Fungal endophytes of high altitude ethnomedicinal plants as a bioresource of industrially imperative enzymes

Endophytic fungi have been in the spotlight as a reservoir of novel agents with diverse bioactivities. Similarity in chemical diversity with the host plant makes them an amenable target for industrial interventions. A wide range of compounds as secondary metabolites and enzymes are manufactured inside the endophytic fungal factory. However, utilization of endophytic fungi as industrially imperative enzyme producers has been a scarce event. The present study was conducted to bio-prospect the fungal endophytes present in the high altitude medicinal plants of Uttarakhand, as industrially imperative enzyme producers. A total of 58 different endophytic isolates were obtained from Pinus sabiniana, Cinnamomum tamala, Cinnamomum verum, Ocimum tenuiorum and Rhododendron arboreum. Endophytic fungal colonization was highest, 31%, in Pinus sabiniana. The pure isolates were further explored for the production of amylases, cellulases, proteases and L-asparaginase. Out of 58 isolates, 40 isolates exhibited potent enzyme productivity. #7PSSTB isolate was considered as superlative contender on account of its relatively higher production of all the three enzymes viz. amylases, cellulases, proteases. Partial purication of #7PSSTB extract showed compelling enzymatic activity corroborating the existence of exogenous enzyme in the extract. Interestingly, #9 RASTB, #11 RASTB and #17 RASTB exhibited the production of therapeutically imperative L-asparaginase enzyme. The present study puts the spotlight on endophytic diversity in the high altitude medicinal plants as a source of enzymes of industrial interest. Production of L-asparaginase paves the way of pharmaceutical intervention to explore anti-oncogenic effects in the endophytic fungal repository of high altitude regions.


Introduction
Endophytes are speci c group of micro-organisms which colonize plants internally without apparent adverse effects. Endophytic fungi are potent source of novel organic compounds with pharmaceutically important biological activities and a high level of biodiversity (Deshmukh et al. 2018). All nonvascular and vascular plants examined until now have been found to harbor endophytic microbes with the potential to produce novel secondary metabolites. Bio-prospecting of endophytes has unraveled new molecules with therapeutic potentials (Strobel and Daisy 2003). Several endophytic fungi have been reported as potent commercially imperative enzyme producers including Alternaria tangelonis, Cladosporium cladosporioides, Curvularia akaii and Fusarium subglutinans (Masumi et al. 2014), Fusarium solani (Uzma et al. 2016) and Aspergillus terreus (Kalyanasundaram et al. 2015).
Among the repository of fungal enzymes viz. cellulase, amylase, protease and L asparaginase have been investigated for broad spectrum effects. Cellulolytic enzymes have been a focal point of research as a mediator of baggase degradation and second generation ethanol production. cellulase enzymes supplementation can improve the enzymatic hydrolysis of lignocellulosic biomass, in terms of speed and hydrolysis yield. Endophytic fungi viz Botryosphaeria sp. and Saccharicola sp. have been investigated for production of cellulase enzyme (Marques et al. 2018). Amylases are one of the prime industrial enzymes that encompass a wide spectrum of functional applications in pharmaceutical, food, textile and detergent industries. Approx. 30% of the total enzyme production globally accounts for amylases (de Souza and e Magalhães 2010). On the other hand, proteases, an enzyme which breaks proteins to smaller constituents, shares two-third of the world enzyme market. Proteases have profound applications in bioremediation, cosmetics, silk degumming, animal cell culture, therapy, diagnosis, pharmaceutical and food industry (Singh et al. 2016).
L-asparaginase (EC 3.5.11. L-asparaginase amidohydrolase) is widely studied in context to its antitumor potential against tumor of lymphoid precursor, acute lymphoblastic leukemia, acute myeloid leukemia and non-Hodgkin's lymphoma. Regular supply of asparagine, maintained by asparagines synthetase, is requisite for making proteins in the cell. Leukemic cells however are de cient in asparagines synthetase and depend solely on circulating blood for the supply of L-asparagine (Brumano et al. 2019). The immunogenic complications associated with its present microbial sources Escherichia coli, Erwinia caratovora limits its medicinal frontier. Administration of bacterial origin L-asparaginase is associated with allergic reactions and anaphylaxis. Thus, exploring the potential of alternate sources for the production of L-asparaginase has put rich chemical diversity of endophytic fungi under limelight. Lasparaginase derived from endophytic fungi have many bene ts over existing preparations like the nonimmunogenic as they are phylogenetically related and posttranslational modi cations are present, being the eukaryotic microbes and residing inside the plant body (Sarquis et al. 2004

Production Of Secondary Metabolites
Each fungal endophyte was subjected to culture ltrate production by inoculating 5 mm mycelial plug of 7 days old active culture into 100 ml pre sterilized PDB medium aseptically and kept on the rotatory shaker at 26 ± 2ºC, 120 rpm for 10 days. After incubation, the culture ltrate rich in bioactive compounds were separated from fungal mass by ltration through Whatman lter paper no. 4 followed by centrifugation at 12,000 rpm for 15 min to get cell free culture ltrate (Lobo 2006

Screening For Extracellular Enzyme Production
Protease assay: For the protease activity, the skim milk agar plates were prepared containing 1% skim milk and 1% agar. 30 µl of each culture ltrate was added into 5 mm wells prepared by sterile cork borer in skim milk agar plates followed by incubation at 37 ˚C for 24 h. Un-inoculated PDB served as control. After incubation, a clear zone around the wells indicates the proteolytic activity which was measured in terms of zone diameter and expressed as Mean ± SD. Cellulase assay: The modi ed agar well diffusion method was employed to assess the cellulase activity of fungal isolates as per the method described by (Marques et al. 2018;Legodi et al. 2019). Brie y, Czapek-Dox agar medium plates supplemented with 1% carboxymethyl cellulose and 1% Agar was prepared. The plates were allowed to solidify for 30 min and 5 mm well were punched out with help of sterile cork borer. The culture ltrate of each fungus was loaded into the wells followed by incubation at 37˚C for 18-24 h. After the incubation, the plates were ooded with aqueous Congo red solution. The appearance of yellow zone around the fungal colony indicated cellulolytic activity. The zone diameter was measured and represented as Mean ± SD.
Amylase activity assay Amylase activity was assessed by preparing the 1% starch agar plate by following the procedure of (Hankin and Anagnostakis 1975). The plates were solidi ed for 30 min and 5 mm well were punched out with help of sterile cork borer. The culture ltrate of each fungus was loaded into the wells followed by incubation at 37 ˚C for 18-24 h. Un-inoculated media served as control. After the incubation, the plates were ooded with the 1% Iodine solution. Appearance of clear zone around the fungal colony indicated amylolytic activity which was measured in terms of zone diameter and represented as Mean ± SD.

Asparaginase Activity assay
Asparaginase production by the fungal endophytes was assessed by modi ed Ditch plate assay (Mahajan et al. 2013). Brie y describing, L-asparaginase-agar (2%) plates were prepared by adding 2% Lasparagine supplemented with 0.009% phenol red. Each plate was divided into four quadrants followed by preparation of 5 mm wells in each quadrant using pre-sterilized cork borer. Further, 30 µl of culture ltrates of each fungal endophyte were dispensed into the wells followed by incubation at 37 °C for 24 h.
After the incubation period, the plates were observed for the pink halo formation around the wells. The zone diameter was recorded and expressed as Mean ± SD.
Mass Production And Partial Puri cation By "Salting Out" For the partial puri cation of desired enzyme, the potential endophytic fungi was subjected to mass fermentation of 1L by inoculating 5 mm active mycelial plug of selected fungal endophyte in 10 Erlenmeyer ask containing 100 ml pre-sterilized PDB medium followed by incubation at 28˚C, 120 rpm for 7-10 days. After the incubation period, cell mass and culture ltrate was separated by ltration through Whatman lter paper No. 4 followed by centrifugation at 10,000 rpm for 10 min at 4 0 C in refrigerated centrifuge. The obtained supernatant was then further subjected for precipitation of enzymatic protein by employing ammonium sulphate salting out method. Brie y, ammonium sulphate was slowly added to the culture broth to achieve saturation with slow and continuous stirring at 4 °C. The mixture was then incubated overnight at 4 °C and the next day protein precipitate was collected by

Statistical Analysis
The statistical analysis was done using analysis of variance with GraphPad Prism 5 software followed by Tukey's post-hoc test (p < 0.05). Data points were obtained from three replicates, and two independent experiments were performed.

