Production optimization and characterization of mannooligosaccharide generating β-mannanase from Aspergillus oryzae

Accepted Manuscript Production optimization and characterization of mannooligosaccharide generating β-mannanase from Aspergillus oryzae Uttam Kumar Jana, Rahul Kumar Suryawanshi, Bhanu Pratap Prajapati, Hemant Soni, Naveen Kango PII: DOI: Reference:

S0960-8524(18)31084-8 https://doi.org/10.1016/j.biortech.2018.07.143 BITE 20273

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

23 May 2018 26 July 2018 27 July 2018

Please cite this article as: Jana, U.K., Suryawanshi, R.K., Prajapati, B.P., Soni, H., Kango, N., Production optimization and characterization of mannooligosaccharide generating β-mannanase from Aspergillus oryzae, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.07.143

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Production optimization and characterization of mannooligosaccharide generating β-mannanase from Aspergillus oryzae Uttam Kumar Jana, Rahul Kumar Suryawanshi, Bhanu Pratap Prajapati, Hemant Soni, Naveen Kango*

Department of Microbiology, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar, MP 470003, India

*e-mail: [email protected]

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Abstract: A multi-tolerant β-mannanase (ManAo) was produced by Aspergillus oryzae on copra meal, a low-cost agro waste. Under statistically optimized conditions, 4.3-fold increase in β-mannanase production (434 U/gds) was obtained. Purified ManAo had MW ~34 kDa and specific activity of 335.85 U/mg with optimum activity at 60°C and at pH 5.0. Activity of ManAo was enhanced by most metal ions and modulators while maximum enhancement was noticed with Ag+ and Triton X-100. Km and Vmax were 2.7 mg/mL and 1388.8 µmol/min/mg for locust bean gum while the enzyme showed lower affinity towards konjac gum (8.8 mg/mL, 555.5 µmol/min/mg). Evaluation of various thermodynamic parameters indicated high-efficiency of the ManAo with activation energy 12.42 KJ/mol and 23.31 KJ/mol towards LBG

and

konjac

gum,

respectively.

End

product

analysis

of

β-mannanase action by fluorescence assisted carbohydrate electrophoresis (FACE) revealed the generation of sugars from DP 1- 4 with some higher DP MOS from different mannans.

KEYWORDS: Aspergillus oryzae, FACE, locust bean gum (LBG), mannooligosaccharides (MOS), zymography

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1. Introduction Mannans, chief component of hemicellulose, occur widely as structural and storage polysaccharide in plants. Locust bean gum (LBG), guar gum (GG), konjac gum (KG), ivory nut mannan and coffee bean mannan are some of the well known mannans (Soni et al., 2016). Some of the agro-industrial wastes, such as palm kernel cake and copra meal have drawn attention as low-cost and abundant mannan-rich substrates (Ahirwar et al., 2016b). Mannans, being heteropolysaccharides, are acted upon by various enzymes including β-1, 4-mannanase (EC 3.2.1.78), α-galactosidase (EC 3.2.1.22), β-mannosidase (EC3.2.1.25) and β-glucosidase (EC 3.2.1.21). Owing to their availability and renewability, mannans make a suitable substrate for biofuel generation in bio-refineries (Shimizu et al., 2015; Shukor et al., 2016). Mannanases are being exploited in mitigating the anti-nutritional effects of mannan-rich feed so that their digestibility is enhanced (Ma et al., 2017). Mannooligosaccharides (MOS) generated due to random action of endo-β-mannanase are used as prebiotics to promote the health of gut microbiota (Srivastava et al., 2017). β-mannanases are being employed in several industries for wide range of applications such as pulp bio-bleaching, detergent augmentation, feed quality upgradation and oil drilling (Kalidas et al., 2017; Srivastava et al., 2014; EcemÖner et al., 2018). Prior studies on β-mannanase production from fungi e.g. Sclerotium rolfsii (Gübitz et al., 1996), Aspergillus terreus FBCC 1369 (Soni et al., 2016), Malbranchea cinnamomea NFCCI 3724 (Ahirwar et al., 2016a), Pyrenophora phaeocomes S-1 (Rastogi et al., 2016), Trichoderma reesei (Ma et al., 2017), Aspergillus oryzae RIB 40 (Sakai et al., 2017), Rhizomucor miehei (Li et al., 2018), Aspergillus tubingensis NKBP-55 (Prajapati et al., 2018) suggest that fungi are prolific β-mannanase producers. Solid state fermentation employing particulate substrates originating from agro-wastes such as copra meal (CM), rice husk, wheat bran, palm kernel cake (PKC), wheat straw is a cost-effective and viable method (Ahirwar et 3

al., 2016b). In India, every year more than 22.5×10 4 ton CM, consisting of solid particulate mannan-rich agro-residues, is generated after oil extraction from coconut fruit (Prajapati et al., 2018). Although, it is rich source of minerals, carbohydrates and amino acids, only 25–30 % of CM is used as fodder while the rest is dumped as waste (Rodsamran and Sothornvit, 2018). In the present study, β-mannanase production by Aspergillus oryzae MTCC 1846 was statistically optimized in solid state fermentation (SSF) using CM as substrate. Purification and characterization of β-mannanase along with enzyme kinetics and thermodynamic properties was done. Also, effective use of purified β-mannanase in hydrolysis of different mannans and liberation of MOS was demonstrated using FACE. 2. Materials and Methods 2.1 Chemicals Mannose (M1), mannobiose (M2), mannotriose (M3) and mannotetraose (M4) were procured from Megazyme, Ireland. Copra meal was obtained from Parker Industry, Tamilnadu, India. Precision plus protein marker was obtained from Biorad, USA. Locust bean gum, 7-amino-1, 3-naphthalene disulfonic acid monopotassium salt monohydrate (ANDS), sodium cyanoborohydride, 3, 5-dinitrosalicylic acid (DNS) and other chemicals and reagents were of analytical grade and purchased from Sigma Aldrich Chemical Co. (USA). 2.2 Microorganism Aspergillus oryzae MTCC 1846, obtained from Microbial Type Culture Collection (MTCC, INDIA) was used in production of β-mannanase. The fungus was cultured on Potato dextrose agar (PDA) at 28°C and maintained on the slants of the same medium at 4°C.

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2.3 β-mannanase production in SSF β-mannanase was produced by cultivating the fungus in 150 mL Erlenmeyer flasks containing CM (3g) moistened with 9 ml (74%) of de-ionized water. Moisture content (%) of the sample was calculated by deducting the dry weight (wd) from the wet weight (ww) using following formula: Moisture content (%) = (ww - wd)/ ww *100 Flasks were autoclaved and inoculated with 3 mycelial discs (6 mm) cut from five day old fungal culture and incubated at 28°C for five days. After incubation, 30 mL citrate buffer (50 mM, pH 5.0) was added and enzyme was extracted by shaking at 140 rpm for 30 min at 4°C. The slurry thus obtained was centrifuged at 9000g for 15 min and cell-free supernatant was used in further studies (Prajapati et al., 2018). 2.4 Optimization of β-mannanase production Independent variables were optimized to maximize the β-mannanase production using central composite design (CCD). Two independent variables, A (moisture) and B (pH), were optimized for β-mannanase production. Design Expert 11.0 was used for designing and analyzing the experimental data. Total 13 experiments based on a 2-factor, 5-level (–α, –1, 0, +1, +α) CCD with 4 trials of factorial design, 4 trials of axial point and 5 replicate trials of central point were conducted to analyze the response pattern and to determine the optimum combination of factors. Response surface model (RSM) was assessed for β-mannanase activity (U/gds). The prediction of optimum independent variables was made by fitting the experimental data using second order polynomial equation.

