Bioprospecting Fungal Glycosyl Hydrolases: Multi-locus Phylogenetic Analysis and Enzyme Activity Proling for Enhanced Biomass Valorization

Lignocelluloses comprise of celluloses and hemicelluloses which can be effectively depolymerized to obtain fermentable sugars using diverse microbial enzymes, for subsequent conversion to various value-added products. Present study reports the bioprospecting of industrially signicant microorganisms and their characterization to attain xylanases with high catalytic eciency. Four potential xylanolytic fungi were identied through distinct primary and secondary screening process of 294 isolates from samples containing plant degrades. Morphological characterization and multigene analysis (ITS rDNA, 18S rDNA, nLSU rDNA, β-tubulin and actin gene) conrmed them Aspergillus niger AUMS56, Aspergillus tubingensis AUMS60, Aspergillus niger AUMS64 and Aspergillus fumigatus AUKEMS24 and their crude xylanase activities through submerged fermentation using corncob were 18.9, 32.29, 30.68 and 15.82 U ml -1 , respectively. AUMS60 and AUMS64 have highest catalytic activity of 1429 U g -1 and 1243 U g -1 , respectively, all having pH and temperature optima of 6.0 and 60°C respectively, where AUMS60 produced single xylanase (Xyn60; 36 kDa) and AUMS64 secreted 2 probable isozymes (Xyn64A and Xyn64B; 33.4 and 19.8 kDa). Maximum saccharication eciency of AUMS60 and AUMS64 were 51.1% (13 h) and 52.2% (24 h) showing enhanced catalytic activity with various cations. Present research reports potential xylanases from indigenous fungi, providing opportunity for development of bio-catalysts concoction (novelty established) for enhanced saccharication of complex lignocelluloses nding specic industrial applications for production of value-added components. Xyn64A and Xyn64B (all above 50%) for production of industrially signicant xylanases with high catalytic activities, multi-locus phylogenetic analysis of selective organisms and partial proling of xylanase enzyme activities for enhanced biomass valorization. These organisms were isolated from diverse waste residues rich in plant debris, through screening on enrichment media and characterized through morphological/physiological characteristics and multigene analysis (internal transcribed spacer (ITS) rDNA, 18S small subunit rDNA, 28S large subunit (nLSU) rDNA, β-tubulin and actin gene sequences). Two isolates selected for further studies AUMS60 (Aspergillus tubingensis) and AUMS64 (Aspergillus niger) were investigated on production and proling of potential extracellular xylanase from them namely Xyn60, Xyn64A and Xyn64B. Proling of physico-chemical characteristics of theses enzymes suggested their industrial signicance. Effect of metal ions, pH and temperature optima and stability suggests great catalytic potential of these enzymes. The presence of xylan intertwined with cellulosic component in the lignocellulosic biomass creates resistance for ecient conversion of biomass into fermentable sugars. Therefore, we evaluated the performance of the xylanase enzymes obtained from AUMS60 and AUMS64 for depolymerization of birchwood xylan. In our study, the saccharication eciency of AUMS60 and AUMS64 xylanases was found to be 51.1% (4.84 mg ml -1 reducing sugars) and 52.2% (4.95 mg ml -1 reducing sugars) in 13 and 24 hours, respectively when used at an enzyme dosage of 100 Units g -1 substrate. A xylanase from S. variabilis MAB3 was reported to achieve maximum saccharication eciency of 51.1% from birchwood xylan in 72 hours by Sanjivkumar et al. (2018). Pleurotus ostreatus xylanase (rXyn162) resulted in the release of 88.4 mg l -1 reducing sugars from oat spelt xylan (Zhuo et al. 2018). In another study by Tu et al. (2019), the levels of xylo-oligosaccharides released via depolymerization of wheat-arabinoxylan, reached 451.4 mg l -1 within 12 hours of incubation using xylanase from Termoascus crustaceus JCM12803. In view of the previous ndings, the enzymes used in our study accomplish better saccharication of birchwood xylan in much less time even while using low dosages of enzyme. Moreover, we have used enzymes that are partially puried (one-step purication). Most of the previous studies have utilized enzymes that have been puried


