An acidothermophilic functionally active novel GH12 family endoglucanase from Aspergillus niger HO: purification, characterization and molecular interaction studies

Abstract

Endoglucanase (EG) from Aspergillus niger HO was sequentially purified through ultrafiltration, ion exchange and size exclusion chromatography to homogeneity, with an overall recovery of 18 %. The purified EG was a monomeric protein with a molecular weight of about 55 kDa. The enzyme was optimally active at pH 3.5 and 70 °C with a half life (t1/2) of 3 h and Km value of 2.5 mg/ml. Metal ions, such as Ca(2+) and Co(2+) helped in enzyme induction, while Hg(2+) and Cu(2+) strongly inhibited the enzyme activity. Peptide mass fingerprinting results revealed that the purified EG is a novel enzyme that belongs to family 12 of glycoside hydrolase (GH12). Molecular docking studies indicated the presence of Glu116 and Glu204 as important determinant residues for the functional interaction with carboxymethylcellulose and showed hydrogen bonding with Asp99, Glu116, Glu204 and hydrophobic interactions with Trp22, Val58, Tyr61, Phe101, Met118, Trp120, Pro129, Ile130, Thr160 and Phe206. Hydrolysis of 2 % CMC with purified acidothermophilic EG at its optimum temperature and pH resulted in complete hydrolysis within 2 h yielding 18 % cellotriose, 72 % cellobiose and 10 % glucose as evident from HPLC analysis. In comparison to most of the EGs reported in literature, EG from A. niger HO exhibited higher thermostability. The acidothermophilic nature of this enzyme makes it potentially useful for industrial applications.

Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis

“…Several reports have indicated that Co 2+ can enhance the activity of endoglucanases from E. coli Rosetta 2 and Aspergillus niger (Rawat et al, 2015; Martin et al, 2014). In the present study, the activity of EglC22b was also increased by the presence of Co 2+ .…”

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

“…Of these, endo-␤-1,4-glucanase plays a key role in cellulose degradation, as it can randomly break down the cellulose backbone internally to release free chain ends. According to the classification of carbohydrate enzymes (http://www.cazy.org/), endo-␤-1,4-glucanases are often found in GH families 6,7,8,9,12,44,45,48,51,74, and 126 (5). Among them, GH12 is a unique group possessing both endoglucanase and xyloglucanase activities (6), and GH12 endoglucanases usually attract attention due to their higher endo-␤-1,3-1,4-glucanase activity.…”

“…A large number of fungal endoglucanases of GH12 have been cloned and extensively studied, including the EGIII from Trichoderma reesei (8), a thermostable Cel12A from Humicola grisea (4), and the highly active EG from Aspergillus niger (9). Of the eight structure-resolved fungal endoglucanases of GH12, two catalytic sites, Glu116 and Glu200, and eight substrate binding sites, Trp7, Trp22, Val57, Tyr111, Ile130, Asn151, Met154, and Phe202 (EGIII numbering), are highly conserved (8).…”

Industrial Applications of Endoglucanase Obtained from Novel and Native Trichoderma atroviride

“…36,37 Metal ions can cause either inhibition of enzyme or activation, based on the nature of the cations. 36 Determining the effect of metal ions on enzyme hydrolysis is important because many industrial applications require their addition at various stages of the process, and the greater part of the enzymes requires a specific metal ion as a cofactor for their catalytic activity. 36,38 Experiments were conducted in the activity method described above with different concentrations of various salt ions.…”

“…36 Determining the effect of metal ions on enzyme hydrolysis is important because many industrial applications require their addition at various stages of the process, and the greater part of the enzymes requires a specific metal ion as a cofactor for their catalytic activity. 36,38 Experiments were conducted in the activity method described above with different concentrations of various salt ions. Effects of the metal ions on the enzyme activity for hydrolysis of CMC are shown in Table 3.…”