GH10 XynF1 and Xyn11A: the predominant xylanase identified in the profiling of extracellular proteome of Aspergillus oryzae LC1

Abstract

Advanced techniques of enzyme production and purification have become prerequisite due to their diverse industrial applications. There is an utmost requirement for screening of new strains capable of synthesising industrially useful enzymes. The present study reports the production and profiling of extracellular proteins expressed by the newly isolated strain of a filamentous fungus, Aspergillus oryzae LC1. The extracellular enzyme production was done by submerged fermentation using Mendel’s and Sternberg’s medium (MSM), and its optimisation was done using one factor at a time (OFAT). The presence of xylanase was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. In addition, the profiling of extracellular proteome of Aspergillus oryzae LC1 was carried out by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS). In this study, media optimisation showed 5.7-fold increase in xylanase activity. The multiple bands observed in zymography revealed the presence of various forms of xylanase. A total of 73 proteins were identified in LC-MS/MS analysis. Functional classification showed that the hydrolytic enzymes consisted of 48% glycoside hydrolase, 11% proteases, 1% polysaccharide lyase and esterase’s, 9% oxidoreductases and 30% other proteins. A total of 26 families of glycosidic hydrolase were detected with other protein families such as serine peptidase, S, LysM, G-D-S-L, M35, carboxyl esterase (CE1), pectate lyase (PL) and oxidoreductases. Among the huge diversity of synergistically acting biomass cleaving enzymes, endo-1, 4-β xylanase with isoforms: xyn F1, xyn B, β xylanase and xyn 11A belonging to GH10 family covered the major portion of the total percentage of identified proteins. As per our knowledge, this is the first report of extracellular proteome analysis of Aspergillus oryzae LC1 suggesting its capability for recombinant expression and evaluation in hemicellulose deconstruction applications.

Introduction

Xylanases (1,4-β-D-xylan xylohydrolase) (EC 3.2.1.8) and β-xylosidases (EC 3.2.1.37) are group of hemicellulases that can hydrolyze xylan (Nagar et al. 2012). Xylan is a branched, heterogeneous polymer consisting the β-1, 4-linked D-xylopyranose units with different side chain residues such as acetyl groups, arabinose, glucuronic acids etc. (Silva et al. 2015). Its complete breakage requires the action of various enzymes due to its branched nature and is more easily attacked than cellulose (Ghio et al. 2016). Other enzymes such as α-L-arabinofuranosidases (EC 3.2.1.55) and acetyl xylan esterases (EC 3.1.1.72) are also efficient for the depolymerisation of xylan (Liao et al. 2014). Xylan represents the most abundant hemicellulosic part of the plant cell wall. Therefore, hemicellulase degradation to sugar molecules is an important step to convert plant biomass to produce fuels and other biochemicals (Chaturvedi and Verma 2013).

The microbial hemicellulases such as xylanases are the most important groups of enzymes having a vast array of applications in industrial processes such as bio conversion of lignocellulose to bio fuel, single cell protein (SCP), bio-bleaching, textile industry, clarification of juices, waste water treatment, and de-inking purposes (Nagar et al. 2010; Kumar et al. 2018).

On industrial scale, the production of xylanases is mainly done by filamentous fungi of the genera Aspergillus and Trichoderma (Park et al. 2002). Previous reports on the production of xylanases by different Aspergillus strains have been extensively demonstrated which includes A. Awamori (Umsza-Guez et al. 2011), A. tubingensis (Pandya and Gupte 2012) and A. niger (Dhillon et al. 2012). Similarly, in our study, a strain of A. oryzae LC1 was reported earlier for xylanase production (Bhardwaj et al. 2017). However, complete insights into the hydrolytic machinery of microorganisms are required which would allow identification and selection of promising targets for bio refinery applications.

Previous reports have demonstrated that the culture conditions greatly influence the induction of isoforms of a specific enzyme, which leads to substantial variations in the secretome of different microorganisms or even within the same species (Girard et al. 2013; Hashemi et al. 2013). In the present study, we have reported differential expression of proteins of the extracellular proteome of A. oryzae LC1 under the optimised culture conditions obtained by OFAT approach. This study has given an insight into the capabilities of A. oryzae LC1 in secreting a variety of important proteins and their recognized industrial application.

