Thermodynamics characterization and potential textile applications of Trichoderma longibrachiatum KT693225 xylanase

Author’s Accepted Manuscript Thermodynamics characterization and potential textile applications of Trichoderma longibrachiatum KT693225 xylanase Abeer A. Abd El Aty, Shireen A.A. Saleh, Basma M. Eid, Nabil A. Ibrahim, Faten A. Mostafa www.elsevier.com/locate/bab

PII: DOI: Reference:

S1878-8181(18)30036-7 https://doi.org/10.1016/j.bcab.2018.02.011 BCAB707

To appear in: Biocatalysis and Agricultural Biotechnology Received date: 14 January 2018 Revised date: 16 February 2018 Accepted date: 17 February 2018 Cite this article as: Abeer A. Abd El Aty, Shireen A.A. Saleh, Basma M. Eid, Nabil A. Ibrahim and Faten A. Mostafa, Thermodynamics characterization and potential textile applications of Trichoderma longibrachiatum KT693225 x y l a n a s e , Biocatalysis and Agricultural Biotechnology, https://doi.org/10.1016/j.bcab.2018.02.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Thermodynamics characterization and potential textile applications of Trichoderma longibrachiatum KT693225 xylanase Abeer A. Abd El Aty 1, 2, Shireen A.A. Saleh 1, Basma M. Eid 3, Nabil A. Ibrahim 3, Faten A. Mostafa 1*

1

Chemistry of Natural and Microbial Products Dept., National Research Centre, Dokki, Giza, Egypt.

2

Biology Department, Faculty of Education, Hafr Al Batin University, Saudi Arabia.

3

Textile Research Division, National Research Center, Dokki, Giza, Egypt.

Abstract Our study was a trial to participate in solving two main problems namely, environmental pollution resulting from accumulation and bad disposal of agro-industrial wastes, and high cost of industrial xylanase enzyme production. This was achieved through successful xylanase production by solid-state fermentation of low cost disposable agricultural wastes by marine fungal isolate Trichoderma longibrachiatum KT693225. The highest xylanase production 7.13±0.11 U.ml-1 was obtained utilizing rice straw (RS) waste after 7days of fermentation. Xylanase was purified by fractional precipitation with ethanol causing 4.24-fold purification. The 75% ethanol fraction was rich in cellulase, pectinase and α-amylase enzymes beside xylanase. The maximal xylanase activity was obtained at 60ºC, pH 5 and 2.5% xylan concentration. The Km and Vmax were calculated to be 20 mg ml-1 and 20 µmol min-1 ml-1, respectively. The thermostability of T.longibrachiatum KT693225 xylanase was indicated by low Ea (activation energy)and high Ed (energy of denaturation). High T1/2 (half life), D-value (decimal reduction time), ΔHº (enthalpy), ΔGº (free energy) and low Kd (denaturation rate constant), ΔSº (entropy) values at 70ºC emphasized high T.longibrachiatum KT693225 xylanase stability. T.longibrachiatum KT693225 xylanase showed high effectiveness at several textile wet-processing stages including desizing, bioscouring and biofinishing of cellulosic fabrics without adding any additives. These findings in this study have great implications for the future applications of xylanases. Keywords: xylanase; thermodynamics; textile applications. Crossponding author Faten A. Mostafa e.mail: [email protected]

1

1.Introduction Microbial enzymes are currently acquiring much attention with rapid development of enzyme technology. Microbial enzymes are preferred due to their economic feasibility, consistency, high yields, ease of product modification and optimization. Also characterized by rapid growth of microbes on inexpensive media, stability, and greater catalytic activity, microbial enzymes play a major role in bio-industries such as food, leather, textiles, animal feed, and in bio-conversions and bio-remediation (Chelikani et al., 2004; Hebeish et al., 2007; Araújo et al., 2008; Shen and Smith, 2015; Hasan et al., 2015). Microbial enzymes have demonstrated many commercially successful textile applications, such as amylases for desizing, cellulases and laccases for denim finishing, and proteases incorporated in detergent formulations. Amylase, pectinase, cellulases, catalase, and protease to remove starch size, glue between the fiber core and the waxes, fabric finishing in denims, degrading residual hydrogen peroxide after the bleaching of cotton, wool treatment, and the degumming of raw silk also known as biopolishing (Rejzek et al., 2011). Xylanases are considered one of the industrially important microbial enzymes, which can catabolize the xylan residues. Over the years the usage of xylanase at the industrial level has increased significantly (Techapun et al., 2003). Since applications of xylanases in commercial sectors are widening, an understanding of its nature and properties for efficient and effective usage becomes crucial. Xylanases extracted from microorganisms has been used for pulp bleaching, waste paper treatment and for fabric bio-processing, such as: bio-bleaching, desizing and bio-scouring of fabrics (Dhiman et al., 2008). The use of xylanases in the textile industry is an example of white industrial biotechnology, which allows the development of environmentally friendly textile wet processing and strategies to improve the final product quality. The consumption of energy and raw-materials, as well as increased awareness of environmental concerns related to the use and disposal of chemicals into landfills, water or release into the air during chemical processing of textiles are the principal reasons for the application of enzymes in finishing of textile materials (Araújo et al., 2008). Today, the utilization of any enzyme in industrial application is judged by some factors. Mainly, costeffectiveness, eco-friendliness, and ease of use (Taibi et al., 2011). Therefore, the present study 2