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2.5 Enzyme assay and protein estimation The activity of β-mannanase was determined using LBG as substrate. LBG solution (0.9 mL; 0.5% w/v in 50 mM citrate buffer, pH 5.0) and 100 µL of appropriately diluted enzyme was incubated for 10 min at 50°C. The enzyme activity was stopped by adding 1.5 mL DNS reagent and boiling for 5 min (Maijala et al., 2012). One unit of β-mannanase activity was defined as the amount of the enzyme required to release 1 µmol of mannose per min under the assay conditions. Protein concentration was determined by Bradford method with bovine serum albumin as standard. 2.6 Purification of β-mannanase Crude enzyme was fractionated with ammonium sulfate saturation (40-90%). After centrifugation, each fraction was dissolved in 50 mM citrate buffer (pH 5.0) and dialyzed against the same buffer using dialysis membrane (cut-off size 10 kDa). Protein content and β-mannanase activity was evaluated for each fraction by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Fractions showing β-mannanase activity were pooled and concentrated by lyophilization. Concentrated enzyme was loaded into Sephadex G-50 column (1 x 45 cm) equilibrated with citrate buffer (pH 7.0). Elution was performed with the same buffer at a flow rate of 1 mL/min. Fractions of 1 mL/min were collected and those with β-mannanase activity were pooled. All the purification procedures were performed at 4°C. Native-PAGE was performed to estimate the molecular weight and enzyme activity using 12% resolving gel containing 0.02% glucomannan. Culture filtrate, protein precipitates and gel filtration fractions were resolved in the gel at 4°C for 3 h at 90V. After electrophoresis, the gel was cut into two parts; first part with protein marker (Biorad, USA) was stained with coomassie brilliant blue R-250 while the second part was incubated at 50°C for 60 sec. After incubation, gel was stained with Congo red (0.1% w/v)

6

for 20 min and de-stained by NaCl (1M) to visualize zone of β-mannanase activity against red color background (Prajapati et al., 2018). 2.8 Effect of temperature and pH on ManAo Optimum temperature of ManAo was determined by conducting enzyme activity in the range of 30-90°C for 10 min. For determining thermostability, the enzyme was pre-incubated at 60, 70 and 80°C upto 90 min. Optimum pH was determined by performing β-mannanase activity with LBG 0.5% (w/v) dissolved in different buffers viz. sodium citrate buffer (pH 3-5), dipotassium hydrogen phthalate/potassium dihydrogen orthophospate (pH 6), Tris-HCl buffer (pH 7-9) and glycine-NaOH (pH 10). pH stability was estimated by incubating ManAo at pH 3, 5 and 8 for 24 h at room temperature. The residual activity of ManAo was estimated against control. 2.9 Effect of metal ions, solvents, surfactants and other compounds on ManAo activity The effect of metal ions, organic solvents, surfactants and other compounds (5 mM or 5%) was determined by pre-incubating pure enzyme with various additives and relative activity of ManAo was measured as described earlier (Prajapati et al., 2018). 2.10 Substrate specificity and kinetic parameters Substrate specificity of ManAo was analyzed with different polysaccharides such as LBG, GG, KG, beech wood xylan, fenugreek gum, carboxymethyl cellulose (CMC) and gum arabic by incubating the enzyme with these substrates (0.5% w/v). Kinetic parameters were determined using different concentrations of LBG (0.125-10 mg/mL) and KG (1-10 mg/mL). Michaelis-Menten constant (Km) and maximum velocity (Vmax) were determined using Lineweaver-Burk double reciprocal plot. 2.11 Thermodynamic characteristics of ManAo 7

Thermodynamic characteristics were evaluated for LBG and KG hydrolysis by ManAo using Eyring absolute rate equation derived from the transition state theory (Regmi et al., 2016). Turn over number, Kcat= Vmax/ [ET]

where, ET=Total enzyme

The enthalpy (∆H), free energy (∆G), entropy of activation (∆S), free energy of substrate binding (∆GE-S), free energy of transition state formation (∆GE-T) and temperature quotient (Q10) were calculated as described earlier (Dixon and Webb, 1979; Regmi et al., 2016). The

activation

energy

(Ea)

was

calculated

using

CALC (http://www.calctool.org/CALC/chem/kinetics/act_en). 2.12 Fluorescence-Assisted Carbohydrate Electrophoresis (FACE) Different mannans (GG, LBG, KG and CM 1% w/v dissolved in citrate buffer, pH 5.0) were incubated with ManAo at 60°C and aliquots were collected at regular time intervals. End product analysis of mannan hydrolysate was carried out using FACE with some modifications (Zhang et al., 2015; Liu et al., 2018). For fluorescent labeling, mannan hydrolysate (10 µL) was mixed with 10 µL of 0.2 M 7-amino-1, 3-naphthalene disulfonic acid monopotassium salt monohydrate (prepared in 15% acetic acid) and incubated in the solution for 1 h at room temperature in dark.