Introduction
Growing concerns over global warming and hike in fuel prices has necessitated usage of lignocellulosic biomass (most abundant natural biopolymer) as a renewable and eco-friendly source of current fuel platform. There is a strong impetus worldwide, for usage of biofuels that are economical and sustainable (Rastogi and Shrivastava 2017).
Holocelluloses (cellulose and all of the hemicellulose) forms major fraction of lignocelluloses along with lignin which can be ideally hydrolyzed to monosaccharides for effective conversion to various value-added products. Traditional thermochemical methods utilized for degradation of lignocelluloses typically generate toxic by-products, therefore emphasis is laid on enzymatic sacchari cation considering the cost and yield in the biore neries (Varghese et al. 2017; Sunkar et al. 2020). Lignocellulosic biomass is naturally degraded by diverse glycosyl hydrolases (GHs) producing microorganisms that can be utilized for several industrial as well as biotechnological applications. Cellulases, xylanases, lipases, pectinases, proteases and ligninolytic enzymes are few of the enzymes being utilized for the improvement of microbial and enzymatic processes for degradation of lignocelluloses (Rastogi and Shrivastava 2020).
Xylanases are ubiquitously found in nature produced by a variety of organisms but fungi secrete much higher levels of xylanases than other microbes in addition to several auxiliary enzymes required for the degradation of the substituted xylan (Polizeli et al. 2005). Xylanase cleaves β-1,4 glycosidic backbone of xylan (major hemicellulosic fraction) generally releasing xylose and xylobiose such as xylo-oligosaccharides as end products (Shrivastava 2020). In the last few decades, microbial xylanolytic enzymes have demonstrated a tremendous biotechnological potential in various elds apart from bioenergy production, including food for improving dough elasticity, animal husbandry for boosting the weight of chicks, clarifying juices for degumming of bers, bread making, pre-bleaching of Kraft pulp, deinking of waste newspapers and wine making (Chadha et al. 2019; Rastogi and Shrivastava 2020). Enzymatic sacchari cation of lignocellulosic biomass is a feasible alternative to physico-chemical treatment but its usage is limited owing to the high cost of enzymes. On the other hand, a vast array of indigenous microorganisms can be exploited for production of desirable glycosyl hydrolases via submerged (SmF) or solid-state fermentation (SSF) by substituting puri ed xylan with lignocellulosic biomass as substrate for providing the necessary nutrients for the microbial growth and the induction of the enzymatic production (Sunkar et al. 2020). SmF system accounts nearly for 90% of total xylanase produced worldwide (Polizeli et al. 2005) and is considered more exible for allowing large-scale fermentations (Taddia et al. 2020).
Filamentous fungi, such as Aspergillus, easily adapt for cultivation on solid substrates due to simulation of lignocelluloses to their natural habitat and hence deliberated as good producers of hydrolytic enzymes for holocellulose degradation (Dias et al. 2018). Presently, the usage of acidic and alkaline xylanases in industries is compromised due to the environmental impact and high energy costs while neutral and weak acidic enzymes with high shelf life are gaining popularity due to reduced energy and expenditure input for their production and functions (Guo et al. 2012). At industrial scale, thermostable enzymes (operating at 45-100°C) not only improve enzyme robustness and enhance higher mass transfer and reaction rates decreasing the amount of enzyme required, but also reduce viscosity to increase the solubility of reactants and products, reduce risk of contamination and improve hydrolytic performance due to long half-lives at high temperatures (Bhalla et al. 2013;Bibra et al. 2018). Therefore, scientists are always exploring new microbial sources having higher activities with novel characteristics to improve e ciency for biomass valorization. Although a plethora of cellulo-xylanolytic strains have been reported, they are far from ful lling our needs.
Present investigation reports bioprospecting of potential microorganisms for production of industrially signi cant xylanases with high catalytic activities, multi-locus phylogenetic analysis of selective organisms and partial pro ling of xylanase enzyme activities for enhanced biomass valorization. These organisms were isolated from diverse waste residues rich in plant debris, through screening on enrichment media and characterized through morphological/physiological characteristics and multigene analysis (internal transcribed spacer (ITS) rDNA, 18S small subunit rDNA, 28S large subunit (nLSU) rDNA, β-tubulin and actin gene sequences). Two isolates selected for further studies AUMS60 (Aspergillus tubingensis) and AUMS64 (Aspergillus niger) were investigated on production and pro ling of potential extracellular xylanase from them namely Xyn60, Xyn64A and Xyn64B. Pro ling of physicochemical characteristics of theses enzymes suggested their industrial signi cance. Effect of metal ions, pH and temperature optima and stability suggests great catalytic potential of these enzymes.