directed towards, the production of the industrially important xylanase from local fungal isolate utilizing agro-residues as a substrate for enzyme economic production with low costs, studying the thermodynamics of produced xylanase and potential applications of the obtained xylanase in the proper textile wet processing. 2.Materials and Methods 2.1.Microorganisms and maintenance The microorganisms Trichoderma longibrachiatum, Alternaria tenussima and Chaetomium globosum have been isolated from marine sources and identified genetically given the accession numbers KT693225 (Abdel Wahab et al., 2018), KM651985 (Abd El Aty et al., 2015) and KM651986 (Shehata and Abd El Aty, 2015), respectively. The fungal isolates were maintained on malt extract agar (MEA) medium at 4°C, contained the following components (g l-1): biomalt 20, agar15, 800 ml sterile sea water and 200 ml distilled water (Abd El Aty et al., 2014). 2.2.Production of xylanase enzyme under solid-state fermentation (SSF) In the screening step, six different agricultural wastes wheat bran (Wb), rice straw (RS), potato peels (PP), artichoke leaves (AL), saw dust (SD) and licorice residue (LR) were used as substrates for xylanase production. Solid state fermentation was carried out in Erlenmeyer flasks (250 ml) with 3gram of the dried solid substrates (oven dried). Moisture was adjusted by addition of 15 ml of sea water to each flask. Each flask was covered with hydrophobic cotton and autoclaved at 121 ◦C for 20 minutes. After cooling, it was inoculated with 1.0 ml inoculum containing (5 × 107spores/ml) of 7 days old culture which was prepared by harvesting the slant of the fungus in 15 ml sterile distilled water. The inoculated flasks were incubated for 7 days at (32 ± 2 ◦C) under static conditions (Mostafa et al., 2014). All the experiments were carried out in duplicate and the average values are reported as mean ± SD calculated using MS Excel. 2.3.Extraction of xylanase enzyme Enzyme extraction was done by adding fifty ml of distilled water and left in a rotary shaker for 60 minutes. Then the mixture was filtered through a cloth and the culture filtrate centrifuged for 15 min at 5,000 rpm and 4°C. The supernatant was used as the crude xylanase enzyme extract (Abd El Aty and Mostafa, 2015). 2.4.Enzymes Determinations 2.4.1.Xylanase activity assay 3

This was done according to the method of Warzywoda et al. (1983). Half ml of diluted enzyme solution was added to 0.5 ml of 1% xylan in 0.05 M acetate buffer pH 5.0. Incubation of the reaction mixture was performed for 30 minutes at 50˚C. The amount of reducing sugar liberated was quantified by the method of Neish (1952) using xylose as standard. One unit of xylanase is defined as the amount of enzyme releasing 1 μmol of xylose equivalents per minute under assay conditions. 2.4.2.Cellulase activity This was done according to the method of Mandels and Weber (1969). 0.5ml of enzyme solution was added to 0.5ml of 1% carboxymethylcellulose(CMC) in 0.05M acetate buffer(pH 5.0). Incubation of the reaction mixture was performed for 30min at 50°C. The released reducing sugars were determined by the method of Neish(1952). One unit of cellulase(IU) was defined as the amount of enzyme releasing 1µmol of glucose per min under the assay conditions. 2.4.3.Pectinase activity This was done according to the method of Silva et al.(2005) by mixing 0.2ml enzyme solution and 0.8ml of citrus pectin(0.5%pectin in 0.05M acetate buffer,pH5.0).Samples were incubated at 50°C for 10.0min and the reducing sugars were determined by Somogyi method. One unit of exo-PGactivity was defined as the amount of enzyme releasing 1μmol of reducing sugars (as galacturonic acid) per min under the assay conditions. 2.4.4.α-amylase activity This was done according to Ahmed et al. (2017) by mixing 0.5 ml of enzyme solution and 0.5 ml of 1% starch solution prepared in acetate buffer (0.05 M, pH 5.0). The mixture was incubated for 20 min at 40°C and the released reducing sugars were determined by Somogyi method (1952). 2.5.Protein assay The protein concentration was determined by the method of Lowry et al. (1951). 2.6.Partial purification of xylanase enzyme The crude xylanase enzyme was partially purified by fractional precipitation with ethanol and acetone (Farinas et al., 2011). The enzyme fractions obtained 25, 50 and 75% concentrations of ethanol and acetone were dried over anhydrous calcium chloride under decreased pressure at room temperature and weighed. Each enzyme fraction was assayed for xylanase activity and protein content. Specific activity and purification fold were calculated. 2.7.Characterization of purified xylanase

4

The optimum conditions for maximal xylanase activity including temperature, pH and substrate concentration were determined by carrying out the reaction at different temperatures 30, 40, 50, 60 and 70ºC. Also, the optimum pH for the reaction was determined by carrying it at different pH 3-7 with 0.05M sodium acetate buffer. Different birchwood xylan concentrations 0.25- 4% for maximum xylanase activity were examined. 2.8.Determination of kinetic and thermodynamic parameters for purified xylanase The Michael’s constant Km and maximum velocity Vmax were determined from Lineweaver-Burk plot using birchwood xylan. The activation energy (Ea) was calculated from Arrhenius plot. The other thermodynamics were calculated with the following equations (1-6) ((Pal and Khanum, 2010) The half-life of the xylanase (T1/2, min-1) was determined from the relationship: T1 / 2= ln 2/Kd

(1)

The D-values (decimal reduction time or time required to preincubate the enzyme at a given temperature to maintain 10% residual activity) was calculated from the following relationship: D- value= ln 10/Kd (2) The activation energy (Ed) for xylanase denaturation was determined by an Arrhenius plot of log denaturation rate constants (ln kd) versus reciprocal of the absolute temperature (K) using the Equation: Slope= -Ed/R

(3)

The change in enthalpy (ΔHº, kJ mol-1), free energy (ΔGº, kJ mol-1) and entropy (ΔSº, J mol-1 K-1) for thermal denaturation of xylanase were determined using the following equation: ΔH° = Ed − RT ,

(4)

ΔGº=-RT.ln(Kd.h/Kb.T) (5) ΔSº=( ΔH°- ΔGº)/T

(6)

