 1, the desalting process using Sephadex G-25 can increase the enzyme activity of the laccase enzyme. The same result can also be observed at the laccase enzyme’s specific activity. The increasing value of specific activity affects the purity of laccase which go up to 2.93 fold.

Laccase pH characterization: Characterization of enzymes against pH is important to obtain the optimum enzyme condition so, that the enzyme can maximize its ability in converting the substrate. The tests were carried out by measuring the enzyme activity under various pH conditions. The results of this test were presented in Fig. 3, laccase enzyme activity at various pH above, the laccase enzyme produced by Trametes versicolor (L.) Lloyd has the highest activity at pH 4. This result showed that the laccase enzyme works optimally at pH 4.

DISCUSSION

Trametes versicolor (L.) Lloyd has shown the reddish-brown colour around the mycelia based on the qualitative assay. This condition indicate that Trametes versicolor (L.) Lloyd can produce laccase enzyme, which the reddish brown colour that emerged on the PDA added with guaiacol was the result of guaiacol oxidation by laccase enzyme. Before the oxidation process occurred, guaiacol has a slightly yellowish colour and then turns brownish after the oxidation has taken place. This result was in line with²⁰, red-brown oxidized zones developed around colonies picked for laccase production. The wider the reddish brown zone indicates the greater concentration of laccase enzyme¹⁴. This laccase enzyme can catalyze the oxidation process of guaiacol which produces a ketone group and H₂O²¹. The enzymatic reaction carried out by laccase is one-electron oxidation. Oxygen is needed as the electron acceptor and then produces water. When oxidation takes place, the substrate will lose one electron and form free phenoxy radicals. These free radicals can do further enzymatic oxidative reactions or non-enzymatic reactions such as hydration and polymerization²². In this case, laccase enzymes convert guaiacol into radical quinine and with the help of oxygen will be converted again into a quinone. The result of that oxidation process initiated the polymerization reaction forming the brown-coloured melanoidin.

Purification of crude laccase enzyme was conducted in 2 steps using the column chromatography method. The basic principle of protein separation with gel filtration column chromatography is the difference in protein molecular weight. The Sephadex gel was used as a stationary phase, while the acetate buffer acted as a mobile phase²³. The first step is the desalting process with Sephadex G-25 gel matrix and then continued to the purification process using Sephadex G-100 gel. The desalting process using Sephadex G-25 involves the removal of salts and smaller molecule size than a protein, in which those molecules will be retained in the gel matrix pores. The bigger molecule size such as proteins will be separated and fractionated first²⁴. Sephadex G-100 was then used to separate protein with a molecular weight of 4-150 kDa²⁵. In this study, laccase was indicated by the highest peak of absorbance in each step of purification. Those obtained samples were then assayed to determine their protein content, enzyme activity and specific activity.

The result of enzyme activity shows that laccase enzymes desalted with Sephadex G-25 have a greater value than the purified ones. The used of Sephadex G-25 increased laccase activity on laccase production from Botrytis cinerea (DSMZ 877)²⁶. The decreased laccase enzyme activity after purification may occur due to the laccase enzyme molecular weight being too small for the molecular weight that was able to pass through the pores of the Sephadex G-100 matrix, which only allow molecules with 4-150 kDa molecular weight. Based on this study, the molecular weight of the laccase enzyme studied does not match the pore size allowed to pass through the Sephadex G-100 matrix. However, the Sephadex G-100 was used for partial laccase purification of Bacillus sp. The AKRC01 with a molecular weight of 61 kDa²⁷. The same method was also carried out for laccase purification from white-rot fungus, Marasmius sp., BBKAV79 using Sephadex G-100¹⁶. An alternative that can be used to solve this problem is to reduce the pore capacity that can be passed by a molecule in the column chromatography system. This can be applied by performing the column chromatography system using Sephadex G-75 which has a molecular fractionation range of 3-80 kDa²⁸. Sephadex G-75 has been used for the purification of laccase from the rice blast fungus, Magnaporthe grisea which resulted in 282-fold purification with a specific enzyme activity of 225.91 U mg^–1 and a yield of 11.92%²⁹. Further investigation of the laccase molecular weight can be determined by the electrophoresis SDS-PAGE method. Thus, it can be said that the laccase enzyme produced by Trametes versicolor (L.) Lloyd used in this study tended to be more effectively purified using the Sephadex G-25 gel matrix.

In this study, laccase purification using Sephadex G-25 increased the specific activity, which was 3.738 U mg^–1 and then increased to 10.966 U mg^–1 with a purity of 2.93 fold after purification. The protein content in the sample decreases along with the purification process. Purification in this experiment was proven by the decreased protein content and increased enzyme purity. The crude laccase applied to the desalting treatment has increased in purification level up to 1.6-2.5 times after precipitation and dialysis³⁰. The same result was also found in research conducted by the use of a gel filtration chromatography purification step increased the enzyme purity to 3.7-fold (28-fold purification) over the cold acetone precipitation obtained above and showed 57% recovery of enzyme activity³¹. The difference in specific activity values obtained compared to the literature can be influenced by the source of the enzyme and the growth media used. The higher the total protein, the lower the specific activity of the sample. This is because there are several protein contaminants besides laccase that inhibit substrate changes in the product. The increased enzyme activity in purified enzymes is due to a decrease in the number of contaminants that prevent the active side of the enzyme from binding to the substrate³².

Laccase in this study has the optimum pH of 4. A low optimum pH can be associated with fungi that adapt to acidic growing conditions. Laccase enzyme produced by fungi is generally at a pH range of 4-6³³. The laccase activity of the ascomycete fungus Nectriella pironii had an optimum pH of 2.0 or 6.0³⁴. Jin et al.³⁵ reported the purified laccases from Trametes versicolor were optimized at a pH range from 5.0-7.0. Enzyme activity is influenced by the degree of acidity (pH) during the enzymatic reaction³⁶. The active side and three-dimensional shape of enzymes can undergo ionic changes caused by variations in pH. The change of structure can reduce the contact between enzyme and substrate so that it will affect the enzyme activity³⁷. When the enzyme is at the optimum pH condition, the proton donor and acceptor groups in the catalytic site of the enzyme are at the desired ionization level so that the conformation of the active site matches the substrate. This condition will stimulate the formation of a maximum complex between the enzyme and the substrate so that the enzyme will be effective in producing products³⁸. Overall, the results of this study showed the potential for partially purified white rot fungus termites versicolor to increase the specific activity with 2.9 times higher purity. So, it can be considered for the production of laccase on a larger scale.

CONCLUSION

This research has obtained the specific activity and the increased purity value of laccase enzyme produced from Trametes versicolor (L.) Lloyd can be effectively purified using Sephadex G-25, with the specific activity of purified laccase was 10.966 U mg^–1 with the purity of 2.93 fold. Laccase enzyme has the optimum pH of 4. This study will inform researchers or industries about a pH condition that is suitable with the laccase enzyme activity which can maximize its application for analytical purposes or everyday life needs.

SIGNIFICANCE STATEMENT

This study was carried out to determine the activity of purified laccase enzyme and the properties of laccase enzyme isolated from T. versicolor by submerged fermentation system. Purification of laccase enzyme in this study by chromatography gel filter column. The principle of protein separation by filter column chromatography gel is the difference in molecular weight of a protein. After undergoing purification, the specific laccase activity of T. versicolor and the degree of purity increased and the optimum pH was obtained.

ACKNOWLEDGMENT

This work was financially supported by the Universitas Padjadjaran Grant (1982/UN6.N/LT/2019) from the Ministry of Research, Technology and Higher Education, Indonesia.