To this, 10 µL of 1 M sodium

cyanoborohydride (prepared in dimethyl sulfoxide) was added and it was further incubated at 40°C for 12 h. Native PAGE was conducted by preparing 40% acrylamide gel in 375 mM Tris-HCl buffer (pH 8.8) without stacking gel. Loading buffer comprised of sucrose (50% w/v) and bromophenol blue (3:2). Sample was mixed with loading buffer in a ratio of 3:5 and then loaded into wells (15 µL). Electrophoresis was performed at 15 mA for ~1.5 h and bands were visualized in UV-transilluminator (310 nm).

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3. Results and Discussion 3.1 Production optimization of ManAo by A. oryzae A. oryzae MTCC 1846 produced 101 U/gds of β-mannanase on CM under unoptimized conditions in SSF.

Optimization of pH and moisture values by RSM led to

4.3-fold increase (434.6 U/gds) (Fig. 1A & 1B). The validation of quadratic model was done under optimum conditions i.e. moisture content 74% and pH 9.0. Experimental value was 434.6 U/gds which indicated the close agreement with predicted β-mannanase production (433.9 U/gds). Recently, 7-fold increment (422 U/gds) in β-mannanase yield was achieved by optimizing pH and moisture content in case of A. terreus FBCC1369 grown on CM (Soni et al., 2016). Optimization of pH and moisture content in case of β-mannanase production by A. niger ATCC 20114 lead to 1.3-fold (1495 nkat/mL) increase on GG (Mohamad et al., 2011). β-mannanase production by M. cinnamomea NFCCI 3724 was increased by 5-fold in SSF conducted on PKC (Ahirwar et al., 2016b). Improved β-mannanase production up to 8-fold (4431.93 U/g) was achieved by P. chrysogenum QML-2 after statistical optimization (Zhang and Sang, 2015). 3.2 Purification of β-mannanase ManAo was purified to apparent homogeneity by ammonium sulfate fractionation followed by gel filtration. 37.79% protein yield recovery was achieved after 70% ammonium sulfate fractionation with specific activity 216.71 U/mg. Gel filtration resulted in 6.92-fold purification (Table 1) and a single band of ~34 kDa (Fig. 2). Purified enzyme hydrolyzed mannan on gel and a clear zone of hydrolysis was seen in the same region. Eriksson and Winell (1968) purified β-mannanase (42 kDa) from a commercial preparation ‘Cellulase 36’ sourced from fungus belonging to Aspergillus niger-oryzae group with 11% yield and 61-fold purification using ammonium sulfate precipitation and Sephadex G-75. A recombinant low 9

molecular weight β-mannanase (19 kDa) from A. oryzae RIB 40 has been reported (Sakai et al., 2017). Regalado et al. (2000) purified a high molecular weight β-mannanase (110 kDa) from A. oryzae CECT 2094 grown on copra paste in SSF. β-mannanases ranging from 18-43 kDa are reported from some fungi including Aspergillus nidulans (43 kDa) (Rosengren et al., 2014), Thielavia arenaria XZ7 (40.8 kDa) (Lu et al., 2013), Penicillium occitanis Pol6 (18 kDa) (Blibech et al., 2010). 3.3 Characterization of β-mannanase 3.3.1 Effect of pH and temperature ManAo had optimum activity at pH 5.0 and retained 60% activity at pH 3.0 after 4 h indicating tolerance for acidic pH (Fig. 3A). Similar pH optimum of β-mannanase was reported for Chaetomium sp. CQ31 (Katrolia et al., 2012), Thielavia arenaria XZ7 (Lu et al., 2013), Aspergillus nidulans XZ3 (Lu et al., 2014), Pholiota adiposa (Ramachandran et al., 2014). ManAo had optimum activity at 60°C (Fig. 3B) which was similar to Myceliophthora thermophila β-mannanase (Katsimpouras et al., 2016) while β-mannanases of T. arenaria XZ7 and A. nidulans XZ3 had 75°C and 80°C as their temperature optima, respectively. 3.3.2 Effect of metal ions, solvents, surfactants and other compounds on ManAo activity ManAo was not only tolerant but its activity was positively altered by some metal ions, surfactants (Triton X-100, SDS, Tween-20 and Tween-80), sulfhydryl reagent (sodium azide) and organic solvents (Table 2). In case of Ag+, its activity increased up to 140% whereas Cu2+ and K+ also enhanced activity upto 130%. ManAo also exhibited tolerance for organic solvents and surfactants and its activity was ameliorated in presence of Triton X-100 (145%) and t-amyl alcohol (130%). Activity of Paenibacillus sp. HY-8 β-mannanase is reported to increase in the presence of Ca2+, Triton X-100 and sodium azide (Kim et al., 2017). Bacillus

