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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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ABSTRACT

An acidophilic-solvent-thermostable endo β-1,4-d-glucanase produced from a potential Trichoderma harzianum strain HZN11 was purified to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with 33.12 fold purification with specific activity of 66.25 U/mg and molecular mass of ~55 kDa. The optimum temperature and pH were 60 °C and 5.5 retaining 76 and 85 % of activity after 3 h, respectively. It showed stability between pH 4.5–6.0 and temperature between 50–70 °C indicating thermostability. Endo β-1,4-d-glucanase was activated by Ca2+ and Mg2+ but inhibited by Hg2+, Pb2+ and Cd2+. The effect of thiol reagents, metal chelators, oxidizing agents and surfactants on enzyme activity has been studied. Purified endo β-1,4-d-glucanase exhibited highest specificity towards carboxymethyl cellulose. Kinetic analysis showed the Km, Vmax and Ki (cellobiose inhibitor) of 2.5 mg/mL, 83.75 U/mg and 0.066 M, respectively. The storage stability of purified endo β-1,4-d-glucanase showed a loss of mere 13 % over a period of 60 days. The hydrolysis efficiency of purified endo β-1,4-d-glucanase mixed with cocktail was demonstrated over commercial 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 sorghum bagasse yielded 4.3 g/L bioethanol (16 h) confirmed by gas chromatography–mass spectrometry (GC–MS). Morphological and structural changes were assessed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. Elemental analysis was carried out by SEM equipped with energy dispersive X-ray technique. These unique properties prove the potentiality of enzyme for biomass conversion to biofuel and other industrial applications.

Electronic supplementary material: The online version of this article (doi:10.1007/s13205-016-0421-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Enzymatic hydrolysis of untreated and pretreated sweet sorghum bagasse with purified endo β-1,4-d-glucanase mixed cocktail and commercial enzyme at 40 °C (a), optimization of temperature and time for enzymatic hydrolysis of alkali pretreated sweet sorghum and sugarcane bagasse with purified endo β-1,4-d-glucanase mixed cocktail (b), and production of ethanol from sweet sorghum bagasse hydrolyzate fermented by Saccharomyces cerevisiae NCIM 3594 (c). Data values represent average of triplicates and error bars represent standard deviation
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Fig3: Enzymatic hydrolysis of untreated and pretreated sweet sorghum bagasse with purified endo β-1,4-d-glucanase mixed cocktail and commercial enzyme at 40 °C (a), optimization of temperature and time for enzymatic hydrolysis of alkali pretreated sweet sorghum and sugarcane bagasse with purified endo β-1,4-d-glucanase mixed cocktail (b), and production of ethanol from sweet sorghum bagasse hydrolyzate fermented by Saccharomyces cerevisiae NCIM 3594 (c). Data values represent average of triplicates and error bars represent standard deviation

Mentions: In the process of production of cellulosic bioethanol, pretreatment and enzymatic hydrolysis is the crucial steps for saccharification and the reducing sugars could be fermented to ethanol. Untreated and alkali pretreated sweet sorghum bagasse and sugarcane bagasse were used to test the ability of the purified endo β-1,4-d-glucanase with other crude hydrolytic enzyme cocktail and commercial cellulase for the production of fermentable sugar by enzymatic hydrolysis. Alkali pretreated sweet sorghum and sugarcane bagasse released higher amounts of reducing sugars as compared to untreated. Maximum reducing sugars of 3.7 and 5.2 g/g were produced from sweet sorghum and sugarcane bagasse, respectively, at 48 h when treated with purified endo β-1,4-d-glucanase mixture of cocktail, in comparison to commercial cellulase which produced 2.4 and 4.3 g/g of reducing sugars with sweet sorghum and sugarcane bagasse, respectively, at 48 h represented in Fig. 3a. Hence, the efficient bioconversion of lignocellulosic biomass necessarily requires the synergetic action of cellulolytic enzymes, depolymerizing and debranching hemicellulolytic enzymes. Enzymatic hydrolysis of the alkali pretreated sweet sorghum and sugarcane bagasse into glucose was optimized. Optimization of hydrolysis time and temperature revealed high amounts of sugars 5.2 g/g at 36 h and 6.8 g/g at 48 h from alkali pretreated sweet sorghum and sugarcane bagasse, respectively, at 50 °C indicated in Fig. 3b. Therefore, these process parameters play an essential role in hydrolysis of lignocelluloses to get efficient yield of reducing sugars. Moreover, short processing times are a key parameter to an economically viable industrial process. Formulation of cellulase enzymes is challenging for hydrolysis process but will not only lower the enzyme loadings but also reduce the capital cost in a cellulosic bioethanol production. In agreement to our results, crude enzyme cocktail from T. harzianum KUC1716 and S. commune KUC9397 could replace the commercial enzymes with approx 80 % hydrolysis yield (Lee et al. 2015). A similar study reported by Patel et al. (2015) on hydrolysis of pretreated maize stover suggests that enzyme cocktail with commercial cellulase results in better sugar release. Vimala et al. (2011) studied the effect of different pretreatment strategies for enzymatic hydrolysis of sorghum straw and found that alkali delignified treatment released higher sugars. To minimize the process cost is of great importance for an economical cellulose-based ethanol production. Even if the new enzyme mixes are said to have improved efficiency, the enzyme cost is still contributing to a big part of the total production cost. The present study indicated that the purified endo β-1,4-d-glucanase with cocktail obtained from Trichoderma harzianum strain HZN11 enhanced the efficiency of biomass hydrolysis.Fig. 3