Material and methods

Fungal culture

Aspergillus oryzae LC1 (Accession no. MG923342) was isolated from leaf sample of plant Lantana camera obtained from Cachar district, Assam, India in our previous work by Bhardwaj et al. (2017). The culture was maintained on potato dextrose agar plates at 4 °C.

Preliminary screening of media for xylanase production

In order to select the suitable media, three different media namely, Czapek Dox Broth (CzDB) (Tallapragada and Venkatesh 2011), Basal Salt Media with wheat bran (BSMwb) (Bakri et al. 2010) and MSM (Shah and Madamwar 2005) were selected (composition mentioned in Table 1 which has been previously reported for xylanase production. Three Erlenmeyer flasks (200-mL media in 500 mL of flask) were inoculated with A.oryzae LC1 spore suspension (4.35 × 10⁷ spores/mL) and incubated at 28 °C in a rotary shaker incubator for 20 days at 100 rpm. Samples were withdrawn at regular interval of 48 h and used for further analysis.

Table 1 The composition of media used in the study for the production of xylanase

Xylanolytic enzyme assay

Xylanase assay of crude enzyme was done using method described by Ghose and Bisaria (1987). The reaction mixture (0.6 mL) containing 1% (w/v) birch wood xylan (0.4 mL) (SIGMA) and crude enzyme (0.2 mL) was incubated at 50 °C for 30 min. The reaction was stopped by adding dinitro salicylic acid (DNSA) reagent (0.6 mL) and boiled for 10 min in water bath. The amount of reducing sugar was determined by measuring absorbance at 540 nm (Miller 1959). Protein estimation was done by Lowry method using BSA (bovine serum albumin) as standard (Lowry et al. 1951).

Optimisation of media components using OFAT approach for enhanced xylanase production

Among the three media used, MSM was selected and inorganic salt concentrations were optimised by OFAT approach for enhanced xylanase production. Five inorganic components (NH4)₂SO₄, KH₂PO₄, urea (NH₂CONH₂), CaCl₂ and MgSO₄·7H₂O were tested in three different concentrations (Table 2).

Table 2 Experimental set-up for one factor at time (OFAT) using Mendel’s and Sternberg’s medium (MSM)

Crude enzyme preparation from the optimised medium concentration obtained from OFAT

Erlenmeyer flask (500 mL of flask) containing 200 mL MSM composed of (g/L) 2.8 (NH₄)₂SO₄; 5.0 KH₂PO₄; 0.7 urea; 0.5 CaCl₂; 0.7 MgSO₄·7H₂O; 1.0 peptone; trace elements (mg/L) 5.0 FeSO₄; 1.6 MnSO₄·H₂O; 1.4 ZnSO₄·7H₂O; 2.0 CoCl₂; 0.1% Tween 80 and 1% xylan used as carbon source; pH 5. The flask was inoculated with A. oryzae LC1 spore suspension (4.35 × 10⁷ spores/mL) and incubated at 28 °C in a rotary shaker at 100 rpm for 10 days. Samples were withdrawn at regular interval and used for further analysis. All experiments were performed in triplicate.

Concentration of extracellular proteome of A. oryzae LC1

Different solvents such as trichloroacetic acid (TCA) (1:10) (Sánchez-Herrera et al. 2007), acetone (2:1) (Giridhar and Chandra 2010), methanol-chloroform (4:1:3) (Fic et al. 2010), ethanol (2:1) (Giridhar and Chandra 2010) and ammonium sulphate (60%) (Bhardwaj et al. 2017) were tested for their ability to precipitate the enzymes from crude enzyme solution with maximum activity retention. All the solvents (chilled) were added to the crude enzyme (5 mL) in defined ratios and for over-night kept at 4 °C. Precipitates were collected by centrifugation at 10,000 rpm for 15 min and dissolved in 100 μL of 0.05 M sodium acetate buffer (pH 5.0), further used for xylanase assay, protein activity and loaded on the SDS-PAGE gel.

