Improvement of the Catalytic Activity of Chitosanase BsCsn46A from Bacillus subtilis by Site-Saturation Mutagenesis of Proline121

Abstract

BsCsn46A, a GH46 family chitosanase from Bacillus subtilis, has great potential for industrial chitooligosaccharide production due to its high activity and stability. In this study, a special amino acid Pro121 was identified not fit in the helix structure, which was located in the opposite side of the active center in BsCsn46A, by the PoPMuSiC algorithm. Then, saturation mutagenesis was performed to explore the role of the site amino acid 121. Compared with the wild type, the specific activity of P121N, P121C, and P121V was increased by 1.69-, 1.97-, and 2.15-fold, respectively. In particular, the specific activity of P121N was increased without loss of thermostability, indicating that replacing the structural stiffness of proline in the helical structure could significantly improve the chitosanase activity. The K m values of P121N, P121C, and P121V decreased significantly, indicating that the affinity between the enzyme–substrate complex was enhanced. Through molecular docking, it was found that the increase of hydrogen bonds and van der Waals force between the enzyme–substrate complex and the removal of unfavorable bonds might be the main reason for the change of enzyme properties. In addition, the optimal temperature of the three mutants changed from 60 to 55 °C. These results indicate that the site 121 plays a critical role in the catalytic activity and enzymatic properties of chitosanase. To our knowledge, the results provide novel data on chitosanase activity and identify an excellent candidate of industrial chitosanase.

Improved Whole-Cell Biocatalyst for the Synthesis of Vitamin E Precursor 2,3,5-Trimethylhydroquinone

“…The increase of hydrogen bonds between the enzyme−substrate complex improved the specific activity of variants P121N, P121C, and P121V of chitosanase BsCsn46A. 29 The kinetic assays revealed that the apparent K m values of L128A and L128K for 2,3,6-TMP were 0.16 ± 0.01 and 0.16 ± 0.02 mM, respectively, and the corresponding k cat / K m values were 2.93 and 3.03 s −1 mM −1 , respectively (Figure S4). The catalytic efficiency of L128A and L128K was higher than that of wild-type MpdA (K m , 0.17 ± 0.01 mM; k cat /K m , 2.84 s −1 mM −1 ).…”

“…Similar results were reported in previous work. The increase of hydrogen bonds between the enzyme–substrate complex improved the specific activity of variants P121N, P121C, and P121V of chitosanase BsCsn46A . The kinetic assays revealed that the apparent K m values of L128A and L128K for 2,3,6-TMP were 0.16 ± 0.01 and 0.16 ± 0.02 mM, respectively, and the corresponding k cat / K m values were 2.93 and 3.03 s –1 mM –1 , respectively (Figure S4).…”

New Insights into Bifunctional Chitosanases with Hydrolysis Activity toward Chito- and Cello-Substrates

“…Chitosanase catalysis represents a promising green solution, and its degradation products can be extensively used in pharmaceuticals, fine chemicals, and functional food. 2,3 Chitosanases are found in many species, such as bacteria, fungi, cyanobacteria, and plants, 4,5 and have been classified into six GH families in the Carbohydrate-Active Enzyme database (www.cazy.org) according to their amino acid sequences: the GH-5, 7, 8, 46, 75, and 80 families. 6,7 GH5, GH7, and GH8 chitosanases have evolved activities against a range of substrates.…”

“…Chitosanase (EC 3.2.1.132), a class of glycoside hydrolases (GHs), hydrolyzes internal β-1,4-glycosidic linkages between N -acetyl glucosamine (GlcNAc) (A-unit) and glucosamine (GlcN) (D-unit) in chitosan, thereby playing crucial roles in the biological cycling of polysaccharides and presenting several biotechnological applications. Chitosanase catalysis represents a promising green solution, and its degradation products can be extensively used in pharmaceuticals, fine chemicals, and functional food. , …”

Engineering the Thermostability of a d-Carbamoylase Based on Ancestral Sequence Reconstruction for the Efficient Synthesis of d-Tryptophan

“…Enormous endeavors have been devoted to engineering enzymes with improved stability. − However, a generally applicable strategy for engineering the thermostability of various enzymes has yet to be established . At present, several tools based on computational calculations and force field analysis have been developed, such as FoldX and Rosetta, which are time- and computation-intensive and rely on accurate protein structures and virtual saturation mutagenesis. − Moreover, when the structural and functional information of the protein is not fully understood, it is difficult to construct a rational design, let alone to predict the epistatic effect of combinatorial mutants. , It can be noted that most of the reported experimental and computational methods depend on site-directed saturation mutagenesis or screening of millions of candidates, which are labor- and computation-intensive. Ancestral sequence reconstruction (ASR) refers to the technique of deriving the amino acid sequences of ancestral enzymes of extinct organisms by computer algorithms.…”