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
Enzymatic hydrolysis of untreated and pretreated sweet sorghum bagasse with purified endo β-1,4-d-glucanase mixed cocktail and commercial enzyme at 40 °C (a), optimization of temperature and time for enzymatic hydrolysis of alkali pretreated sweet sorghum and sugarcane bagasse with purified endo β-1,4-d-glucanase mixed cocktail (b), and production of ethanol from sweet sorghum bagasse hydrolyzate fermented by Saccharomyces cerevisiae NCIM 3594 (c). Data values represent average of triplicates and error bars represent standard deviation
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Fig3: Enzymatic hydrolysis of untreated and pretreated sweet sorghum bagasse with purified endo β-1,4-d-glucanase mixed cocktail and commercial enzyme at 40 °C (a), optimization of temperature and time for enzymatic hydrolysis of alkali pretreated sweet sorghum and sugarcane bagasse with purified endo β-1,4-d-glucanase mixed cocktail (b), and production of ethanol from sweet sorghum bagasse hydrolyzate fermented by Saccharomyces cerevisiae NCIM 3594 (c). Data values represent average of triplicates and error bars represent standard deviation
Mentions: In the process of production of cellulosic bioethanol, pretreatment and enzymatic hydrolysis is the crucial steps for saccharification and the reducing sugars could be fermented to ethanol. Untreated and alkali pretreated sweet sorghum bagasse and sugarcane bagasse were used to test the ability of the purified endo β-1,4-d-glucanase with other crude hydrolytic enzyme cocktail and commercial cellulase for the production of fermentable sugar by enzymatic hydrolysis. Alkali pretreated sweet sorghum and sugarcane bagasse released higher amounts of reducing sugars as compared to untreated. Maximum reducing sugars of 3.7 and 5.2 g/g were produced from sweet sorghum and sugarcane bagasse, respectively, at 48 h when treated with purified endo β-1,4-d-glucanase mixture of cocktail, in comparison to commercial cellulase which produced 2.4 and 4.3 g/g of reducing sugars with sweet sorghum and sugarcane bagasse, respectively, at 48 h represented in Fig. 3a. Hence, the efficient bioconversion of lignocellulosic biomass necessarily requires the synergetic action of cellulolytic enzymes, depolymerizing and debranching hemicellulolytic enzymes. Enzymatic hydrolysis of the alkali pretreated sweet sorghum and sugarcane bagasse into glucose was optimized. Optimization of hydrolysis time and temperature revealed high amounts of sugars 5.2 g/g at 36 h and 6.8 g/g at 48 h from alkali pretreated sweet sorghum and sugarcane bagasse, respectively, at 50 °C indicated in Fig. 3b. Therefore, these process parameters play an essential role in hydrolysis of lignocelluloses to get efficient yield of reducing sugars. Moreover, short processing times are a key parameter to an economically viable industrial process. Formulation of cellulase enzymes is challenging for hydrolysis process but will not only lower the enzyme loadings but also reduce the capital cost in a cellulosic bioethanol production. In agreement to our results, crude enzyme cocktail from T. harzianum KUC1716 and S. commune KUC9397 could replace the commercial enzymes with approx 80 % hydrolysis yield (Lee et al. 2015). A similar study reported by Patel et al. (2015) on hydrolysis of pretreated maize stover suggests that enzyme cocktail with commercial cellulase results in better sugar release. Vimala et al. (2011) studied the effect of different pretreatment strategies for enzymatic hydrolysis of sorghum straw and found that alkali delignified treatment released higher sugars. To minimize the process cost is of great importance for an economical cellulose-based ethanol production. Even if the new enzyme mixes are said to have improved efficiency, the enzyme cost is still contributing to a big part of the total production cost. The present study indicated that the purified endo β-1,4-d-glucanase with cocktail obtained from Trichoderma harzianum strain HZN11 enhanced the efficiency of biomass hydrolysis.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

An acidophilic-solvent-thermostable endo β-1,4-d-glucanase produced from a potential Trichoderma harzianum strain HZN11 was purified to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with 33.12 fold purification with specific activity of 66.25 U/mg and molecular mass of ~55 kDa. The optimum temperature and pH were 60 °C and 5.5 retaining 76 and 85 % of activity after 3 h, respectively. It showed stability between pH 4.5–6.0 and temperature between 50–70 °C indicating thermostability. Endo β-1,4-d-glucanase was activated by Ca2+ and Mg2+ but inhibited by Hg2+, Pb2+ and Cd2+. The effect of thiol reagents, metal chelators, oxidizing agents and surfactants on enzyme activity has been studied. Purified endo β-1,4-d-glucanase exhibited highest specificity towards carboxymethyl cellulose. Kinetic analysis showed the Km, Vmax and Ki (cellobiose inhibitor) of 2.5 mg/mL, 83.75 U/mg and 0.066 M, respectively. The storage stability of purified endo β-1,4-d-glucanase showed a loss of mere 13 % over a period of 60 days. The hydrolysis efficiency of purified endo β-1,4-d-glucanase mixed with cocktail was demonstrated over commercial 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 sorghum bagasse yielded 4.3 g/L bioethanol (16 h) confirmed by gas chromatography–mass spectrometry (GC–MS). Morphological and structural changes were assessed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. Elemental analysis was carried out by SEM equipped with energy dispersive X-ray technique. These unique properties prove the potentiality of enzyme for biomass conversion to biofuel and other industrial applications.

Electronic supplementary material: The online version of this article (doi:10.1007/s13205-016-0421-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus