The present study was undertaken to investigate a newly isolated α-amylase from a fungus of endophytic origin, Preussia minima, and the potential opportunities it may offer as an additive, particularly for the detergent industry. The purification of α-amylase was performed from crude extracts of culture and approximately 7-fold greater enzyme concentration than the crude enzyme with 55.11% recovery was achieved. Since specific activity is affected by the different conditions used in fermentation and purification steps, various values of activity were reported [19, 20]. The lower purification yield of α-amylase could be attributed to higher loss of enzyme during downstream processing or, alternatively, lower initial protein concentration of the crude extract used for the purification process . The activity of α-amylase can be increased using various activators .
Enzyme assays were performed of P. minima culture supernatants following incubation in the second hydrolase-inducing medium  at different temperatures and pH. The optimum temperature and pH based on specific enzyme activity were 25°C and 9, respectively; however, activity at low temperature was also notable. The optimum temperature and pH conditions of amylase production are likely to reflect the climatic conditions found in environments inhabited by the original host plant . The name Eremophila means ‘desert-loving’ and this genus is usually found throughout Australia, predominantly in arid conditions.
Specific activity (138 U/ mg) at pH 9 was remarkable in comparison with activities previously reported [8, 9, 18, 21–24]. These results were confirmed by zymography. α-amylases from most fungi exhibits pH optima in the acid to neutral range; however, this enzyme appears to be exceptional with a pH optimum of 9.0. Though slightly alkaline, α-amylases have been reported to be stable in a wide pH range . Amylase activity under these conditions of low optimum temperature and alkaline pH optimum would be desirable for the application of this enzyme in the detergent industry as an additive to remove starch-based stains .
The effect of metal ions on total amylase activity was studied. Results demonstrated that CaCl2 and MnCl2 increased the total amylase activity in comparison to the control, confirming previous reports which indicated that amylases are mostly metalloenymes and require calcium and manganese ions for activity, structural integrity and stability [7, 9, 18]. Calcium enhances amylase activity by its interaction with negatively charged amino acid residues such as aspartic and glutamic acids . Magnesium and sodium ions were found to inhibit amylase activity and similar observations were made by Varalakshmi et al.  and Reyed . The results obtained with MnCl2 suggest that this salt can be a strong candidate as a culture additive to increase enzyme production. The effects of different nitrogen and carbon sources suggested that inclusion of starch and L-asparagine (in the standard medium) produced higher specific activity (138 U/mg). Replacement with three different carbon (maltose, cellulose and glucose) and nitrogen sources (ammonium nitrate, yeast extract, peptone and tryptophan) did not increase production of amylase. These results were confirmed by zymography. We observed that fungal growth was superior when the medium was supplemented with specific carbon and nitrogen sources, which reflected the reported levels of enzyme production. This suggests that enzyme activity is linked to biomass production. Many previous reports showed the same effect on enzyme activity by changing the conditions of fermentation [8, 9, 21, 24]. Since fungi do not naturally produce enzymes at levels high enough for commercial purposes, fermentation is undertaken to increase the secretion of target enzymes to levels that are economically sustainable. Consequently, environmental screening programs are used to seek enzymes from various environments, with the view to express these enzymes in highly secreting production hosts . In the present study, different sources of carbon and nitrogen showed significant effects in both amylase production and enzyme activity.
In the bioreactor studies, total enzyme activity (212 U/ml) was comparable to that in shaker cultures (214 U/ml). Thus, from the current studies in the bioreactor, it was concluded that the process for the production of α-amylase from P. minima can be optimized successfully in a bioreactor with no loss in enzyme activity, which has significant implications for practical applications in industry.
The secretome of P. minima was analysed using gel electrophoresis and mass spectrometry. As the genome sequence was unavailable, cross-species identification and zymography assisted the analysis. Most of the identified proteins within the P. minima secretome are enzymes involved in the degradation of plant cell wall polymers (starch, cellulose, lignin, pectin and proteins). A diverse range of other enzymes, as well as some proteins with unknown functions, were also identified from the secretome study. Two of the protein spots on the 2D gel were identified as α-amylase. However, a few spots could be seen clustered around the same molecular weight of one of the spots (spot 26) but with slightly different pI values, which could possibly be isoenzymes of α-amylase. Amylase activity was shown by sharp common bands on the 1D amylase zymogram (approximately 70 kDa) at the approximate molecular weight of the identified amylase spot 26 on the 2D gel. The prominent spot was identified as α-amylase based on LC-ESI-MS/MS data from a protein spot at approximately 70 kDa, pI 6 on the 2D gel.
To aid metabolism and subsequent survival under different environmental conditions, endophytic fungi secrete several polymer-degrading enzymes, many of which were identified in the P. minima secretome. One such polymer is xylan, which is abundant in the leaves of dicotyledons such as Eremopholia species. This requires the action of numerous enzymes such as endo- 1, 4- β-xylanases, for its complete degradation. It acts by cleaving the xylose sugar backbone. Xylanase was also found in the secretome of other fungi, including Podospora anserine and Doratomyces stemonitis. Cellulose forms an integral part of plant cell wall where it is covalently linked with lignin. The presence of cellulase in the secretome of P. minima implies the need of the enzyme to break down plant material by the fungus to obtain nutrients. Another enzyme identified in the P. minima secretome was pectinase, which is involved in the degradation of pectin, an indigestible polysaccharide in leaves. The presence of pectin-degrading enzymes in the secretome of P. minima could be reflective of high levels of pectin contained within E. longifolia leaves. In previous studies, pectinase was identified in other industrial fungi such as Aspergillus sp. and D. stemonitis. Mannase was also found in the secretome of P. minima. Mannan forms a component of the cell wall matrix of dicotyledonous leaves and the mannase acts by cleaving within the mannose backbone of the mannan polymer. Two different kinds of proteases were found in the secretome of P. minima that could allow the fungus to increase the efficiency of the degradation of the plant cell wall matrix. Metalloproteases are endoproteases that cleave within amino acid chains and enable fungi to utilize proteins during the digestion process. Proteases have been found in the secretomes of other filamentous fungi, including Aspergillus oryzae, Aspergillus niger, Botrytis cinerea and Trichoderma reesei.
As the genome of P. minima has not been sequenced, all protein assignments in P. minima secretome were made by cross-species identification based on sequence similarities to proteins from other fungal and bacterial species in the NCBI database. The greatest number of assignments (nine) was to proteins from Bacillus subtilis. Many of the other proteins were from fungi Magnaporthe oryzae and Aspergillus sp. Over twenty different types of enzymes have been identified in the secretome of P. minima as a result of our work, though α-amylase dominated the secretome. The other proteins were mainly enzymes that break down cellulose, lignin, pectin and protein which is reflective of the fungal endophytic lifestyle. However, there were many small protein spots that were left unidentified as good quality MS/MS spectra could not be assigned confidently to any known protein in the NCBI database. These unidentified proteins might be enzymes that have complementary activity to the enzymes already identified in the secretome, thereby, increasing their access to their target substrates.