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sp. N16-5 β-mannanase was inhibited in the presence of Zn2+ and Fe2+ (Ma et al., 2004). β-mannanase from Penicillium pinophilum C1 and Bacillus subtilis G1 were inhibited by SDS (Cai et al., 2011; Hang et al., 2012). Triton X-100 and other surfactants influence the enzyme structure due to polymerization in structural moiety of the protein and presence of negatively charged acidic amino acids in protein helps to preserve interaction by SDS/surfactant (Lin et al., 1995; Zhang et al., 2012). Earlier reports suggested that Hg2+ and Ag+ had inhibitory effect on β-mannanase activity (Katrolia et al., 2012; Lu et al., 2014; Ma et al., 2004; Li et al., 2012), however, ManAo activity was stimulated in presence of Hg2+ (116%) and Ag+ (140%). Heavy metal compounds such as mercuric chloride and silver nitrate are enzyme inhibitors and both affect the enzyme folding by binding with sulfhydryl (S-H-) groups. Mercuric chloride forms mercaptide with sulfhydryl group and partially unfolds the enzyme (Printz and Gounaris, 1972; Snodgrass et al., 1960). Owing to the presence of more sulfhydryl groups in the enzyme, the denaturating effect of Hg2+ and Ag+ is minimized. In our findings, 5 mM of Hg2+ and Ag+ did not affect the enzyme negatively; however, 10 mM concentration of Hg2+ and Ag+ reduced 50% activity of the mannanase. 3.3.3 Substrate specificity and kinetic parameters ManAo showed higher affinity towards LBG and KG but had lower affinity for GG. It showed no activity with gum arabic, fenugreek gum and CMC under assay conditions. Kinetic parameters, Km and Vmax for LBG and KG were 2.77 mg/mL, 1388.8 µmol/min/mg and 8.8 mg/mL, 555.5 µmol/min/mg, respectively. Based on Lineweaver–Burk plot, Kcat and Kcat/Km values of ManAo toward LBG were 3858.02 S-1 and 1388.8 mL S-1mg-1, respectively. The

affinity

β-mannanases

of such

ManAo

towards

as

Gloeophyllum

LBG

was

trabeum

higher

than

CBS900.73

other (3.71

fungal mg/mL),

A. terreus FBCC 1369 (5.9 mg/mL), Penicillium oxalicum GZ-2 (7.6 mg/mL) and Penicillium freii F63 (7.8 mg/mL) (Wang et al., 2016; Soni et al., 2016; Liao et al., 2014; 11

Wang et al., 2012). ManAo had higher Kcat value compared to mannanases from A. oryzae RIB 40 (Sakai et al., 2017) and P. occitanis Pol6 (Blibech et al., 2010). Results indicated high substrate affinity and catalytic efficiency of ManAo towards GG, LBG and KG as compared to other fungal β-mannanases. 3.3.4 Thermodynamic analysis of mannan hydrolysis by ManAo Thermodynamic characteristics of purified ManAo such as activation energy (Ea), enthalpy (∆H), Gibb’s free energy (∆G), and entropy (∆S) with LBG and KG as substrate are given in Table 3. Entropy in case of LBG hydrolysis was much lower than KG which means the binding property of enzyme favor galactomannan (LBG) over glucomannan (KG). In addition, ManAo had high substrate to product transition property than binding to the substrate because ∆GE-S values were positive in both cases where ∆GE-T was negative (Table 3). The activation energy of ManAo for both LBG and KG were 12.42 KJ/mol and 23.31 KJ/mol were much lower in comparison to Bacillus sp. CFR1601 (87.4 kcalmol-1) (Srivastava et al., 2016) and Sphingobacterium sp. GN25 β-mannanase (36.0 kJ/mol) (Zhang et al., 2015). Temperature quotient (Q10) is a measure of temperature sensitivity of an enzymatic reaction upon the increase of temperature by 10°C. The Q10 of ManAo was 1.15 and 1.30 for LBG and KG, respectively. In both the cases the enzyme showed thermal independence (Q10 1.0-1.5) due to increase in the temperature, maximum activity (Q10 2.0-4.0) and lower activity (Q10 0.2-0.8). ManAo had Q10 values similar to Bacillus sp. CSB39 (Q10 1.32) (Regmi et al., 2016), Bacillus subtilis subsp. inaquosorum CSB31 (Q10 1.40) (Regmi et al., 2017) and Sphingobacterium sp. GN25 (Q10 1.6) (Zhang et al., 2015).