Materials And Methods
Agro-residues, chemicals and sample collection Agro-residues obtained from local farms were washed thoroughly, followed by oven drying and grinding to obtain particles of uniform size 1 mm, to be used as carbon source during screening and fermentation process. Xylanase activity was determined by monitoring the production of reducing sugars by 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) with minor modi cations using xylose as standard according to Bailey et al. (1992). One unit of xylanase activity was de ned as the amount of enzyme that catalyzes the release of 1 µmol of xylose equivalent per minute under de ned assay conditions. CMCase activity was determined according to Ghose (1987), with 1% CMC as substrate. One unit of CMCase activity was de ned as the amount of enzyme that releases 1 μmol of glucose per ml per minute.

Identi cation of fungal isolates
Xylanolytic and cellulolytic microbial strains were examined microscopically using Lactophenol cotton blue (Leck 1999) and Grams stain (Bartholomew and Mittwer 1952)  Enzyme production, partial puri cation and molecular characterization Enzyme production from strains AUMS60 and AUMS64 was carried out as mentioned above with fermentation media supplemented with 3% (w/v) corn cob (pH 7); at 40°C on a rotary shaker (110 rpm) for 7 days. Filtered cell free extract was concentrated 10-fold via ultra ltration membrane with a molecular weight cutoff of 10-kDa (Millipore TFE system, Bedford, MA, USA). Xylanase activity of all samples were determined as mentioned previously and protein quanti cation was done through Lowry's method using BSA as standard (Lowry et al. 1951).
Electrophoretic analysis of crude and partially puri ed samples was done through native and SDS PAGE (Ornstein and

Biochemical characterization of partially puri ed enzymes
Effect of pH on partially puri ed enzymes from AUMS60 and AUMS64 was studied at pH 5-8 at 40°C, 50°C and 60°C (Sodium acetate and Tris chloride buffer, 0.05 mol l -1 ). Stability at pH 5 and 6 were studied for 120 h at 40°C. Temperature optima was determined by studying catalytic e ciency from 30°C to 90°C. Thermostability was studied at 40°C, 50°C and 60°C. Effect of selective metal ions and chemicals (NaCl, KCl, CaCl 2 , CuSO 4 , MgSO 4 , FeSO 4 , EDTA and SDS) in dose of 1 and 10 mM was studied by incubation at 50°C for 30 min with respective analyte.
Effect of enzyme dose (20-100 U g -1 of substrate) was studied on sacchari cation of puri ed substrate (1% (w/v) birchwood xylan prepared in Acetate buffer, pH 6.0) upto 24 h with incubation at 40°C in rotary shaker at varying speed of 100-140 rpm.

Screening and selection of GHs producing biocatalysts
Out of 294 indigenous bacterial and fungal strains isolated from samples at temperature ranging from 20-60°C, 46% (135) and 12% (35) were cellulolytic and xylanolytic respectively (Fig. 1a). Among all glycoside hydrolase positive strains 69% were bacteria and 31% were fungi with most of them having optimum growth temperature of 40-50°C ( Fig.   1b and 1c), complimenting the site of sample collection.

