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Purification and characterization of endo β-1,4-d-glucanase from Trichoderma harzianum strain HZN11 and its application in production of bioethanol from sweet sorghum bagasse

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Purification and characterization of endo β-1,4-d-glucanase from Trichoderma harzianum strain HZN11 and its application in production of bioethanol from sweet sorghum bagasse
           1 3 3 Biotech  ISSN 2190-572XVolume 6Number 1 3 Biotech (2016) 6:1-10DOI 10.1007/s13205-016-0421-y Purification and characterization of endo-1,4-d-glucanase from Trichodermaharzianum  strain HZN11 and itsapplication in production of bioethanol rom sweet sorghum bagasse Zabin K. Bagewadi, Sikandar I. Mulla & Harichandra Z. Ninnekar           1 3 Your article is published under the CreativeCommons Attribution license which allowsusers to read, copy, distribute and makederivative works, as long as the author ofthe srcinal work is cited. You may self-archive this article on your own website, aninstitutional repository or funder’s repositoryand make it publicly available immediately.  ORIGINAL ARTICLE Purification and characterization of endo  b -1,4- D -glucanasefrom  Trichoderma harzianum  strain HZN11 and its applicationin production of bioethanol from sweet sorghum bagasse Zabin K. Bagewadi 1 • Sikandar I. Mulla 1 • Harichandra Z. Ninnekar 1 Received: 15 January 2016/Accepted: 27 March 2016   The Author(s) 2016. This article is published with open access at Abstract  An acidophilic-solvent-thermostable endo  b -1,4- D -glucanase produced from a potential  Trichodermaharzianum  strain HZN11 was purified to homogeneity byDEAE-Sepharose and Sephadex G-100 chromatographywith 33.12 fold purification with specific activity of 66.25U/mg and molecular mass of       * 55 kDa. The optimumtemperature and pH were 60   C and 5.5 retaining 76 and85 % of activity after 3 h, respectively. It showed stabilitybetween pH 4.5–6.0 and temperature between 50–70   Cindicating thermostability. Endo  b -1,4- D -glucanase wasactivated by Ca 2 ? and Mg 2 ? but inhibited by Hg 2 ? , Pb 2 ? and Cd 2 ? . The effect of thiol reagents, metal chelators,oxidizing agents and surfactants on enzyme activity hasbeen studied. Purified endo  b -1,4- D -glucanase exhibitedhighest specificity towards carboxymethyl cellulose.Kinetic analysis showed the  K  m ,  V  max  and  K  i  (cellobioseinhibitor) of 2.5 mg/mL, 83.75 U/mg and 0.066 M,respectively. The storage stability of purified endo  b -1,4- D -glucanase showed a loss of mere 13 % over a period of     60 days. The hydrolysis efficiency of purified endo  b -1,4- D -glucanase mixed with cocktail was demonstrated overcommercial enzyme. Optimized enzymatic hydrolysis of     sweet sorghum and sugarcane bagasse released 5.2 g/g(36 h) and 6.8 g/g (48 h) of reducing sugars, respectively.Separate hydrolysis and fermentation of sweet sorghumbagasse yielded 4.3 g/L bioethanol (16 h) confirmed by gaschromatography–mass spectrometry (GC–MS). Morpho-logical and structural changes were assessed by scanningelectron microscopy (SEM) and Fourier transform infraredspectroscopy. Elemental analysis was carried out by SEMequipped with energy dispersive X-ray technique. Theseunique properties prove the potentiality of enzyme forbiomass conversion to biofuel and other industrialapplications. Keywords  Endo  b -1,4- D -glucanase    Trichodermaharzianum  strain HZN11    Purification    Characterization   Enzymatic hydrolysis    Bioethanol Introduction In the current scenario, the major concerns are towards thediminishing of fossil fuels which have forced the energyindustries and researchers to develop alternatives to theexisting fuels (Bentsen and Felby 2012). One of theattractive sustainable substitutes is the microbial produc-tion of bioethanol from lignocellulosic wastes as it is cost-effective and renewable (Ren et al. 2009). Plant biomassconstitute of cellulose which is the major organicpolysaccharide found in the biosphere (Bhat and Bhat1997) and is renewable. Biodegradation of plant basedbiomass requires cellulose and hemicellulose saccharifyingenzymes. For example, cellulases participate in sacchari-fication of biomass for bioethanol production (Dhillon et al.2011), by mainly acting on  b -1,4-glycosidic bonds of cel-lulose. Cellulolytic enzymes have been classified as:endoglucanase (endo-1,4- D -glucanase, EG), cellobiohy-drolase (exo-1,4- D -glucanase, CBH) and glucosidase (1,4- D -glucosidase, BG) (Saha 2004), which have been shown toact synergistically for effective degradation (Lynd et al. Electronic supplementary material  The online version of thisarticle (doi:10.1007/s13205-016-0421-y) contains supplementarymaterial, which is available to authorized users. &  Harichandra Z. 1 Department of Biochemistry, Karnatak University,Dharwad 580 003, Karnataka, India  1 3 3 Biotech (2016) 6:101 DOI 10.1007/s13205-016-0421-y  2002) whereas xylanases (1,4- b - D -xylanohydrolase)hydrolyze xylan, a major component of hemicellulose(Zhang et al. 2011). The fungal endoglucanases finds itsapplications in biomass bioconversions, pulp and paper,textile, detergents, starch processing, grain alcohol fer-mentation, brewery, wine making, extraction of fruit andvegetable juices (Karmakar and Ray 2011; Kuhad et al.2011). These applications certainly require endoglucanaseswith industrial attributes like thermostability, stability atvarying pH, substrate specificities (Bhat 2000), solventtolerant, detergent compatibility, chemical stability, etc.Solid state fermentation (SSF) for cellulase production isan advantageous process as it reduces the capital invest-ment with easy operating conditions (Pandey et al. 1999).Cellulose saccharification can be carried out by separatehydrolysis and fermentation (SHF) process with an easeof optimizing the enzymatic hydrolysis conditions (Zhuet al. 2012) for ethanol production. Ethanol quantifica-tion can be achieved by employing methods like GC–MS. Desirable for better specificity, few mass spectro-metric (MS) methods for ethanol analysis have beenreported (Tiscione et al. 2011). Insights of molecularlevel changes and functional groups in the lignocellu-losic material at various fermentation steps could bestudied by employing FTIR (Adapa et al. 2011; Sim et al.2012), morphological changes by SEM and substrateelemental analysis by higher throughput techniques likeSEM equipped with EDX technique.Keeping in view the industrial applications of the endo b -1,4- D -glucanase, this study was carried out to purify andcharacterize a novel endo  b -1,4- D -glucanase from  Tricho-derma harzianum  strain HZN11. Enzymatic hydrolysis andethanol fermentation was successfully achieved. Further,sweet sorghum bagasse was molecularly characterized withtechniques like FTIR, SEM and SEM/EDX. Materials and methods Chemicals, substrate and culture All the chemicals and media components used were pro-cured from HiMedia, Sigma-Aldrich (USA) and Merck (USA). Sweet sorghum stalks were collected fromUniversity of agricultural sciences, Dharwad.  Saccha-romyces cerevisiae  NCIM 3594 was procured fromNational Collection of Industrial Microorganisms (NCIM). Fungal strain and production of endo b -1,4- D -glucanase Trichoderma harzianum  strain HZN11 previously isolatedfrom forest soil was identified based on 18S rDNAsequencing (data was not shown). The nucleotide sequenceof the strain was deposited to NCBI (National Center forBiotechnology Information) GenBank with accessionnumber KP050786. The newly isolated  Trichoderma har- zianum  strain HZN11 is maintained at the Department of     Biochemistry, Karnatak University, Dharwad on potatodextrose agar (PDA) enriched with carboxymethyl cellu-lose (CMC) at 4   C. Endo  b -1,4- D -glucanase was producedby  Trichoderma harzianum  