Results
Fungal endophytes were isolated from medicinal plants A total of 58 different fungal endophytes were isolated from various medicinal parts (Table 1, Fig. 1). Maximum fungal endophytes were recovered from Pinus sp. (31.0%) followed by C. tamala (27.5%) and R. arboreum (25.8%). However least fungal colonization was observed in C. verum (5.1%). The host tissue of each plant sample exhibited a variation in colonization of the endophytic myco ora. Further, colonization in different parts of plants was estimated. It was observed that maximum fungal colonization was observed in stem (62.1%) followed by leaf (24.1%). However, bark and stem internal tissues were least colonized by endophytes (Fig. 2).

Screening Of Amylase Producing Endophytic Fungal Isolates
In the amylolytic screening assay, only 5 endophytes exhibited the amylase producing potential. As per One-way ANOVA analysis [F (38,76) = 469.2, < 0.001] and Tukey's post hoc analysis, #1 RASTB was found to be potent amylase producer with zone size of 13.0 mm followed by #5 RASTB with zone size of 12.3 mm (Fig. 4a-b). However, least activity was observed in #14b PSSTB.

Screening Of Cellulase Producing Fungal Endophytic Isolates
Cellulose is imperative in leather, detergents and food processing industries. In the cellulolytic activity, 15 endophytes out of 58 showed positive results. As per One-way ANOVA analysis [F (38,76) = 1473, < 0.001] and Tukey's post hoc analysis, maximum activity was recorded in #7 PSSTB with a zone size of 17.0 mm followed by #16a PSSTB with zone size of 16.3 mm. However least activity was recorded in #26RASTB and #27RASTB with zone size of 8.0 mm (Fig. 4c-f).

Screening Of L-asparaginase Producing Fungal Endophytes
The isolated endophytic fungal isolates were screened for the production of L-asparaginase enzyme. In the L-asparaginase screening assay, 9 endophytes were found to be potent L-asparaginase producer.

Bioactivity Screening Of Partially Puri ed Protein
Further the protein puri cation was done from the endophytic fungal isolates using salting out method and evaluated for enzyme activities. The partially puri ed protein of #7PSSTB, opted due to consistent best results in all the three screenings, was again subjected for bioactivity pro ling. In protease, amylase and cellulase activity assay, zone size of 16 mm, 12 mm and 16 mm respectively was obtained (Fig. 6).  (Table 1). Maximum colonization of endophytic fungi was observed in Pinus sabiniana and stem as an explant source (Fig. 2). Further, the isolates were screened for production of industrially and medicinally useful enzymes such as amylases, cellulases, proteases and L-asparaginase. Out of the 58 isolated endophytic fungi 40 isolates were found to be enzyme producers with varying degree of productivity whereas 18 isolates did no showed any enzymatic activity. #7PSSTB isolate was selected as the best candidate for further screening owing to its potential of relatively higher production of all the three enzymes viz. amylases, cellulases, proteases ( Availability of data and materials

Discussion
The data pertaining to this article are incorporated in the manuscript.

Competing interest
There is no actual or potential competing interest.

Funding
Not applicable      (a-c) L-asparaginase enzyme production by culture ltrates obtained from 10 days old of different fungal endophytes as indicated by dark pink color halo around the well over Asparagine-phenol red agar plate.
Maximum L-asparaginase production was observed in #9 RASTB with a zone size of 20.3 mm.

Figure 6
Bioactivity pro ling of partially puri ed protein of #7PSSTB. The cultural ltrate of most promising candidate #7PSSTB was processed for partial puri cation of protein by salting out. The bioactivity pro ling exhibited (a) Amylolytic activity (b) Protease activity (c) Cellulase activity with a zone size of 16 mm, 12 mm and 16 mm respectively

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Graphicalabstract.tif