Where Ed is the activation energy for denaturation, T is the corresponding absolute temperature (K), R is the gas constant (8.314 J mol-1 K-1), h is the Planck constant (6.626 X 10-34 J min), Kb is the Boltzman constant (1.38 X 10-23 J K-1) and Kd is the deactivation rate constant (min-1). 2.9.Textile applications of Trichoderma longibrachiatum KT693225 xylanase Different types of fabrics were used throughout this work. Table (1) illustrates the specifications of the used fabrics 2.9.1.Enzymatic desizing 5

Desizing of starch-sized cotton fabric was carried out by using different concentrations of cocktail of enzymes containing xylanase (00.00-60.00 U.l-1) and α-amylase (00.00-25.00 U.l-1), material-to-liquor ratio 1:20 at 70oC and pH 6 for 60 minutes then increased the temperature up to 80oC for 15 minutes in presence of agitation, followed by thoroughly hot rinsing. Weight loss of fabric (WL) was determined according to ASTM (D3776-79). Wettability was assessed according to AATCC (79-1992). Desizing efficiency was assessed by using the TEGEWA scale method, violet scale shade (Ibrahim et al., 2004). The surface morphologies of the untreated and treated fabric samples were observed with JEOLJXA 840A scanning electron microscope, Japan. 2.9.2.Bioscouring of cellulosic fabrics Grey linen and knitted fabrics were bioscoured in presence of cocktail of enzymes containing xylanase (00.00-6.00 U.40 ml-1), pectinase (00.00-3.00 U.40 ml-1) and cellulase (00.00-3.00 U.40 ml-1) at material-to-liquor ratio 1:20 at 70oC and pH 6 for 60 min then raising the temperature up to 80oC for 15 min with continous agitation, followed by thoroughly hot rinsing. Weight loss of fabric (WL) was determined according to ASTM (D3776-79). Wettability was assessed according to AATCC (791992). The surface morphologies of the untreated and treated fabric samples were investigated using scanning electron microscope (SEM) JEAOL JXA-840A (Mostafa et al., 2016). 2.9.3.Biofinishing of cellulosic fabrics Bleached cotton, and linen as well as indigo-dyed cotton fabrics were biofinished using xylanase rich cocktail enzymes (1.0 % based on the weight of fabric, owf), material-to-liquor ratio 1:20 at 70oC and pH 6 for 60 minutes. After the biofinishing treatment the treated fabric samples were washed at 90 oC for 15 minutes at pH 9 to inhibit the enzyme, neutralized and thoroughly rinsed. Finally, the fabrics were dried in ambient conditions. Surface roughness (SR) was measured according to JIS B0031-1994 standard, using surface roughness measuring instrument (SE-1700, Japan). Stiffness (St) of treated fabrics was evaluated according to ASTM (D3388-1996). The color strength of untreated and treated denim fabric samples, expressed as K/S, was calculated by the following equation, Kubelka-Munk equation (Judd and Wyszeck, 1975): K/S = (1-R)²/2R where R = surface reflectance, K: light absorption, S: light scattering. The surface morphologies of the untreated and treated fabric samples were observed investigated using scanning electron microscope (SEM) JEAOL JXA-840A. 2.10. Statistical analysis Data analysis was carried out with Microsoft Excel. All data are presented as the average of the triplicate measurements (±) standard deviation. 6

3.Results and discussion 3.1.Screening for xylanase production by SSF One of the most critical obstacles that utilization of microbial industrial xylanase faces is the high cost of xylan as substrate for xylanase production. In order to overcome this problem, this study investigated the utilization of disposal agricultural wastes i.e. wheat bran, rice straw, potato peels, artichoke leaves, saw dust and licorice residue as xylan containing substrates. Also, three marine fungal isolates have been tested for their ability to produce xylanase after 7 days utilizing these agricultural wastes by SSF. The results as shown in Fig. (1) indicated that, xylanase production differed according to two main factors the fungal isolate and the agricultural waste. i.e. T. longibrachiatum was able to produce xylanase utilizing all the agricultural wastes nearly similar to that reported by Sanghvi et al. (2014) for xylanase production by halophilic bacterium-OKH. While, A. tenussima could not utilize PP, AL, LR. C. globosum produced xylanase utilizing rice straw only. The highest xylanase production was obtained by T. longibrachiatum 7.13±0.11 U.ml-1 on RS higher than that produced by Aspergillus terreus 1.86 U.ml-1 utilizing molokhia stalks (Ahmed et al., 2016). The utilization of agricultural wastes for xylanase production was in the following order RS>PP>WB>SD>AL>LR. While Mander et al. (2014) and Mostafa et al. (2014) reported the maximum xylanase production by Streptomyces griseofuscus and Aspergillus flavus, respectively on WB. Wheat straw was found to be a good inducer for xylanase production (Goswami et al., 2013). 3.2.Purification of T. longibrachiatum xylanase The data represented in Table (2) showed that the fractional precipitation T. longibrachiatum with ethanol and acetone gave six fractions. The fractions obtained with ethanol fractional precipitation contained xylanase activity better than those obtained with acetone. As shown in Table (2) among the three ethanol precipitated fractions, the 50% fraction possessed the lowest activity (6.09 U. fraction-1) and the lowest protein (30.72 mg.fraction-1) giving specific activity 0.20 U/mg, the 75% fraction possessed the highest activity (26.48 U.fraction-1) and the highest protein (55.97 mg.fraction-1) giving specific activity 0.47 U/mg achieving 4.24-fold purification. Many authors used the fractional precipitation with ammonium sulphate saturation as traditional method for partial purification of xylanase. e.g. Kamble and Jadhav (2012) partially purified xylanase from Bacillus arseniciselenatis DSM 15340 with 35–80% ammonium sulphate saturation with 1.37-fold purification only nearly similar to that partially purified from Aspergillus niger with 1.85-fold-purification (Ahmad et al., 7

2013). Also, xylanase from Bacillus brevis was partially purified with 30-60% ammonium sulphate saturation causing 5.6-fold purification (Goswami et al., 2013) resembling that partially purified from Bacillus circulans with 5.42-fold purification (Pithadiya et al., 2016). It was also noticeable that our 75%ethanol fraction was rich in pectinase, amylase and cellulase enzymes which made this fraction more effective for textile industry application. The proceeding work was done on 75% ethanol fraction. 3.3.Characterization of Trichoderma longibrachiatum KT693225 xylanase The optimum conditions including reaction temperature, pH and substrate concentration to obtain the maximum xylanase activity were examined.

