Improvement of enzyme activity of β-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues

Abstract

β-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG, BG, and BG were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG was 2.11-fold higher than that of wild-type BG, whereas those of BG and BG were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BG (pH 5.0 and 40 °C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (k /K) of BG were 1.92- and 2.12-fold greater than those of BG at 40 °C, respectively. The catalytic efficiency of BG increased to 3.09-fold that of BG at 60 °C. These increases in the thermostability and catalytic efficiency of BG might be useful for the hydrolysis of β-glucans to produce fermentable sugars. Of the six mutated residues of BG, five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α/α-double-barrel structure is essential for enzyme activity.

Marine‐polysaccharide degrading enzymes: Status and prospects

“…Presently, structural information is only available for eight chitosanases from four families, GH7 (PDB ID: 1CEL), GH8 (PDB IDs: 1V5D and 5XD0), GH46 (PDB IDs: 1QGI, 4OLT, 1CHK, and 4ILY), and GH80 (PDB ID: 5B4S) (Adachi et al., 2004; Baek et al., 2017; Divne et al., 1994; Lyu et al., 2014; Marcotte, Monzingo, Ernst, Brzezinski, & Robertus, 1996; Saito et al., 1999; Takasuka et al., 2014; Yorinaga, Kumasaka, Yamamoto, Hamada, & Kawamukai, 2017). The enzyme belonging to GH7 showed a two‐domain organization with a large catalytic domain connected to a small cellulose‐binding domain via a glycosylated linker peptide (Figure 4A) (Divne et al., 1994).…”

“…X4 and Bacillus sp. K17, the catalytic sites are on the scaffold of a double‐α 6 ‐α 6 ‐barrel (Figure 4B and 4C) (Adachi et al., 2004; Baek et al., 2017). The other reported structures share overall similarity with each other, although the sequence identities between them are <20%.…”

Characterization of a GH8 β-1,4-Glucanase from Bacillus subtilis B111 and Its Saccharification Potential for Agricultural Straws

“…3.2.1.4) [ 21 ], licheninase (E.C. 3.2.1.73) [ 22 ], endo-1,4-β-xylanase (E.C. 3.2.1.8) [ 23 ], and reducing-end-xylose releasing exo-oligoxylanase (E.C.…”

Purification, characterization, immobilization and applications of an enzybiotic β-1,3–1,4-glucanase produced from halotolerant marine Halomonas meridiana ES021

“…X4 and Bacillus sp. SJ-10 cultures (Baek et al 2017 ; Tak et al 2019 ), and for both wild type and mutant β-1,3–1,4-glucanase purified from Bacillus subtilis MA139 (Pei et al 2015 ). Also, it was higher than that reported for recombinant β-1,3–1,4-glucanase purified from Saccharophagus degradans (30 °C) (Lafond et al 2016 ), and Bacillus velezensis ZJ20 (35 °C) (Xu et al 2016 ).…”