Table 1.
Origin and zone indices of L‒asparaginase, L‒glutaminase, and urease production of the endophytic fungal isolates used in this study.
Table 2.
One‒way ANOVA results indicated significant differences (p < 0.05) among fungal isolates in the production of L‒asparaginase, L‒glutaminase, and urease. All experiments were cunducted in triplicate. The sample size for L‒asparaginase production consisted of 62 isolates and for both L‒glutaminase and urease production was 49 isolates.
Fig 1.
Screening of endophytic fungal isolates for L‒asparaginase, L‒glutaminase, and urease production on MCD agar culture medium supplemented with bromothymol blue (BTB) after five days of incubation at 25°C under continuous dark conditions.
Labels “a,” “b,” and “c” indicate MCD agar supplemented with L‒asparagine, L‒glutamine, and urea as the sole nitrogen sources, respectively. Label “d” represents isolates grown on MCD agar culture medium without any nitrogen source.
Fig 2.
L‒asparaginase-producing endophytic fungal isolates and their L‒glutaminase and urease co‒activities.
Columns represent the mean zone indices for L‒asparaginase, L‒glutaminase, and urease after five days of incubation at 25°C under continuous dark conditions on MCD culture medium supplemented with L‒asparagine, L‒glutamine, and urea. Error bars indicate the standard error.
Fig 3.
Heatmap of best‒performing endophytic fungal isolates for L‒asparaginase production.
According to the heatmap, among the 11 endophytic fungal isolates that produced high levels of L‒asparaginase, Alternaria brassicae C and Aureobasidium pullulans IH1‒2 showed no L‒glutaminase production. In addition, A. brassicae C produced less urease compared to Au. pullulans IH1‒2.
Fig 4.
Standard curve constructed using various concentrations of ammonium sulfate solution.
The regression equation and R² value are displayed within the plot area. An R² vale close to 1 indicates that the regression model is appropriate for estimating the amount of ammonia released during enzyme activity.
Table 3.
One–way ANOVA results indicated a significant difference (p-value = 3.0 × 10-12) among fungal isolates in L–asparaginase activity. The sample size consisted of 12 endophytic fungal isolates and the experiment was conducted in triplicate.
Fig 5.
L‒asparaginase activity of selected endophytic fungal isolates under the submerged fermentation conditions.
Isolates were grouped using Duncan’s post hoc test at a significance level of 0.05 (p‒value = 3.0 × 10-12). Letters above the columns indicate grouping; isolates sharing the same letter belong to the same statistical group. Error bars represent the standard error.
Table 4.
Result of the Pearson correlation test. Pearson correlation close to 1 and sig. (2‒tailed) ≤ 0.05 (p‒value = 0.016) displayed a significant strong direct correlation between zone index in solid culture media and enzyme activity in submerged fermentation.
Fig 6.
Scatterplot of correlation between zone index in solid culture media and enzyme activity in submerged fermentation.
The regression equation and corresponding R² value are displayed in the plot erea.
Table 5.
One–way ANOVA results showed no significant difference (p–value = 0.184) in L–asparaginase activity among fungal isolates cultured in MCD culture medium without L–asparagine as the sole nitrogen source. However, a significant difference (p–value = 1.6 × 10−10) was observed among the isolates when L–asparagine was supplemented into MCD culture medium.
Fig 7.
L‒asparaginase activity of examined endophytic fungal isolates in MCD culture medium with and without L‒asparagine.
The sample size consisted of five endophytic fungal isolates. Each experiment was performed in triplicate. “ns” indicates no significant difference; “**” denotes a significant difference (p < 0.01) between enzyme activities in culture media with and without L‒asparagine, based on an independent samples t-test. The exact p‒value of enzyme activity in each isolate was determined in the ANOVA table (S2 Table). Error bars represent the standard error.
Fig 8.
L‒asparaginase production by selected endophytic fungal isolates grown on ten different culture media.
Columns represent the culture media, labeled as follows: a) Citrate agar; b) Mineral salts agar; c) Brown agar; d) Sucrose proline agar; e) Elliott agar; f) Asthana and Hawker culture medium A; g) Dox agar; h) Kuehner basal culture medium; i) Cerelose ammonium nitrate agar; and j) Piefer, Humphrey, and Acree culture medium. Rows correspond to the fungal isolates.
Fig 9.
Duncan’s post hoc test of L–asparaginase production by selected endophytic fungi grown on ten different culture media at a significance level of 0.05: A) Sucrose proline agar (p–value = –3.0 × 10-36); B) Mineral salts agar (p–value = 3.0 × 10-30); C) Asthana and Hawker culture medium A (p–value = 3.0 × 10-33); D) Elliott agar (p–value = 2.0 × 10-33); E) Brown agar (p–value = 5.0 × 10-32); F) Dox agar (p–value = 1.0 × 10-31); G) Cerelose ammonium nitrate agar (p–value = 2.0 × 10-31); H) Citrate agar (p–value = 5.0 × 10-32); I) Kuehner basal culture medium (p–value = 1.0 × 1032); and J) Piefer, Humphrey, and Acree culture medium (p–value = 2.0 × 10-28). MCD (K) (p–value = 5.0 × 10-32) was used as the basal culture medium. Each group indicated by a lowercase letter.
Isolates sharing the same letter belong to the same group. The sample size consisted of 12 endophytic fungal isolates and the experiments were performed in triplicate. Error bars represent the standard error.
Fig 10.
Heatmap illustrating L‒asparaginase production by endophytic fungal isolates on different culture media.
Columns represent the culture media, numbered 1 to 10 as follows: 1) Sucrose proline agar; 2) Mineral salts agar; 3) Asthana and Hawker culture medium A; 4) Elliott agar; 5) Brown agar; 6) Dox agar; 7) Cerelose ammonium nitrate agar; 8) Citrate agar; 9) Kuehner basal culture medium; and 10) Piefer, Humphrey, and Acree culture medium. MCD was used as the basal culture medium. Rows correspond to the tested fungal isolates.