3.3.1.Optimum temperature Xylanase from T. longibrachiatum was found to be stable from 30-60ºC as shown in Fig. (2, a) recording the maximum activity

at 60ºC (7.47U.ml-1) similar to the xylanases from different

Streptomyces species (Mander et al., 2014; Kaneko et al., 2000; Ninawe et al., 2008; Deesukon et al., 2011) and Aspergillus niger (Ahmad et al., 2013). Recording the maximal activity at 60ºC emphasized that T. longibrachiatum xylanase is mesophilic enzyme according to Polizeli et al. (2005). Therefore, it can be considered as promising and strong candidate for future industrial applications mainly in pulp bleaching technology. Our result was higher than that reported for other xylanases (Pithadiya et al., 2016; Monisha et al., 2009; Roy and Rowshanul, 2009; Sharma et al., 2013; Dobrev et al., 2012; Shakoori et al., 2015). 3.3.2.Optimum pH The optimum pH for T. longibrachiatum xylanase activity was 5.00 Fig. (2, b) coinciding with those reported for other fungal xylanases (Dobrev and Zhekova, 2012; Silva et al., 2015; Ahmed et al., 2012). T. longibrachiatum xylanase can be applied in biotechnological process as biomass hydrolysis and as animal feed additives since these applications requires xylanases active within pH range 4.8-5.5 (Subramaniyan and Prema, 2002). 3.4.Kinetics and thermodynamics determination Xylanase from T. longibrachiatum showed the maximal activity with 2.5% oat xylan similar to xylanase from bacterial isolate XPB-GS02 (Shakoori et al., 2015). The Km and Vmax as shown in Fig. (2, c) were found to be 20 mg.ml-1 and 20 µmol.min-1.ml-1, respectively. Km and Vmax values differ depending on xylanase source and xylan type. e.g. Km and Vmax for xylanase from, Streptomyces sp. 8

CS624 for beechwood xylan were 5.61 mg.ml-1 and 74.62 mmol.min−1.mg−1 while for birchwood xylan were 9.79 mg.ml-1 and57.47 mmol min−1mg−1, respectively (Mander et al., 2014), from Trichoderma inhamatum for oat splets were of 14.5mg.ml-1 and 2680.2 U.mg-1, 1.6 mg.ml-1 and 462.2 U.mg-1 for birchwood xylan, respectively. For Bacillus arseniciselenatis DSM 15340 xylanase they were determined to be 5.26 mg.ml-1 and 277.7 µmol.min-1.mg-1, respectively (Kamble and Jadhav, 2012). The values of Km (5.75 mg.ml-1) and Vmax (1.33 U.ml-1) for xylanase from B. brevis were determined (Goswami et al., 2013). Km and Vmax of the recombinant xylanase was found to be 8.17 mg.ml-1 and 3.7 U.ml-1, respectively (Goswami and Rawat, 2015). The suitability of any enzyme for industrial applications is judged by its thermodynamic parameters including Kd, T1/2 and D-values. As shown in table (3), the rate of deactivation Kd at 50, 60 and 70ºC was lower than that reported for other xylanases (Pal and Khanum, 2010; Sugumaran et al., 2012; Robledo et al., 2014). Also,the half-life values T1/2 and D-value at 50, 60 and 70ºC were higher than those reported for other xylanases (Pal and Khanum, 2010; Robledo et al., 2014; Gawande and Kamat, 1998; Benedetti et al., 2013) reflecting the high stability of Trichoderma longibrachiatum KT693225 xylanase. Also, The thermal stability of any enzyme is reflected by low Ea activation energy and high Ed requiring low energy for product formation and high energy for enzyme denaturation, respectively. The Ea of Trichoderma longibrachiatum KT693225 xylanase was calculated from Fig. (3a) to be 3.775 Kcal.mol-1 lower than other xylanases (Pal and Khanum, 2010; Sugumaran et al., 2012; Robledo et al., 2014; Gawande and Kamat, 1998; Benedetti et al., 2013). Ed of T. longibrachiatum xylanase was 62.05 KJ.mol-1 as shown in Fig. (3b) higher than Aspergillus niger DFR-5 xylanase 56.86 KJ.mol-1 (Pal and Khanum, 2010). Thermostability was indicated by high ΔHº change in enthalpy and ΔGº free energy and low ΔSº entropy for thermal denaturation as shown in table (3). It should be noticed that ΔGº values, which are measures of the spontaneity of the inactivation processes, are higher than the ΔHº values. This is due to the negative entropic contribution during the inactivation process as stated by Tanaka and Hoshino (2002). Negative values for ΔSº in all temperature measured indicated that there are aggregation processes taking place during thermal inactivation (Dumitraşcu et al., 2012). 3.5.Potential textile applications 3.5.1.Desizing of starch-sized fabrics As mentioned before our 75% ethanol fraction was rich in xylanase, pectinase and amylase enzymes. As shown in Table (4) the increase in xylanase and amylase units have desirable remarkable effect on 9