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3.4 Generation of sugars from different mannans by ManAo The hydrolysis products generated by ManAo action on various mannans and CM were analyzed by FACE. As seen in the image, the enzyme liberated high amounts of M1, M2, M3 and high DP oligomers from LBG (Fig. 4A). Similarly, GG was hydrolyzed into M1, M2, M3 and M4 equivalents with several high DP MOS (Fig. 4B). The major product in case of KG were M1 and M2 with traces of M3 and M4 (Fig. 4C) while CM when subjected to hydrolysis yielded only M1 and M2 as the major products. High DP MOS were not observed in case of KG and CM. FACE was very effective in resolving the sugars of varying DP as distinct

bands.

Neosartorya

fischeri

β-mannanase

effectively

hydrolyzed

various

galactomannans liberating M2 and GM4 as the major products (Yang et al., 2015). β-mannanase from Clostridium thermocellum ATCC 27405 hydrolyzed carob galactomannan into DP 1-5 sugars after 24 h (Ghosh et al., 2013). In the present study, ManAo hydrolyzed both galactomannans (LBG and guar gum) as well as glucomannan (KG) effectively into MOS. 4. Conclusion Enhanced production of A. oryzae β-mannanase was achieved by RSM on low-cost agro-residue, CM. Characterization of purified β-mannanase revealed its multi-tolerant nature. The enzyme was functional in the presence of broad range of chemicals which signifies that it can be used in a variety of industrial applications. ManAo hydrolyzed different indigestible mannans to useful prebiotic MOS while mannose can be utilized in bio-refineries for generation of second generation biofuel. 6. Abbreviations

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FACE, fluorescence assisted carbohydrate electrophoresis; MOS, mannooligosaccharide; LBG, locust bean gum; GG, guar gum; KG, konjac gum; CM, copra meal; PKC, palm kernel cake; SSF, solid state fermentation; ANDS, 7-amino-1, 3-naphthalene disulfonic acid monopotassium salt monohydrate; CCD, central composite design; RSM, response surface method; CMC, carboxymethyl cellulose; DP, degree of polymerization. 7. Acknowledgements Author UKJ is grateful to UGC, New Delhi for providing financial assistance as PhD scholarship. BPP and RKS acknowledge financial support from UGC, New Delhi as national fellowship. Authors thank Sophisticated Instrumentation Centre (SIC), Dr. Harisingh Gour Vishwavidyalaya, Sagar, India for instrumentation facilities. 8. Appendix A. Supplementary data Supplementary data related with this study can be found in the electronic version of this article. 9. References 1. Ahirwar, S., Soni, H., Rawat, H.K., Ganaie, M.A., Pranaw, K., Kango, N., 2016a. Production optimization and functional characterization of thermostable β-mannanase from Malbranchea cinnamomea NFCCI 3724 and its applicability in mannotetraose (M4) generation. J. Taiwan. Inst. Chem. Eng. 63, 344-353. 2. Ahirwar, S., Soni, H., Rawat, H.K., Prajapati, B.P., Kango, N., 2016b. Experimental design of response surface methodology used for utilization of palm kernel cake as solid substrate for optimized production of fungal mannanase. Mycology. 7(3), 143-153.