Molecular weight determination by SDS-PAGE and activity staining by zymography

The molecular weights of the enzymes were determined by SDS-PAGE that was done by the method described earlier by Laemmli (1970) using 12% polyacrylamide. The sample obtained from optimised and unoptimised medium with maximum xylanase activity on 6th day and 14th day was loaded on SDS-PAGE gel. The each well was loaded with 30 μg of protein and the gel was analyzed by Coomassie Brilliant Blue R-250 staining method. For the detection of xylanase activity, zymography was performed, where 60-μg protein samples were applied to the native PAGE without boiling followed by the addition of 0.1% (w/v) birch wood xylan into the liquid polyacrylamide gel solution. Subsequently, the gel was incubated in 0.05 M acetate buffer, pH 5 at 50 °C for 1 h. The gel was then stained with 0.1% (w/v) Congo red solution for 30 min at room temperature followed by washing with 1 M NaCl. Congo red de-staining reaction was stopped by immersing the gel in 0.1% (v/v) glacial acetic acid solution.

Sample preparation for LC-MS analysis

Crude enzyme (200 mL) obtained using optimised MSM media by A. oryzae LC1 after 6 days of growth was used for precipitation of enzyme by adding TCA in ratio 1:10. The mixture was stored overnight at 4 °C and the precipitate was collected by centrifugation at 10,000 rpm for 15 min. After subsequent washing with chilled ethanol and acetone (100%), the pellet was dissolved in 2 mL of 0.05 M sodium acetate buffer (pH 5.0). Xylanase activity and protein contents were calculated and the sample was further used for LC-MS analysis.

Preperation for MS analysis

The sample was prepared as per Korwar et al. (2015) where tryptic digestion using 100 μg of protein obtained from TCA was diluted with 100 μL of NH₄HCO₃ buffer (50 mM) containing 0.1% RapiGest. The mixture was incubated at 80 °C for the solubilisation of complete proteome. The reduction of denatured proteins was done using DTT (0.1 M) at 60 °C for 15 min. Later, the alkylation was done using iodoacetamide (0.2 M) at room temperature for 30 min in dark. Proteomic grade trypsin was used for protein digestion at 1:50 (enzyme/substrate) ratio at 37 °C for overnight. The reaction was stopped by the addtion of concentrated HCl and incubated at 37 °C for 10 min. Later, the mixture was centrifuged, and the desalting of peptides was done by using C18 Zip tip (Millipore, MA, USA) followed by its concentration using vacuum centrifuge. Peptide digest was stored at 20 °C until further use.

Extracellular proteome analysis by using LC-MS analysis

The instrument-specific methods and settings (LC-HR/AM Q-Exactive Orbitrap) were done as described by Korwar et al. (2015) for the construction of the fragment ion library and the quantification of glycated peptides. LC-MS analysis was performed at National Chemical Laboratory (NCL), Pune, India. Accela 1250 UHPLC (Thermo Fisher Scientific) equipped with a Hypersil Gold C18-reverse phase column (150 * 2.1 mm, 1.9 μm) was used for separating peptide digests (2.5 μg). The column along with the mobile phase A (100% water, 0.1% formic acid (FA) 98%, mobile phase B (100% Acetonitrile (ACN), 0.1% FA) 2%, at flow rate 350 μL/min was used to load sample. The run time was 40 min linear gradient of 2 to 40% mobile phase B, the column temperature was 40 °C and auto sampler at 4 °C. In full MS/dd-MS2 acquisition sample analysis was done on hybrid quadruple Q-Exactive Orbitrap MS. The parameters of the instrument were as follows, spray voltage 4200 V, capillary temperature 320 °C, heater temperature 200 °C, S-lens RF value 55, sheath and auxiliary gases pressure were 30 and 8 psi, respectively. The final data were processed by using Proteome Discoverer, Version 1.4.0.288 (Thermo Fisher Scientific). A computer algorithm for database search known as SEQUEST HT was used for peptide identification. The protein database UniProt was used for data search.