the performance properties of desized fabric samples. The positive effect of xylanase and α-amylase rich fraction on fabric samples was indicated by the high loss in weight of the samples due to the ability of crude enzyme to degrade the starch size by α-amylase component as well as the removal of non-cellulosic constituents by xylanase Csiszar et al. (2001) such as the surface fibrils, seed coat fragments and other natural impurities of cotton. Moreover, results indicate an enhancement in the violet scale as shown in Fig. (4a, b) from 1 to 4 and improvement in the wettability of the treated samples due to ability of α-amylase to degrade the starch components from the fabric surface during the mechanical action as reported by some authors (Dhiman et al., 2008; Ibrahim et al., 2004; Battan et al., 2012). On the other hand, SEM images of sized and enzymatic desized fabric samples are presented in Fig. (5a, b). It can be clearly seen that fabric surfaces become cleaner in case of desized cotton fabric Fig. 5b compared with the untreated ones Fig.5a, reflecting the positive effect of enzymatic treatment on removal of the deposited impurities including the starch size and some noncellulosic impurities simultaneously. 3.5.2.Bioscouring of grey cellulosic fabrics The presence of pectinase enzyme component in the 75% ethanol fraction along with the other enzymes, i.e. xylanse and cellulase brought about a noticeable improvement in the tested performance properties including weight loss (WL), wettability and reducing sugar. As shown in Table (5). Dramatically increase in WL and liberation of reducing sugar was accompanied with increasing the xylanase, cellulase and pectinase cocktail enzymes dose confirming the successful removal of noncellulosic impurities from the nominated cellulosic substrates. The differences among the treated substrates in the extent of weight loss, wettability as well as the liberated reducing sugars are attributed to the type, structure, and construction of the cellulosic fabric i.e. linen or cotton, woven or knitted and the type of knitted fabrics single Jersey 20/1 or Peque 24/1, fabric weight, amount, location and extent of distribution of the noncellulosic/ hydrophobic impurities as well as the availability and accessibility of these impurities like pectins, waxes and oils to enzymatic attack (Ibrahim et al., 2004; Csiszar et al., 2001; Battan et al., 2012; Ibrahim et al., 2005; Ibrahim et al., 2008). 3.5.3.Bio-polishing of cellulosic fabric The effect of cocktail enzymes on the biopolishing process is given in Table (6). It is clear that: i) enzymatic treatment was accompanied by loss in weight regardless of the used substrate. ii) all the treated samples showed a noticeable improve in the surface smoothness as well as remarkable decrease in their stiffness properties compared with the untreated ones, and iii) in case of indigo-dyed 10

cotton fabrics, the color strength was slightly decreased by enzymatic treatment owing to the partial removal of dye fragments during the removal of the cellulosic fibrils at the surface. The enhancement in the tested performance properties of the treated cellulosic fabrics is attributed to the presence of cellulase and xylanase enzymes in the enzymes mixture that had the ability to degrade and remove the cellulosic fibrils at the surface with the help of the existed stainless steel balls during the mechanical action. Additionally, the change in the surface morphology before and after enzymatic treatment was illustrated in Fig. (6, a-f). It can be clearly seen that the surface become smoother and cleaner after biofinishing process which attributed to the removal of the protruding cellulosic fibrils at the surface during enzymatic treatment. There are no cracks or cavities in the enzymatic treated fabrics which confirms that the enzymatic treatment didn't cause any serious damage in the cellulosic fibers, i.e surface treatment. In case of indigo dyed fabrics Fig. (6 e&f), the biopolished fabric samples (Fig. 6f) showed partial removal of some of the dyed fiber fragments from the fabric surface comparing with the untreated one (Fig. 6e). 4.Conclusion Trichoderma longibrachiatum KT693225 is a good producer for cocktail of industrially potent enzymes xylanase, pectinase, cellulase and α-amylase utilizing different low cost agricultural wastes. T. longibrachiatum KT693225xylanase was mesophilic enzyme. i.e. active over a wide temperature range 40-60ºC showing maximum activity at 60ºC. T. longibrachiatum KT693225 xylanase had remarkable thermodynamic parameters including low Ea, Kd, ΔSº and high Ed, T1/2, ΔHº, ΔGº. These parameters were good evidences for T.longibrachiatum KT693225xylanase utilization in biotechnological applications. T.longibrachiatum KT693225xylanase, amylase, pectinase and cellulase showed high effectiveness in desizing of starch-sized fabrics, bioscouring of grey cellulosic fabrics and biopolishing of cellulosic fabric.

Acknowledgment This work was financially supported by National Research Centre, Cairo, Egypt, project number P100221.

11

References Abd El Aty, A.A., El-Shamy, A.R., Atalla, S.M.M., El-Diwany, A.I., Hamed, E.R., 2015. Screening of fungal isolates for laccase enzyme production from marine sources. RJPBCS 6, 221–228. Abd El Aty, A.A., Mostafa, F.A., 2015. Production and characterization of fungal α-amylase from marine Alternaria alternata utilizing lignocellulosic wastes and its application. Res. J. Pharm. Biol. Chem. Sci. 6(3), 813-825. Abd El Aty, A.A., Wehaidy, H.R., Mostafa, F.A., 2014. Optimization of inulinase production from low cost substrates using Plackett–Burman and Taguchi methods. Carbohyd. Poly. 102, 261– 268. Abdel Wahab, W.A., Abd El Aty, A.A., Mostafa, F.A., 2018. Improvement of catalytic, thermodynamics and antifungal activity of constitutive Trichoderma longibrachiatum KT693225 exochitinase by covalent coupling to oxidized polysaccharides. Int J Biol Macromol. 112:179-187. Ahmad, Z., Butt, M.S., Riaz, M., 2013. Partial purification and characterization of xylanase produced from Aspergillus niger using wheat bran. Pak. J. Agri. Sci., 50 (3), 433-437. Ahmed, S., Imdad, S.S., Jamil, A., 2012. Comparative study for the kinetics of extracellular xylanases from Trichoderma harzianum and Chaetomium thermophilum Elec. J. Biotechnol. doi: 10.2225/vol15-issue3-fulltext-2 Ahmed, S.A., Mostafa, F.A., Helmy, W.A., Abdel-Naby, M.A. 2017. Improvement of bacterial αamylase production and application using two steps statistical factorial design. Biocatalys. Agri. Biotechnol. 10, 224-233.