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3. Blibech, M., Ghorbel, R.E., Fakhfakh, I., Ntarima, P., Piens, K., Bacha, A.B., Chaabouni, S.E., 2010. Purification and characterization of a low molecular weight of βmannanase from Penicillium occitanis Pol6. Appl. Biochem. Biotechnol. 160(4), 12271240. 4. Cai, H., Shi, P., Luo, H., Bai, Y., Huang, H., Yang, P., Yao, B., 2011. Acidic βmannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application. J. Biosci. Bioeng. 112(6), 551-557. 5. Dixon, M., Webb, E.C., 1979. Enzyme kinetics, 3rd edition, Academic Press, New York. 6. EcemÖner, B., Akyol, Ç., Bozan, M., Ince, O., Aydin, S., Ince, B., 2018. Bioaugmentation with Clostridium thermocellum to enhance the anaerobic biodegradation of lignocellulosic agricultural residues. Bioresour. Technol. 249, 620-625. 7. Eriksson, K.E., Winell, M., 1968. Purification and characterisation of a fungal βmannanase. Acta Chem. Scand., 22, 1924-1934. 8. Ghosh, A., Luís, A.S., Brás, J.L., Fontes, C.M., Goyal, A., 2013. Thermostable recombinant β-(1→4)-mannanase from C. thermocellum: biochemical characterization and manno-oligosaccharides production. J. Agric. Food Chem. 61(50), 12333-12344. 9. Gübitz, G., Hayn, M., Sommerauer, M., Steiner, W., 1996. Mannan-degrading enzymes from Sclerotium rolfsii: Characterisation and synergism of two endo β-mannanases and a β-mannosidase. Bioresour. Technol. 58(2), 127-135. 10. Hang, T.T., Quyen, D.T., Dao, T.T., Le, S., Nguyen, T., 2012. Cloning, high-level expression, purification, and properties of a novel endo-β-1, 4-mannanase from Bacillus subtilis G1 in Pichia pastoris. J. Microbiol. Biotechnol. 22, 331-338. 15

11. Kalidas, N.R., Saminathan, M., Ismail, I.S., Abas, F., Maity, P., Islam, S.S., Shaari, K., 2017. Structural characterization and evaluation of prebiotic activity of oil palm kernel cake mannanoligosaccharides. Food Chem. 234, 348-355. 12. Katrolia, P., Zhou, P., Zhang, P., Yan, Q., Li, Y., Jiang, Z., Xu, H., 2012. High level expression of a novel β-mannanase from Chaetomium sp. exhibiting efficient mannan hydrolysis. Carbohydr. Polym. 87(1), 480-490. 13. Katsimpouras, C., Dimarogona, M., Petropoulos, P., Christakopoulos, P., Topakas, E., 2016. A thermostable GH26 endo-β-mannanase from Myceliophthora thermophila capable of enhancing lignocellulose degradation. Appl. Microbiol. Biotechnol. 100(19), 8385-8397. 14. Kim, D.Y., Chung, C.W., Cho, H.Y., Rhee, Y.H., Shin, D.H., Son, K.H., Park, H.Y., 2017.

Biocatalytic characterization of an

endo-β-1,4-mannanase produced by

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21

Figure captions: Figure 1. Production optimization of ManAo from A. oryzae. A: 3D response surface plot and B: Contour plot for the effect of moisture content and pH on ManAo production. Figure 2. Native-PAGE and zymography of ManAo. MW: molecular marker, Native PAGE: Crude enzyme (L1), 70% ammonium sulfate fraction (L2), Sephadex G-50 fraction (L3), Zymography: Crude enzyme (L4), Sephadex G-50 fraction (L5). Figure 3. Temperature and pH profile of A. oryzae ManAo (A) pH optima and stability (B) temperature optima and stability. Figure 4. FACE analysis of different mannan hydrolysates after ManAo treatment (A) LBG (B) guar gum (C) konjac gum. For (A) & (B), L1: 1 h, L2: 2 h, L3: 3 h, L4: 4 h, L5: 24 h and for (C) L1: 24 h, L2: 4 h, L3: 3 h, L4: 2 h, L5: 1 h incubation period. L6: Standards: M1, M2, M3 and M4.

Figure 1

Figure 2

KDa MW L1 250 75 50 37

25 20

15

L2

L3

L4

L5

Figure 3

Time (min)

A

0

50

100

150

200

250

300

120

Relative activity (%)

100

80

pH pH Optima Optima StabilityatatpH Stability pH33 Stability Stability at pH 5 Stability Stability at pH 8

60

40

20

0 4

6

8

10

12

pH Time (min)

B

0

20

40

60

80

Temperature Optima Stability at 60°C Stability at 70°C Stability at 80°C

100

Relative activity (%)

100

120

80

60

40

20

0 30

40

50

60

70

Temperature (°C)