strain HZN11 in SSF usingalkali pretreated sweet sorghum bagasse as substrate. SSFwas carried out in 250 mL Erlenmeyer flasks containing10 g of pretreated substrate in Mandels–Weber mediumcontaining (g/L) urea 0.3; ammonium sulfate 1.4; KH 2 PO 4 0.3; CaCl 2  0.3; MgSO 4 .7H 2 O 0.3; protease peptone 1.0;lactose 10; and (mg/L) FeSO 4 .7H 2 O 5.0; MnSO 4 .7H 2 O1.6; ZnSO 4 .7H 2 O 1.4; CoCl 2  2; Tween-80 0.1 %; and pH 6with 70 % moisture content. Sterilized flasks were inocu-lated with 4 mL spore suspension and incubated at 35   Cunder static condition for 7 days. The crude enzyme wasextracted with 50 mM sodium acetate buffer, pH 6 with 1:2solid to liquid ratio under shaking (150 rpm) at 35   C for30 min, followed by filtration through muslin cloth. Thefiltrate was centrifuged at 8000 rpm for 20 min at 4   C.The clear supernatant was used as crude enzyme forpurification. Enzyme assay and protein determination Endo  b -1,4- D -glucanase hydrolyzes CMC to produce freecarboxymethyl glucose units. Endo  b -1,4- D -glucanaseactivity was estimated using CMC as substrate understandard conditions according to Standard InternationalUnion of Pure and Applied Chemistry (IUPAC) methoddescribed by Ghose (1987). The reducing sugars releasedfrom the reaction were determined according to Miller(1959) by dinitrosalicylic acid (DNS) method. In the aboveassay, one unit (U) of enzyme was defined as the amount of     enzyme that released 1  l mol of the glucose per minuteunder standard assay conditions (30 min incubation at50   C with 50 mM acetate buffer pH 6.0). The concen-trations of soluble proteins were estimated according toLowry et al. (1951) using bovine serum albumin (BSA) asthe standard. Purification of endo  b -1,4- D -glucanase and molecularmass determination Endo  b -1,4- D -glucanase produced from sweet sorghumbagasse by  Trichoderma harzianum  strain HZN11 wassubjected for purification. The cell debris was removed byvacuum filtration (Millipore India Ltd.) and the enzymeprotein was subjected to fractionation by (NH 4 ) 2 SO 4  (70 %w/v). The precipitate was centrifuged at 8000 rpm for  101 Page 2 of 10 3 Biotech (2016) 6:101  1 3  15 min at 4   C. Enzyme was resuspended in a minimumvolume of 50 mM sodium acetate buffer (pH 6.0) anddialyzed against the same buffer for 6 h at 4   C and lyo-philized. Total proteins and endo  b -1,4- D -glucanase activ-ity of partially purified fraction were determined before andafter dialysis. Endo  b -1,4- D -glucanase was further purifiedby column chromatography. The lyophilized enzyme wasdissolved in 50 mM sodium acetate buffer (pH 6.0). Theenzyme protein was loaded onto DEAE-Sepharose column(40  9  2 cm) pre-equilibrated with 50 mM sodium acetatebuffer (pH 6.0). The flow rate was maintained at 1 mL/minand eluted with gradient of 0.1–1 M NaCl. The pooledfractions were dialyzed and lyophilized. The fractions werere-dissolved in buffer and loaded onto the Sephadex G-100column (30  9  2 cm) pre-equilibrated and eluted with50 mM sodium acetate buffer (pH 6.0) at a flow rate of     0.5 mL/min. The fractions were analyzed for proteins byA 280  method and endo  b -1,4- D -glucanase activities weredetermined. The resulting concentrated active endo  b -1,4- D -glucanase fractions were pooled and used for furthercharacterization (Bakare et al. 2005). The apparentmolecular weight of the purified endo  b -1,4- D -glucanasewas determined by sodium dedocylsulphate polyacry-lamide gel electrophoresis (SDS-PAGE) with proteinmolecular weight ladder [lysozyme (14.3 kDa),  b -lac-toglobulin (20 kDa), carbonic anhydrase (29 kDa), oval-bumin (43 kDa), bovine serum albumin (66 kDa) andphosphorylase B (97.4 kDa) run along with sample],according to the method described by Laemmli (1970).Protein bands were visualized by staining with coomassiebrilliant blue R-250. Characterization of purified endo  b -1,4- D -glucanase Optimum pH of the purified endo  b -1,4- D -glucanase wasdetermined by incubating the enzyme at different pHranging from pH 3–11 using 0.1 M buffers of sodiumcitrate (pH 3–4.5), sodium acetate (pH 5–6), sodiumphosphate (pH 6.5–7.5), Tris–HCl (pH 8.0–9.5), and gly-cine–NaOH (pH 10.0–11.0). The stability of pH wasdetermined at optimum range of pH 5–6 for 3 h and therelative activity was calculated. Optimum temperature wasevaluated in range of 20–85   C and the thermo stability of     endo  b -1,4- D -glucanase was assessed between optimumtemperature ranges of 50–65   C for 3 h at pH 5.5 bymeasuring the relative activity. Effect of various metal ionslike Co 2 1 , Zn 2 1 , Ca 2 1 , Mg 2 1 , K  1 , Na 1 , Cu 2 1 , Hg 2 1 ,Fe 2 1 , Pb 2 1 , Ni 2 1 , Mn 2 1 and Cd 2 1 was determined atdifferent concentrations (1–10 mM). Endo  b -1,4- D -glu-canase relative activity was evaluated in the presence of     various additives like dithiothreitol (DTT),  b -mercap-toethanol, ethylene diamine tetra acetic acid (EDTA), urea,phenyl methyl sulphonyl fluoride (PMSF),  N  -bromosuccinimide, dimethyl sulfoxide (DMSO), iodoac-etamide,  p -chloromercuribenzoate (  p -CMB) and 1,10-phenanthroline, at various concentrations (1–10 mM).Relative activities of endo  b -1,4- D -glucanase in the pres-ence of various detergents like sodium dodecyl sulfate(SDS), sodium tetraborate, and commercial detergents(Tide, Ariel and Surf Excel), surfactants like tween-20,tween-40, tween-80 and triton X-100 and oxidizing agentslike sodium perborate, sodium hypochlorite and hydrogenperoxide at varying concentrations (0.1–1 %) were deter-mined. Endo  b -1,4- D -glucanase stability in the presence of     various organic solvents like glycerol, ethanol, methanol,acetone, formic acid, propanol, petroleum ether, iso-propanol, benzene, cyclohexane, hexane, butanol andtoluene at different concentrations (10–30 %) were evalu-ated (control was 100 %). Substrate specificity of endo  b -1,4- D -glucanase was tested against variety of substrates like1 % microcystalline cellulose, CMC, chitin, cellobiose,starch, filter paper, PNP- a -galactopyranoside, PNP-glu-copyranoside, PNP-cellobioside, brichwood xylan and oatspelt xylan. Endo  b -1,4- D -glucanase kinetics with CMCconcentration range of 2.5–30 mg/mL was studied andkinetic parameters  K  m  and  V  max  were determined by lineartransformations of the Michaelis–Menten model to Line-weaver–Burk. Inhibition of endo  b -1,4- D -glucanase withcellobiose was studied at 5 mM and 10 mM and inhibitionconstant  K  i  were determined by Lineweaver–Burk plot.The storage stability of the purified endo  b -1,4- D -glucanasewas monitored for 60 days. Enzymatic hydrolysis and ethanol fermentation Enzymatic hydrolysis of untreated and alkali pretreatedsweet sorghum bagasse and sugarcane bagasse was studiedusing purified endo  b -1,4- D -glucanase (53 U/g) mixed withcrude multi-enzyme cocktail (exoglucanase 15 U/g, filterpaper activity (FP) 15 U/g, cellobiase 18 U/g, xylanase1740 U/g and  b -glucosidase 13 U/g) produced by  Tricho-derma harzianum  strain HZN11 from sweet sorghumbagasse and commercial cellulase from  Trichoderma  sps.(9 FP U/mL) individually. Reaction constituted of 2 %substrate in 50 mM sodium acetate buffer pH 5.5, 0.1 %tween-40 and filter sterilized enzyme in a volume of 30 mLincubated at 40   C at 150 rpm. The reaction was fortifiedwith 0.005 % sodium azide. Samples were withdrawn,centrifuged at 8000 rpm for 15 min and the clear super-natants were analyzed for reducing sugars according to themethod described by Miller (1959). Controls were kept foreach reaction with heat-inactivated enzyme. Parameterssuch as hydrolysis time (12–72 h) and temperature(40–60   C) for enzymatic hydrolysis of alkali pretreatedsweet sorghum bagasse and sugarcane bagasse was opti-mized. SHF experiments were designed in which 3 Biotech (2016) 6:101 Page 3 of 10 101  1 3
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