Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS

Abstract

Background: Environmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents.

Results: An α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae.

Conclusions: The findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin.

Figure 1

Electrophoretic analysis of crude and purified samples of EL-14. Lane M: Precision Plus Protein™ Kaleidoscope™ standards (250–10 kDa); Lane 1: 12% SDS-PAGE of the crude sample; Lane 2: 12% SDS-PAGE of the sample after TCA concentration; Lane 3: 12% SDS-PAGE of the sample after gel filtration; Lane 4: 12% SDS-PAGE of the purified α-amylase after anion exchange; Lane 5: 12% amylotic zymogram of purified α-amylase developed from protein staining with Lugol’s solution. Zymography was performed to confirm that the purified sample comprised a single band of 70 kDa.

Figure 2

Effect of pH and temperature on specific amylase activity of EL-14 and standard. (A) EL-14 was grown at different pH and specific amylase activity was determined. (B) EL-14 was grown at different temperatures and specific amylase activity was determined. (C) standards (A. oryzae and A. niger) were grown at the same conditions to compare their activity with P. minima. The crude extracellular sample (EL-14) exhibited the highest activity at 25°C and pH 9, significantly more than standard. Means followed by the same letter within a column are not significantly different at P < 0.01 according to the Duncan multiple range test.

Figure 3

Qualitative assessment of amylase activity by zymography. Zymography was performed to confirm that α-amylase activity at pH 9 and 25°C was optimal, in comparison with standards (A. oryzae and A. niger). M = molecular weight markers. A common band was detected at around 70 kDa for P. minima.

Figure 4

Effect of metal ions and different carbon and nitrogen sources on amylase activity. (A) The relative enzyme activities were measured at optimum of pH and temperature and enzyme activity without metal ions was taken as 100%. (B) Effects of different carbon and nitrogen sources at optimum conditions (25°C and pH 9) on α-amylase production of P. minima. The hydrolase-inducing medium of Nyugen et al. [11] was used as a control. Where nitrogen sources were replaced, starch was the carbon source (as per the control medium); where carbon sources were replaced, L-asparagine was the nitrogen source (as per the control medium). Means followed by the same letter within a column are not significantly different at P < 0.01 according to the Duncan multiple range test.

Figure 5

MS and MS/MS spectrum on LC-ESI-MS/MS. (A) Tryptic digest peptide spectrum of purified 70 kDa band from P. minima.(B) MS/MS spectrum of precursor peak (543.4). The data are representative of three independent experiments.

Figure 6

Two-dimensional gel electropherogram and its amylase zymogram. (A) 2D gel was scanned with a Typhoon FLA 9000 laser scanner (GE Healthcare) using a no emissions filter, PMT 600, Laser Red (633) and normal sensitivity. Crude extracellular proteins (approx. 200 μg) from the supernatant of P. minima following growth in the first hydrolase-inducing medium for 7 days were used to perform 2D gel electrophoresis. Proteins were visualized on a 12% SDS-PAGE gel stained with Coomassie Blue and identified by LC-ESI-MS/MS as indicated in Table 3. (B) Amylase zymogram (pI 4 to 7) produced after 2D gel electrophoresis of protein using crude sample. No amylase activity was detected above around pI 6. Protein in spots 26 and 7 could be assigned to α-amylase in the NCBI nr database by LC-MS/MS analysis (Table 3). Two spots beside spot 26 could be isomers of amylase with different pI.