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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

SDS-PAGE with lane a: purified endo β-1,4-d-glucanase and lane b: Molecular weight markers
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Fig1: SDS-PAGE with lane a: purified endo β-1,4-d-glucanase and lane b: Molecular weight markers

Mentions: The purified endo β-1,4-d-glucanase showed a single protein band on SDS-PAGE with molecular weight ~55 kDa (Fig. 1) and was found to be a monomeric protein from native gel (data not shown). There are reports of varying molecular mass of endo β-1,4-d-glucanase from different organisms, 29 kDa from Aspergillus niger AT-3 (Dutt and Kumar 2014) and 62 kDa from P. betulinus (Valaskova and Baldrian 2006).Fig. 1


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
SDS-PAGE with lane a: purified endo β-1,4-d-glucanase and lane b: Molecular weight markers
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829572&req=5

Fig1: SDS-PAGE with lane a: purified endo β-1,4-d-glucanase and lane b: Molecular weight markers
Mentions: The purified endo β-1,4-d-glucanase showed a single protein band on SDS-PAGE with molecular weight ~55 kDa (Fig. 1) and was found to be a monomeric protein from native gel (data not shown). There are reports of varying molecular mass of endo β-1,4-d-glucanase from different organisms, 29 kDa from Aspergillus niger AT-3 (Dutt and Kumar 2014) and 62 kDa from P. betulinus (Valaskova and Baldrian 2006).Fig. 1

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