12

Ahmed, S.A., Saleh, S.A.A., Mostafa, F.A., Abd El Aty, A.A., Ammar, H.A.M., 2016. Characterization and valuable applications of xylanase from endophytic fungus Aspergillus terreus KP900973 isolated from Corchorus olitorius. Biocatalys. Agri. Biotechnol. 7, 134– 144.

Araújo, R., Casal, M., Cavaco-Paulo, A., 2008. Application of enzymes for textile fibres processing. Biocatal. Biotrans 26(5), 332-349. Battan, B., Dhiman, S.S., Ahlawat, S., Mahajan, R., Sharma, J., 2012. Application of thermostable xylanase of Bacillus pumilus in textile processing. Ind. J. Microbiol. 52(2), 222-229. Benedetti, A.C.E., Da Costa, E.D., Aragon, C.C., Dos Santos, A.F., Goulart, A.J., Angelis, D.A., Monti, R., 2013. Low cost carbon sources for the production of a thermostable xylanase by Aspergillus niger. J. Basic Appl. Pharm. Sci. 34(1),25-31. Chelikani, P., Fita, I., Loewen, P.C., 2004. Diversity of structures and properties among catalases. Cell Mol. Life Sci. 61(2),192-208. Csiszár, E., Urbánszki, K., Szakács, G., 2001. Biotreatment of desized cotton fabric by commercial cellulase and xylanase enzymes. J. Mol. Catal. B: Enz .11(4–6), 1065-1072. Deesukon, W., Nishimura, Y., Harada, N., Sakamoto, T., Sukhumsirichart, W., 2011. Purification, characterization and gene cloning of two forms of a ther-mostable endo-xylanase from Streptomyces sp. SWU10. Proc. Biochem. 46,2255–62. Dhiman, S.S., Sharma, J., Battan, B., 2008. Industrial applications and future prospects of microbial xylanases: A review. Bioresource 3(4), 1377-1402. Dobrev, G., Zhekova, B., 2012. Purification and characterization of endoxylanase Xln-2 from Aspergillus niger B03. Turk. J. Biol. 36, 7-13.

13

Dumitraşcu, L., Stănciuc, N., Stanciu, S., Râpeanu, G., 2012.Thermal inactivation of lactoperoxidase in goat, sheep and bovine milk–A comparative kinetic and thermodynamic study. J. Food Eng. 113(1), 47-52. Farinas1, C.S., Scarpelini, L.M., Miranda, E.A., Bertucci Neto, V., 2011. Evolution of operational parameters of the precipitation of endogluconase and xylanase produced by solid state fermentation of Aspergillus niger. Brazilian J. Chem. Engi. 28, 17-26. Gawande, P.V., Kamat, M.Y., 1998. Preparation, characterization and application of Aspergillus sp. xylanase immobilized on Eudragit S100. J. Biotechnol. 66, 165-175. Goswami, G.K., Pathak, R.R., Krishnamohan, M., Ramesh, B., 2013. Production, partial purification and biochemical characterization of thermostable xylanase from Bacillus brevis. Biomed. Pharmacol. J. 6(2), 435-440. Goswami, G.K., Rawat, S., 2015. Production and biochemical characterization of alkaline, thermostable xylanase secreted by recombinant Escherichia coli. Int. J. Pure App. Biosci. 3 (2), 534-541. Hasan, M., Nabi, F., Mahmud, R., 2015. Benefits of enzymatic process in textile wet processing. Int J Fiber Text. Res. 5(2), 16-19. Hebeish, A., Ibrahim, N.A., 2007. The impact of frontier sciences on textile industry: Review article. Colourage Ann. 54, 41-55. Ibrahim, N.A., El-Hossamy, M., Hashem, M.M., Refai, R., Eid, B.M., 2008. Novel pretreatment processes to promote linen-containing fabrics properties. Carbohyd. Poly. 74 (4), 880-891. Ibrahim, N.A., El-Hossamy, M., Morsy, M.S., Eid, B.M., 2004. Optimization and modification of enzymatic desizing of starch-size. Poly-Plastics Technol. Eng. 43(2), 519-538.

14

Ibrahim, N.A., Abd Allah, S.Z., Hassan, T.M., Borham, H.A.T., 2005. Economical and ecological biotreatment / half bleaching of cotton-containing knit fabrics on Industrial scale. Poly Plastic Technol. Eng. 44 (5), 881-899. Judd, D., Wyszeck, G., 1975. Color in business science and industry, (3rd ed.), New York: John Wiley & Sons. Kamble, R.D., Jadhav A.R., 2012. Isolation, purification, and characterization of xylanase produced by a new species of Bacillus in solid state fermentation. Int. J. Microbiol 8 pages. Kaneko, S., Kuno, A., Muramatsu, M., Iwamatsu, S., Kusakabe, I., Hayashi, K., 2000. Purification and characterization of a family G/11 beta-xylanase from Streptomyces olivaceoviridis E-86. Biosci. Biotechnol. Biochem. 64, 447–51. Lowry, O.H. , Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Mandels, M., Weber, J., 1969.The production of cellulases. Adv. Chem. Ser. 95, 391–414. Mander, P., Choia, Y.H., Pradeep, G.C., Choia, Y.S., Honga, J.H., Chob, S.S., Yoo, J.C., 2014. Biochemical characterization of xylanase produced from Streptomyces sp. CS624 using an agro-residue substrate. Proc. Biochem. 49, 451–456. Monisha, R., Uma, M.V., Murthy, V.K., 2009. Partial purification and characterization of Bacillus pumilus xylanase from soil source. Kathmandu University J. Sci. Eng. Technol. 5(II), 137148. Mostafa, F.A., Abd El Aty, A.A., Hamed, E.R., Eid, B.M., Ibrahim, N.A., 2015. Enzymatic, kinetic and anti-microbial studies on Aspergillus terreus culture filtrate and Allium cepa seeds extract and their potent applications. Biocatalys. Agri. Biotechnol. 5, 116–122.