80

90

100

Figure 4

L1

L2

L3

L4

L5

L1

L6

A

L2

L3

L4

L5

L6

B

High DP

High DP M4

M4

M3 M3 M2

M2

M1

C

L1

L2

L3

M1

L4

L5

L6 High DP M4 M3

M2

M1

Table legends

Table 1: Purification profile of ManAo from A. oryzae MTCC 1846 Table 2: Effect of metal ions, solvents, surfactants and other compounds on the activity of ManAo. Table 3: Kinetic and thermodynamic properties of ManAo

Table 1

22

Purification Step

Total

Total

Specific

protein

activity

activity

(mg)

(U)

(U/mg)

Crude

172.5

5234.53

Ammonium sulfate fractionation (70%)

9.13

Sephadex G-50

1.08

Purification fold

Yield recovery (%)

30.34

1

100

1978.58

216.71

7.14

37.79

362.72

335.85

11.06

6.92

Table 2

23

Chemicals

Concentration

Control

a

Relative activity (%) 100

Zn2+

5 mM

109.12±13.35

Fe2+

5 mM

107.99±25.15

Hg2+

5 mM

116.82±17.51

Hg2+

10 mM

55.92±12.51

Cu2+

5 mM

131.26±16.34

K+

5 mM

130.31±3.98

Rb+

5 mM

118.21±17.56

Mn2+

5 mM

122.37±27.25

Ag+

5 mM

140.53±21.52

Ag+

10 mM

33.21±18.52

Mg2+

5 mM

113.09±20.92

Na+

5 mM

132.02±16.69

Ca2+

5 mM

126.15±12.28

(NH4)2SO4

5 mM

119.79±18.04

Hexane

5% (v/v)

120.09±17.63

Toluene

5% (v/v)

117.06±23.31

Methanol

5% (v/v)

118.58±32.14

Acetonitrile

5% (v/v)

108.17±24.91

t-amyl alcohol

5% (v/v)

134.29±28.05

Acetone

5% (v/v)

113.47±21.27

Glycerol

5% (v/v)

97.38±31.40

Propan-1-ol

5% (v/v)

122.75±23.81

Aniline

5% (v/v)

124.45±20.97

Chloroform

5% (v/v)

123.12±4.76

24

Triton X-100

5% (v/v)

145.09±33.63

SDS

5% (v/v)

128.61±26.38

Tween-80

5% (v/v)

122.55±1.18

Tween-20

5% (v/v)

125.96±32.29

Urea

5 mM

113.66±18.45

EDTA

5 mM

123.12±11.65

Sodium azide

5 mM

136.75±23.00

a

Values are mean ±standard deviation (n=3).

Table 3

Parameters

Properties

Substrate Locust bean gum

Konjac Gum

Vmax

1388.8 µmol/min/mg

555.5 µmol/min/mg

Km

2.7 mg/mL

8.8 mg/mL

Kcat

3858.02 S-1

1543.21 S-1

1388.8 mL mg-1 S-1

173.61 mL mg-1 S-1

12.42 KJ/mol

23.31 KJ/mol

Kcat/Km Ea

25

∆H

9.7 KJ/mol

20.63 KJ/mol

∆G

10.02 KJ/mol

7.56 KJ/mol

∆S

-0.911 J mol-1 K-1

40.44 J mol-1 K-1

∆GE-S

2.743 KJ/mol

5.867 KJ/mol

∆GE-T

-19.43 KJ/mol

-13.84 KJ/mol

1.154

1.308

Q10

26

Highlights 1. 4.3-fold increase in β-mannanase production (434 U/gds) by A. oryzae was obtained on copra meal 2. β-mannanase was multi-tolerant with Km and Vmax as 2.7 mg/mL and 1388.8 µmol/min/mg 3. FACE analysis revealed generation of M1, M2, M3 and high DP mannooligosaccharides

27

Graphical Abstract

Production of β-mannanase

Aspergillus oryzae MTCC 1846

L1

L2

L3

L4

L5

Production optimization by RSM

KDa MW L1 250

L6

β-mannanase

M7 M6 M5

L2

L3

L4

L5

75 50 37

M4

Mannan hydrolysis

M3 M2

25 20

Time (min) 0

20

40

60

80

100

120

15

M1

L2

L3

L4

L5

Re lativ e activ ity (% )

L1

FACE analysis of hydrolysate

100

80

L6

Purification of β-mannanase

60

40

20

0

30

40

50

60

70

80

90

100

pH and temperature optima

28