Phylogenetic analysis
Maximum likelihood phylogenetic tree, generated using ITS region of 40 related sequences through MEGA X and Tamura-Nei model computed the evolutionary distances. The bootstrap consensus tree inferred from 1000 replicates (Felsenstein 1985) was taken to represent the evolutionary history of the taxa analyzed as shown in Supplementary  Fig. S1. Strains AUMS56 and AUMS64 formed a sister node to Aspergillus niger while AUMS60 formed sister node to Aspergillus tubingensis. AUKEMS24 formed a separate clade with sister node to Aspergillus fumigatus. Multigene Bayesian analysis involved sequences of the ITS and nLSU rDNA genes from 39 strains. The aligned dataset consisted of 575 and 860 nucleotides from the ITS and nLSU rDNA gene sequences, respectively. Convergence was assumed as an average standard deviation of split frequencies of 0.004545 was achieved following 3000000 generations. From the generated phylogenetic tree (Fig. 2 (Fig. 3).
Temperature optima for AUMS60 xylanase was determined as 60°C, while xylanases from AUMS64 showed almost same activity at 60°C and 70°C (Fig. 4a). Although all xylanases in the study signi cantly lost their activity at 80°C and above. pH optima for all the enzymes at varied temperature range was determined as 6.0 (Fig. 4b).
Stability of partially puri ed xylanases determined at pH 5 and 6 for 120 hours showed 90% retention in activity till 96 h at pH 6 while 70% activity at pH 5 for AUMS60. Xylanases from AUMS64 were also comparatively stable (>70% residual activity) at both pH 5 and 6 (Fig. 4c).
All xylanases, Xyn60, Xyn64A and Xyn64B worked at pH 6.0, were stable at 40°C for more than 120 h, with considerable loss at 50°C and almost complete loss in activity at 60°C (Fig. 4c and 4d). Although temperature optima for all xylanases was 60°C, they were highly unstable at that temperature. A comparative analysis of xylanases exhibiting differing pH and thermal stability over a wide range reported in previous studies from Aspergillus species is shown in Table 2.  Table 3).

Study of metal ions and chemicals on xylanases suggested
Study of sacchari cation e ciency of all xylanases observed in our investigation suggested maximum sacchari cation with enzyme dose of 100 U g -1 of substrate at 140 rpm, showing 51.1% at 13 h and 52.2% at 24 h incubation for AUMS60 and AUMS64, respectively (Fig. 5).