Results and discussion

Xylanase production in three different media

Xylanase production was tested with three different media. Among them, MSM gave maximum xylanase activity (225 ± 1.9 IU/mL) as compared to other media where BSMwb showed 50 ± 1.8 IU/mL while CzDB showed 10 ± 2.3 IU/mL (Fig. 1).The levels of extracellular xylanase produced by A. oryzae LC1 were significantly higher in comparison to the xylanase produced by A. terreus FSS129 (113 IU/mL) previously reported by Bakri et al. 2010, and comparable to the xylanase produced by A. foetidus MTCC 4898 (210 U/mL) (Shah and Madamwar 2005), A. oryzae MD4 (440 IU/mL) (Chutani and Sharma 2015).

Fig. 1

Xylanase production using three different media, i.e. CzDB, BSMwb and MSM from Aspergillus oryzae LC1. Flasks were incubated at 28 °C in a rotary shaker incubator for 20 days at 100 rpm

Optimisation of inorganic components of MSM using OFAT for enhancing xylanase production

The effective concentrations of inorganic components of MSM for xylanase production were determined by studying the variables. The xylanase production by using inorganic components was obtained on 8th day with an optimum concentration of (NH₄)₂SO₄ (2.8 g/L) 852.6 IU/mL; KH₂PO₄ (5 g/L) 1005.1 IU/mL; urea (1.0 g/L) 1094.9 IU/mL; CaCl₂ (0.5 g/L) 1292.3 IU/mL and MgSO₄·7H₂O (0.7 g/L) 852.6 IU/mL. All five variables had independent effect while keeping other components constant for xylanase production (> 850 IU/mL) (Fig. 2).

Fig. 2

Production of xylanase enzyme using Mendel’s and Sternberg’s medium (MSM) by Aspergillus oryzae LC1 using one factor at time (OFAT) approach under variable concentration of inorganic salts a ammonium sulphate ((NH₄)₂SO₄), b potassium di-hydrogen phosphate (KH₂PO₄), c urea (NH₂CONH₂), d calcium chloride (CaCl₂), e magnesium sulphate (MgSO₄·7H₂O). Flasks incubated at 28 °C in a rotary shaker incubator for 12 days at 100 rpm

Validation of the experimental composition obtained after optimisation using OFAT approach

The initial concentration of MSM (g/L) was 0.1% v/v tween 80; 1.4 (NH₄)₂SO₄; 2.0 KH₂PO₄; 0.3 urea; 0.3 CaCl₂; 0.3 MgSO₄·7H₂O; trace elements (mg/L) 5.0 FeSO₄; 1.6 MnSO₄·H₂O; 1.4 ZnSO₄·7H₂O; pH 5 with 1% xylan used as carbon source. The final concentration obtained from OFAT was (g/L) 2.8 (NH₄)₂SO₄; 5.0 KH₂PO₄; 0.7 urea; 0.5 CaCl₂; 0.7 MgSO₄·7H₂O; keeping other components constant. For validating the optimum combination of the inorganic components, confirmation experiments were carried out. The un-optimised showed 225 ± 1.9 IU/mL of xylanase production on 14th day of incubation, whereas the selected optimum combination of five variables from OFAT resulted in the production of xylanase up to 1290 ± 2.3 IU/mL on the 6th day of incubation which is 5.7-fold higher than the un-optimised condition. The extracellular xylanase produced by A. oryzae LC1 was considerably higher than the xylanase produced by T. viride IR05 using OFAT approach (Irfan et al. 2014). The optimised media leads to higher activity and decrease in the incubation time as compared to unoptimised media.

Concentration of crude enzyme using different solvents

Five different solvents were tested for precipitation of crude enzyme solution obtained from A. oryzae LC1. Among them, TCA-precipitated sample showed highest xylanase activity of 3051.3 IU/mL with protein 25.6 mg/mL and specific activity 119.4 IU/mg as compared to other precipitation methods (Table 3). Concentration of proteins using TCA has been reported previously (Sánchez-Herrera et al. 2007; Fukuda et al. 2010; Watanabe et al. 2011), and it does not affect the native property of enzyme.

