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.

Acidithiobacillus caldus, a typical sulfur oxidizer, derives the majority of its energy from sulfur oxidation. And the essential enzyme for sulfide oxidation catalysis is sulfide quinone oxidoreductase (SQR), an ancient flavoprotein. Here, the catalytic mechanism of SQR generated from A. caldus was investigated (SQR^Ac). According to phylogenetic study, SQR^Ac (ACAty RS11315) is closely related to SQR (BAD99305) of Acidithiobacillus ferrooxidans NASF-1 and is classified as a type I Sqr enzyme. SQR^Ac heterologously produced in Escherichia coli exhibits the distinctive absorption peaks (375, 450 nm) of the flavoproteins family of proteins in its absorption spectrum. Utilizing site-directed mutagenesis, the function of conserved cysteines in the catalytic pathway was determined. Based on the sulfide quinone redox reactions in vitro of SQR^Ac and variations, Cys160 and Cys356 have been identified as enzyme-active residues. Mutation of another cysteine present in all type I SQRs (Cys128) decreased enzyme activity by 56%, indicating that this residue plays an important but non-essential role in enzyme function. In addition, the binding affinities of SQR^Ac, the visualization of its 3D structure, and the interaction between receptors and ligands were investigated. Finally, a suitable sulfide quinone redox catalytic mechanism for A. caldus was proposed.

l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesize β-hydroxy-α-amino acids with unsatisfactory enzyme activity. Here, we expressed the l-threonine aldolase from Pseudomonas putida KT2440 (l-PpTA) in Escherichia coli BL21 (DE3) and improved the activity and thermostability by protein engineering. Five amino acid residues (Ser10, His89, Asp93, Arg177, and Arg321) located in the substrate-binding pocket were selected and for mutation. Eight mutants (D93A, D93G, D93M, D93F, D93S, D93Q, D93Y and D93H) with increased enzyme activity were identified and their k _cat/K _M values showed about 1–7-fold higher than wild-type. Among all the variants, D93H showed the highest catalytic efficiency with 2925 and 4515 s⁻¹ mM⁻¹ of k _cat/K _M values toward l-threonine and l-allo-threonine, respectively. In addition, circular dichroism spectrum exhibited that the melting temperature of D93H (54.2 °C) was 5 °C higher than wild-type (49.2 °C). Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate, which facilitated the proton extraction and promote the enzymatic reaction. This work affords a candidate for the synthesis of β-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.

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.

N-acetylneuraminic acid (NeuAc) is an important nutrient that plays a key role in brain development in infants NeuAc is mainly produced by extraction from natural resources such as edible birds’s nests, crucian eggs, caviar and human breast milk. The extraction process is complicated, resulting in the disadvantages of low NeuAc content and low recovery rate. In this study, a crude enzyme immobilization-based cell-free system (CEICFS) was developed for efficient NeuAc biosynthesis. First, N-terminal coding sequences that improved the expression levels of N-acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid aldolase (NanA) were obtained by high-throughput screening. And these sequences resulted in up to 1.5-fold (1.2-fold) increase in AGE (NanA) enzyme levels. And then, a CEICFS for NeuAc biosynthesis was proposed by directly immobilizing crude enzyme containing AGE and NanA on amino resin. Subsequently, NeuAc production from GlcNAc using CEICFS in one reactor was carried out, resulting 68 g/L of NeuAc and the highest productivity of 6.8 g/L/h. Further, the enzyme activity was still higher than 75% after five repeated uses. The functional properties of CEICFS were studied and compared to those of the free enzyme, immobilization can extend the application of enzyme to some harsh environments, such as low temperature and acidic environment. Therefore, CEICFS with excellent heat resistance, storage stability and reusability exhibit great potential for industrial application.

Trehalose is a disaccharide with many applications in cosmetics, refrigeration, and food. Trehalose synthase is a significant enzyme in trehalose production. Escherichia coli is usually used to express this enzyme heterologously. Since E. coli is a pathogenic strain, we chose Corynebacterium glutamicum ATCC13032 as an engineering strain in this study for food safety reasons. Because of its poor permeability, we constructed two recombinant C. glutamicum strains using two anchor proteins, PorH, and short-length NCgl1337, to anchor trehalose synthase from Streptomyces coelicolor on the cell surface and synthesize trehalose directly from maltose. Studies on enzymatic properties indicated that NCgl1337S–ScTreSK246A had better enzyme activity and thermal stability than the free enzyme. After optimizing the whole-cell transformation, the optimal transformation condition was 35 °C, pH 7.0, and OD₆₀₀ of 30. Under this condition, the conversion rate of 300 g/L maltose reached 69.5% in a 5 L fermentor. The relative conversion rate was still above 75% after repeated five times.

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.