Discussion
Extensive isolation and screening procedure based on various identi cation techniques suggested AUMS56 (Aspergillus niger), AUMS60 (Aspergillus tubingensis), AUMS64 (Aspergillus niger) and AUKEMS24 (Aspergillus fumigatus) as most potential extracellular xylanase producers from all indigenous isolates taken for study. Complete identi cation of these organisms to species level was done through multigene analysis. AUMS60 and AUMS64 were found to be most ideal extracellular xylanase producers among 294 strains studied and formed a discrete clade with Aspergillus tubingensis and Aspergillus niger with 100% Bayesian Inference posterior probability. Multigene analysis could not be carried out by involving the sequences of 18S rDNA and actin genes because of lack of availability of resources in the database. AUMS60, AUMS64 and AUMS56 were both cellulolytic and xylanolytic, whereas AUKEMS24 was only xylanolytic.
A cocktail of biocatalysts producing cellulases and xylanases simultaneously alludes to be an ideal approach towards the sacchari cation of complex lignocelluloses to obtain simpler sugars in biore neries. AUMS60 and AUMS64 could produce xylanase with crude extract activity of 19.69 and 17.32 U ml -1 , respectively over 3 days of incubation using corncob as substrate. Though a signi cant xylanase production was obtained within 3 days, it persistently increased reaching maximum xylanase activity of 28.58 U ml -1 (1429 U g -1 ) and 24.86 U ml -1 (1243 U g -1 ) for AUMS60 and AUMS64, respectively over 10 days. The activity was further enhanced to 32.29 U ml -1 (AUMS60) and 30.68 U ml -1 (AUMS64) in only 7 days upon changing parameters such as pH and concentration of corn cob (though not optimized).
All these xylanases had pH optima as 6.0 and temperature optima as 60℃ and were stable at both pH 5.0 and 6.0 for more than 120 h, but completely lost temperature tolerance even at 50℃ after 48 h of incubation.
Corncob has signi cant amount of xylan content as compared to numerous other industrially signi cant agricultural residues (wheat bran, rice husk etc), thereby making it a potential substrate for xylanase production (Knob et al. 2014).
Moreover, the abundance and easy availability of corncob in India facilitated for the screening of xylanolytic microbes. ) that presented optimal temperatures ranging from 50-60°C. The enzymes obtained in this study showed pH stability at both pH 5 and 6 over long periods of time (more than 72 hours), which is highly signi cant for their use in industrial processes operating for longer durations. Moreover, the enzymes work perfectly around pH 6, which is the optimal pH for many industrial processes. Enzymes from AUMS60 and AUMS64 showed high temperature stability at 40℃ (greater than 120 h) and could also retain about 80% activity for 4 h and 90% activity for 24 h at 50℃, respectively. The results were more encouraging as compared to various other studies as mentioned in Table 2. Even if the pH stability is slightly less, the storage temperature for our enzymes (40℃) is higher than most of the studies (0-25℃) which indicate their application at even high temperatures.
As reported through previous studies, cations have enhancing or inhibitory action on enzyme catalysis, thus was observed for xylanases in our study with sodium and potassium resulting in 80-100% increase in residual enzyme activity and cupric ion showing potential inhibition. This study is ideally performed with puri ed enzymes and have been reported for xylanase from A. terreus S9, which was strongly inhibited in the presence of (10 mM) Hg 2+ and Cu 2+ while Mg 2+ , Fe 2+ , Co 2+ and EDTA caused slight to moderate inhibition, but Ca 2+ , Mn 2+ and K + promoted the activity (Sharma et al. 2018). Xylanases from A. clavatus NRRL1 were found to show halotolerance, exhibiting a relative activity of 110% in the presence of 10 mM NaCl and maintaining more than 90% relative activity over the NaCl range of 0.04-0.2 M (Pasin et al. 2020). Activity of partially puri ed xylanase from Aspergillus oryzae LC1 increased in presence of Fe +2 , Mg +2 , Mn + , Co + and Ag 2+ but inhibited by CuSO 4 , HgCl 2 , ZnCl 2 and EDTA (Bhardwaj et al. 2019).
The presence of xylan intertwined with cellulosic component in the lignocellulosic biomass creates resistance for e cient conversion of biomass into fermentable sugars. Therefore, we evaluated the performance of the xylanase enzymes obtained from AUMS60 and AUMS64 for depolymerization of birchwood xylan. In our study, the sacchari cation e ciency of AUMS60 and AUMS64 xylanases was found to be 51.1% (4.84 mg ml -1 reducing sugars) and 52.2% (4.95 mg ml -1 reducing sugars) in 13 and 24 hours, respectively when used at an enzyme dosage of 100 Units g -1 substrate. A xylanase from S. variabilis MAB3 was reported to achieve maximum sacchari cation e ciency of 51.1% from birchwood xylan in 72 hours by Sanjivkumar  There is a surge of interest in the investigation for xylanolytic microorganism for their use in various industries. Two potent fungal strains of Aspergillus sp. in this study produced signi cant levels of glycosyl hydrolases on raw corn cob as substrate and the xylanases produced were of high catalytic strength. Pro ling of physico-chemical characteristics of these enzymes namely Xyn60, Xyn64A and Xyn64B suggested their industrial signi cance. Effect of metal ions, pH and temperature optima and stability, catalytic strength of the enzymes provides great scope for further studying enzymatic cocktail for enhanced biomass valorization that can be highly bene cial since utilizing these microbes not only helps in waste reduction and management but also leads to development of cost-effective processes for production of enzymes and successive value-added components. To the best of our knowledge we report that the enzymes in our study hydrolyzed birchwood xylan with higher sacchari cation e ciency than most of the previous ndings. Based on this work, process optimization (e.g. enzymatic hydrolysis of substrate, dosages of AUMS60 and AUMS64 xylanases) may be ensued to improve the transformation rates of hemicellulose from varied lignocelluloses.
Our ndings suggest that these enzymes are better candidates for production of reducing sugar from complex polysaccharides, enabling their use in the various industries.

Declarations
MR conducted experiments, analyzed data and wrote the manuscript. SS contributed in supervision, funding acquisition, review and editing. PS advised for nal correspondence of the manuscript. All authors read and approved the manuscript.

Funding information
The work here was funded by DST-SERB, New Delhi, India (project-grant number YSS/2015/002072) received by SS.
Compliance with ethical standards Con ict of interest

Supplementary Files
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