Table 3 Selection of precipitation method for the maximum retention of protein from the crude enzyme solution of A. oryzae LC1

SDS-PAGE and zymography

Among all the precipitation methods, TCA showed multiple bands in SDS-PAGE gel signifying the precipitation of maximum protein, whereas other methods showed comparatively less number of bands. The unoptimised medium showed highest xylanase activity on 14th day of incubation, whereas optimised medium on 6th day. In order to confirm the production of maximum xylanase in less time with optimised medium, samples were loaded on SDS PAGE gel and the maximum number of bands with good intensity were observed on 6th day of optimised medium when compared to unoptimised medium on the same day, whereas on 14th day, number of bands were less with low intensity. The presence of multiple forms of xylanases were further confirmed by zymography and their molecular weight was estimated in the range of 23–35 kDa (Fig. 3).

Fig. 3

SDS-PAGE and zymography of TCA precipitated crude xylanase: (M) Marker; (1) the optimised medium on 14th day; (2) the unoptimised medium on 14th day; (3) the optimised medium on 6th day; (D) the unoptimised medium on 6th day; (5) zymography of sample obtained from 6th day of optimised medium

Analysis of extracellular proteome of A. oryzae LC1

The extracellular proteome of the cell consists of soluble secreted proteins which play major role in biological processes of the cell (Braaksma et al. 2010). LCMS can be utilised to study the proteome analysis of various enzyme secreted by the fungus (Tiwari et al. 2014; Singh et al. 2015; Jeon et al. 2017). In present study, the LCMS analysis showed the presence of 73 proteins in the extracellular portion, which were detected and identified from database searches (UniProtKB and Pubmed). LC-MS analysis results showed the presence of various hemicellulases, cellulases, proteases, esterase’s and other enzymes (Table 4). The detected proteins had a molecular weight ranging from 10 to 118 kDa and isoelectric points (pI) from 3 to 7. According to proteome analysis, 35.5% of proteins have a molecular weight below 40 kDa and the molecular weight of 62.5% proteins ranged from 40 to 117 kDa and isoelectric points (pI) from 4 to 6.5. The classification of the identified protein has been depicted in Fig. 4 and consisted of the major proportion of glycoside hydrolase (48%), which is a known superfamily of cellulases, hemicellulases and other accessory enzymes are involved in the hydrolysis of polysaccharides.

Table 4 Hydrolytic enzymes detected in the extracellular proteome of A. oryzae LC1

Fig. 4

Functional classification of proteins detected LC-MS analysis of A. oryzae LC1 secretome

Other hydrolytic enzymes such as proteases (11%), oxidoreductases (9%) and a lesser amount of esterase’s, lyase and other minor proteins (30%) were also observed. Proteases play a major role in the assimilation of nitrogen from complex organic nitrogen source used for enzyme induction. The inclusion of proteases as auxiliary enzymes in novel enzyme mixtures which can be directly utilised by lignocellulosic biomass and has been previously reported by Duck et al. (2003). Fungal oxidoreductases can serve as a promising tool for the production of fine chemicals for pharmaceuticals, flavours and fragrances (Straathof et al. 2002). Among the known pectinases, pectin lyase (PL) is the only pectic enzyme that can cleave the alpha-1,4 bonds of highly esterified pectin’s without prior action of other enzymes (Ferreira et al. 2010).

