Lignocellulosic grass biomass is potential substrate for economical and sustainable bioethanol production. However, the processing cost of bioethanol that majorly includes the hydrolysis of cellulose by cellulases is still a major concern for its industrial production. Thus, knowledge on the sequence to the structural study of cellulase enzyme with consideration of its catalytic region can give important information for effective enzyme engineering and consequently towards enhanced bioethanol production from Pennisetum sp. Therefore, in this study, sequence conservativeness of different cellulosic site among a group of endoglucanase family of cellulase from previously isolated Aspergillus species has been determined. Furthermore, comparative molecular modeling of the endoglucanase from eight different Aspergillus species including Aspergillus fumigatus was conducted and the obtained structures revealed a high degree of difference in their conformational folds. Analysis from InterProScan revealed that the modeled endoglucanase has similar types of domains and share homology with protein family, such as glycoside hydrolase family-61 and fungal cellulose binding domain. Furthermore, molecular docking and interaction studies demonstrated the presence of residues in the endoglucanase of A. fumigatus viz. His20, His88, Asp96, Ala99, Ser100, Ser101, His102, His169, Glu170, Arg173, Glu178, and Tyr218 that are responsible in forming the substrate interaction. An interesting molecular phenomenon, i.e., catalytic promiscuity has been noted for all the substrate bound complexes of A. fumigatus endoglucanase which also depicts the degree of ligand binding efficacy of the studied enzyme. The molecular interaction study, binding energy analysis and molecular dynamics simulation, demonstrated that heteromeric substrate XylGlc3 is more strongly interacting with the receptor enzyme. Overall, the present findings revealed that important amino acid residues can help in increasing the specificity of endoglucanase from A. fumigatus towards hydrolysis of Pennisetum sp. and other biomass that has an adequate amount of XylGlc3, for possible industrial applications.
Aspartate 4-decarboxylase (ASD) has been modified to obtain the catalytic ability of the unnatural substrate l-3-methylaspartate. However, the mechanism remains to be clarified. In the present study, the semi-rational modification was used to identify key residues of importance for the activity towards l-3-methylasparte. The ASD from Pseudomonas dacunhae 21192 (PdASD) was used as a template, which showed better activity than the other two ASDs. Four residues proved to be critical for the activity towards l-3-methylasparte, with three located in the active site and one on the surface. Combinatorial variants were constructed to analyze the role of each mutation. The enzymatic properties of the combined variants were determined and compared. The residue at the 17th position was a member of the substrate entrance gate and contributed to the activity by reducing the steric hindrance. The residue at the 37th position was necessary for activity. Two mutations, I288 and V382, exhibited strong epistatic interactions on the activity of ASD. Structural changes in the active site were analyzed by molecular dynamics simulations, and it is proposed that the increased activity of PdASD variants is related to a suitable binding pocket for the substrate. These results provide new evidence for the mechanism of β-decarboxylation, which lays the foundation for enhancing the activity of ASD.
As an important industrial enzyme, protease is widely used in feed, food and other fields. At present, the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production. In this study, Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method. To improve the production of protease, atmospheric room temperature plasma (ARTP) mutagenesis was used in the first round, protease activity reached 315.0 U/mL. Then, to enhance production of protease, 60Co-γ irradiation was used for combined mutagenesis, leading to protease activity of B. amyloliquefaciens FMME ZK003 up to 355.0 U/mL. Furthermore, to realize the efficient production of protease, after optimization of fermentation conditions, protease activity was increased to 456.9 U/mL. Finally, protease activity of B. amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter. These results indicate that B. amyloliquefaciens can efficiently produce protease, which provides a good foundation for the industrial production of protease.
Brucella melitensis 7α-hydroxysteroid dehydrogenase (Bm7α-HSDH) catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. In this work, we investigated the effects of terminal modification (His-tags location and terminal truncation) on its catalytic efficiency and thermostability. Compared with C-terminal His-tagged Bm7α-HSDH (C-Bm7α-HSDH), N-Bm7α-HSDH showed a 3.6-fold higher k cat and a 1.3-fold lower K m, resulting in a 7.0-fold higher k cat/K m value toward chenodeoxycholic acid. Circular dichroism spectroscopy indicated that the melting temperature of N-Bm7α-HSDH (46.13 °C) was 3.0 °C lower than that of C-Bm7α-HSDH (49.13 °C). N-Bm7α-HSDH produced 7-oxolithocholic acid in the highest yield of 96.7% in 4 h, whereas the C-Bm7α-HSDH gave 96.4% in 10 h. Moreover, amino acids truncation and His-tag cleave experiments confirmed the C-terminal residues played key roles in catalytic functions. Molecular dynamics simulations further indicated C-terminal His-tagged modification could deform the substrate-binding region to disrupt the enzyme–substrate interactions and catalytic motion. However, the N-terminal His-tag hardly affected the catalytic efficiency due to its location far from the active site of the enzyme. This study provides structural insights into the terminus modifications of hydroxysteroid dehydrogenase on steroid substrate recognition and stabilization, thus affecting its catalytic functions.
Nigerose is a kind of rare disaccharide connected by an α-1,3 glucosidic bond, which is a potential probiotic due to its anti-digestive properties and beneficial functions. This study identified and characterized a novel GH65 glycoside phosphorylase derived from Anaerosporobacter mobilis (AmNP). This new protein could specifically catalyze the phospholysis of nigerose to generate glucose and glucose-1-phosphate in the presence of phosphate, indicating it was a typical nigerose phosphorylase. Compared to the previously reported nigerose phosphorylases, AmNP exhibited lower affinity towards nigerose in phosphorolysis reaction and higher affinity towards glucose in reverse phosphorolysis reaction, which indicated that AmNP might be superior in the synthetic capability of disaccharide. Then AmNP was employed to synergize with maltose phosphorylase from Lactobacillus brevis (LbMP) to catalyze the synthesis of nigerose using maltose as the substrate. After optimization of reaction conditions, the highest nigerose yield reached 132.0 g/L with a 66.3% conversion rate, which was higher than ever reported cases using the same reaction pathway to our knowledge. These findings on AmNP in this work were expected to provide a new candidate for large-scale enzymatic synthesis of nigerose and have important theoretical significance for studying nigerose phosphorylase.