15

Mostafa, F.A., Abd El Aty, A.A., Wehaidy, H.R., 2014. Improved xylanase production by mixing low cost wastes and novel co-culture of three marine-derived fungi in solid state fermentation. Int. J. Curr. Microbiol. App. Sci. 3(7), 336-349. Neish, A.C., 1952. Analytical methods for bacterial fermentation, Repor no., 468-3, National Council of Canada, Second Revision, November, P. 34. Ninawe, S., Kapoor, M., Kuhad, R.C., 2008. Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32. Bioresour. Technol. 99, 1252–8. Pal, A., Khanum, M.Y., 2010. Characterizing and improving the thermostability of purified xylanase from Aspergillus niger DFR-5 grown on solid-state-medium. J. Biochem. Tech. 2(4), 203-209. Pithadiya, D., Nandha, D., Thakkar, A., 2016. Partial purification and optimization of xylanase from Bacillus circulans. Archives App. Sci. Res. 8 (2), 1-10. Polizeli, M.L., Rizzatti, A.C., Monti, R., Terenzi, H.F., Jorge, J.A., Amorim, D.S., 2005. Xylanases from fungi: Properties and industrial applications. Appl. Microbiol. Biotechnol. 67(5), 577591. Rejzek, M., Stevenson, C.E., Southard, A.M., 2011. Chemical genetics and cereal starch metabolism: structural basis of the non-covalent and covalent inhibition of barley 𝛽-amylase. Mol. Biosys. 7 (3), 718–730. Robledo, A., Aguilar, C.N., Cerda, R.E.B., Esquivel, J.C.C., Hernández, M.A.C., Sáenz, J.C.M ., 2014. Kinetic and thermodynamic parameters of the thermostable xylanase production. Int. J. Res. Agri. Food Sci. 1(6), 16-23. Roy, N., Rowshanul, H.M., 2009. Isolation and characterization of xylanase producing strain of Bacillus cereus from soil. Iran. J. Microbiol. 1 (2), 49 – 53.

16

Sanghvi, G., Jivrajani, M., Patel, N., Jivrajani, H., Bhaskara, G.B., Patel, S., 2014. Purification and characterization of haloalkaline, organic solvent stable xylanase from newly isolated halophilic Bacterium-OKH. Int. Scholarly Res. Notices, 10 pages. Shakoori, F.R., Shokat, M., Saleem, F., Riaz, T., 2015. Screening, optimization and characterization of xylanase by locally isolated bacteria. Punjab Univ. J. Zool. 30 (2), 65-71. Sharma, M., Mehta, S., Kumar, A., 2013. Purification and characterization of alkaline xylanase secreted from Paenibacillus macquariensis. Adv. Microbiol. 3, 32-41. Shen, J., Smith, E., 2015. Enzymatic treatments for sustainable textile processing, In Woodhead Publishing Series in Textiles, edited by Richard Blackburn, Woodhead Publishing, 119-133. Shehata, A.N., Abd El Aty, A.A., 2015. Improved production and partial characterization of chitosanase from a newly isolated Chaetomium globosum KM651986 and its application for chitosan oligosaccharides. J. Chem. Pharm. Res. 7(1):727-740. Silva, D., Tokuioshi, K., Martins, E.S., daSilva, R., Gomes, E., 2005. Production of pectinase by solid state fermentation with Penicillium viridicatum RFC3. Proc. Biochem. 40, 2885–2889. Silva, L.A.O., Terrasan, C.R.F., Carmona, E.C., 2015. Purification and characterization of xylanases from Trichoderma inhamatum. Elect. J. Biotechnol. 18, 307–313. Somogyi, M., 1945. A new reagent for the determination of sugars. J. Biol. Chem. 160, 61–68. Subramaniyan, S., Prema, P., 2002. Biotechnology of microbial xylanases: Enzymology, molecular biology and application. Critical Rev. Biotechnol. 22 (1), 33-46. Sugumaran, K.R., Srivastava, S.N., Ponnusami, V., 2012. Kinetic and thermodynamic characterization of Pleurotus eryngii MTCC 1798 xylanase and Fusarium oxysporum MTCC 3300 xylanase: A comparision. Int. J. Chem. Sci. 10(3), 1626-1636. Taibi, Z., Saoudi, B., Boudelaa, M., Trigui, H., Belghith, H., Gargouri, A., Ladjama, A., 2011. Purification and biochemical characterization of a highly thermostable xylanase from

17

Actinomadura sp. strain Cpt20 isolated from poultry compost. Appl. Biochem. Biotechnol. 166(3), 663-679. Tanaka, A., Hoshino, E., 2002. Calcium-binding parameter of Bacillus amyloliquefaciens α-amylase determined by inactivation kinetics. Biochem. J. 364, 635-639. Techapun, C., Poosaran, N., Watanabe, M., Sasaki, K., 2003. Thermostable and alkaline-tolerant microbial cellulase free xylanases produced from agricultural wastes and the properties required for use in pulp bleaching bioprocesses: A review. Proc. Biochem. 38, 1327-1340. Warzywoda, M., Ferre, V., Pourquie, J., 1983. Development of a culture medium for large-scale production of cellulolytic enzymes by Trichoderma reesei. Biotechnol. Bioengi. 25, 30053010.

Table (1) Specifications of the used fabrics. Type

Structure

Weight (g/m2)

Starch-sized cotton

woven

Plain 1/1

185

Grey linen

woven

Plain 1/1

220

Grey cotton

knitted Jersey 1/20 210

Grey cotton

knitted Peque 1/24 185

Bleached cotton

woven

Plain 1/1

130

Bleached linen

woven

Plain 1/1

200

3/1z

390

Substrate

Indigo-dyed cotton (jeans article) woven

18

Table (2) Partial purification of Trichoderma longibrachiatum KT693225 xylanase by fractional precipitation with ethanol and acetone. Fraction concentrati on

Protein content of fraction (mg.fraction-1)

Recovered protein (%)

Total activity -1

(U.fraction )

Recovered

Specific

activity

activity (U.mg

(%)

protein-1)

-fold purification

Aceo

Ethan

Aceto

Ethan

Aceto

Ethan

Aceto

Ethan

Aceto

Ethan

Aceto

Ethan

ne

ol

ne

ol

ne

ol

ne

ol

ne

ol

ne

ol

25

51.55

43.87

8.861

7.54

18.21

15.09

28.03

23.22

0.35

0.34

3.16

3.08

50

27.36

30.72

4.702

5.28

0.00

6.09

0.00

9.38

0.00

0.20

0.00

1.79

75

45.31

55.97

7.788

9.619

15.15

26.48

23.32

40.75

0.33

0.47

2.99

4.24

%

Culture filtrate contain 64.97 U xylanase activity and 581.76 mg protein

19

Table (3) Kinetics and thermodynamics of Trichoderma longibrachiatum KT693225 xylanase

Property

Value

Km (mg.ml-1) -1

20 -1

Vmax (µmol.min .ml )

20

Ea (Kcal.mol-1)

3.775

-1

Ed (KJ.mol )

62.05

T1/2 (min) 50ºC

326.90

60ºC

220.35

70ºC

84.21

Kd (min-1) 50ºC

0.0021

60ºC

0.0031

70ºC

0.0082

20

D-value (min) 50ºC

1086.15

60ºC

732.12

70ºC

279.79

ΔHº (KJ.mol-1) 50ºC

59.36

60ºC

59.28

70ºC

59.19

ΔGº (KJ.mol-1) 50ºC

95.85

60ºC

97.81

70ºC

98.09

ΔSº (J.mol-1.K-1) 50ºC

-112.97

60ºC

-115.72

70ºC

-113.39

21

Table (4) Effect of xylanase concentration on desizing efficiency of starch-sized grey fabric samples Enzyme concentration WL

(U.l-1)

%

Violet Scale

Wettability sec.

xylanase

α-amylase

00.00

00.00

0.45

<120

1

15.00

6.25

1.4

20

2

30.00

12.50

4.46

10

2

60.00

25.00

5.47

7

4

22

Table (5) Effect of enzymes cocktail treatment on the efficiency of the bioscouring process Substrate

Enzyme concentration

WL

Wettability

Reducing

U.40 ml-1

%

sec.

sugar mg.40 ml-1

pectinase xylanase

Linen

Single Jersey 1/20

Peque 1/24

cellulase

00.00

00.00

00.00

3.00

> 120

00.00

1.50

3.00

1.50

5.82

15

984

3.00

6.00

3.00

13.40

7

3408

00.00

00.00

00.00

2.00

>120

00.00

1.50

3.00

1.50

2.00

60

2472

3.00

6.00

3.00

13.99

45

6454

00.00

00.00

00.00

1.70

> 120

00.00

1.50

3.00

1.50

2.50

25

984

3.00

6.00

3.00

10.33

10

9408

23

Table (6) Effect of enzymes cocktail treatment on performance properties of cellulosic fabric WL

Roughness

Stiffness

(%)

(μm)

(mg)

blank

00.00

23.76

1246

--

without

0.57

17.79

997

--

with

1.45

15.36

641

--

blank

00.00

27.88

4272

--

without

1.81

24.38

3838

--

with

2.15

22.48

3560

--

blank

00.00

23.37

7120

11.82

without

1.28

21.79

6764

10.98

with

2.07

20.35

6408

10.70

Substrate Cotton (white)

Linen (white)

Indigo-dyed fabric

24

K/S

T.longibrachiatum

Xylanase activity U/ml

A.tenussima C.globosum

Agricultural wastes

Fig. (1) Xylanase production by three marine isolates on different agricultural wastes

b

Xylanase activity U/ml

Xylanase activity U/ml

a

pH

Reaction temperature

c

1/V

y = 1.2118x + 0.05 R² = 0.9397

1/Vmax 1lKm

1/S

Fig. (2): a,Xylanase reaction at different temperature. b, Xylanase reaction at different pH.c, Lineweaver-Burk plot

a

Log of relative activity

y = -0.8295x + 4.5489 R² = 0.9697

1000/Tk

1000/Tk

ln Kd

b

y = -5.1278x + 9.6924 R² = 0.9894

Fig. (3) Arrhenius plot for, a. activation energy (Ea), b. energy of denaturation (Ed)of T. longibrachiatum KT693225 xylanase.

a

b

Fig. (4) violet scale of untreated (a) and xylanase treated fabric samples (b).

a

b

Fig. (5): SEM images of untreated (a) and xylanase treated fabric samples(b).

a

b

c

d

e

f

Fig. (6) SEM images of treated and enzymatic treatment of cotton (a&b), linen (c&d) and indigo dyed cotton (e&f) samples.

Highlights  Utilization of disposal agricultural wastes in industrial enzymes production.  Purification of T. longibrachiatum KT693225 xylanase by fractional precipitation  Studying the kinetics of T. longibrachiatum xylanase Km and Vmax.  Emphasizing thermostability and suitability for industrial applications.  Studying thermodynamics of T.longibrachiatum xylanase (T1/2, Kd, ΔHº, ΔGº, ΔSº).  T. longibrachiatum can be used as an intermediate step in bioethanol production .  T.longibrachiatum enzymes showed effectiveness in textile wetprocessing stages.