The glycoside hydrolyses (GH) were further classified on the basis of sequence homology of GH families which belonged to 26 different families (Fig. 5). About 28.3% of the proteins belonged to GH10 which houses endo-1, 4-beta-xylanase F; endo-1, 4-beta-xylanase B (Table 5), beta-xylanase, endo-1, 4-beta-xylanase (EC: 3.2.1.8), and 14.4% belonged to GH75, encompassing endo chitosanase C (EC: 3.2.1.132). The GH3 containing beta-glucosidase-related glycosidase with several domain proteins constituted 10.2% of total hydrolase. Other families such as GH2 (3.9%), GH7 (9.7%), GH13 (8%) housing alpha-amylase, GH15 (11.5%), GH17 (1%), GH28 (1.7%), GH61 (1.2%), GH62 (5.1%) were also detected in the analysis. However, GH families less than 1% such as GH5, GH6, GH12, GH16, GH18, GH20, GH31, GH32, GH35, GH43, GH47, GH53, GH67, GH72 and a very rare protein GH125 were also observed. The presence of these GH families can be further exploitated for overexpression of respective GH gene based on type of application involved (Fig. 6). GH 10 family xylanase showed xylooligosaccharides (e.g. xylobiose, xylotriose, xylotetraose and xylopentose) production by the hydrolysis of xylan and agricultural residues in the study reported by Kumar and Satyanarayana (2011, 2013).

Fig. 5

Distribution of various glycoside hydrolase (GH) families in the exracellular secretome of A. oryzae LC1

Table 5 List of various forms of xylanase identified in LC-MS/MS analysis

Fig. 6

Chromatogram obtained from LC-MS/MS analysis: largest peak (value 41.95) showing the presence of more xylanase than the other proteins

Besides several polysaccharides side chain hydrolysing enzymes, other enzymes, such as esterase’s, belonged to CATH super family SGNH hydrolase super family, CE1 (carbohydrate esterase), type B carboxyl esterase were also detected. Their presence is significant as they can act synergistically with xylanases for the degradation of hemicelluloses (Biely et al. 2014). Various polysaccharide lyase belonging to PL1, PL2 and PL3 families, oxidoreductases and other proteins were also observed in the analysis. Extracellular proteome analysis of Talaromyces amestolkiae, a cellulose-producing fungi, showed the secretion of high level of β-glucosidase. β-1,4-endoglucanase, exoglucanase and β-glucosidase with different carbon sources (Eugenio et al. 2017). Transcriptomic, extracellular proteome and active secretome analysis of Nicotiana benthamiana showed a large and diverse protease range (Grosse-Holz et al. 2018). Champer et al. (2016) reported fungi-specific proteins in the cytosol, cell wall and secretome of 13 different fungi which do not share significant homology with human proteins, for the development of vaccines or drug targets. A unique lignin modification pattern is associated with extracellular proteome of Phlebia radiate and showed a synergy of ligninolytic enzymes in the selective degradation pattern (Bule et al. 2016). Hence, extracellular proteome analysis showed presence of variety of enzymes and proteins expressed by A. oryzae LC1 which can have appreciable industrial applications. This is also evident from the results obtained in our previous work by Bhardwaj et al. (2017), where crude enzyme was capable of hydrolysing several lignocellulosic agro-residues such as rice straw, wheat straw, pearl millet husk, wheat bran, groundnut shell etc.

Conclusion

OFAT approach has considerably enhanced the production of xylanase activity. This study provides new insights in the ability of a newly isolated strain A. oryzae LC1 to produce various forms of xylanases. Strain A. oryzae LC1 is a good producer of xylanase and can also produce other polysaccharide side chain hydrolysing enzymes. Endo-1, 4- β xylanase named as Xyn F1, Xyn B, β xylanase and Xyn 11A belonging to GH10 family were the main xylanase identified in the LC-MS/MS analysis. The other enzymes such as proteases, polysaccharide lyase, esterase’s and oxidoreductases can synergistically act with xylanases for hemicellulose degradation leading to the production of value-added products for industrial and medical purposes. The potential of these enzyme cocktail obtained by A. oryzae LC1 can bring about considerable conversion of lignocellulosic polysaccharides to sugar monomers. The xylanase and its hydrolytic enzyme complex can be effectively used in the food industry, paper and pulp industry, production of ethanol and xylitol. It can also be used in animal feed as well as in pharmaceutical analysis. As xylanase and enzyme cocktail have its utility in various bioprocess industries and multiple biotechnological industries, the future prospect would involve further enhancement via protein engineering and expression studies.