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.
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 BG2-6, BG2-10, and BG5-28were 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 BG5-28was 2.11-fold higher than that of wild-type BGwt, whereas those of BG2-6and BG2-10were 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 BGwt(pH 5.0 and 40 °C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (kcat/Km) of BG5-28were 1.92- and 2.12-fold greater than those of BGwtat 40 °C, respectively. The catalytic efficiency of BG5-28increased to 3.09-fold that of BGwtat 60 °C. These increases in the thermostability and catalytic efficiency of BG5-28might be useful for the hydrolysis of β-glucans to produce fermentable sugars. Of the six mutated residues of BG5-28, 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 α6/α6-double-barrel structure is essential for enzyme activity.
Department of Pharmacy, Sunchon National University, Suncheon, 57922, Republic of Korea.|Department of Agricultural Chemistry, Sunchon National University, Suncheon, 57922, Republic of Korea.
Seung Cheol
SC
Baek
Department of Biological Sciences, Konkuk University, Gwangjin-gu, Seoul, 05029, Republic of Korea.
Thien-Hoang
TH
Ho
Department of Pharmacy, Sunchon National University, Suncheon, 57922, Republic of Korea.
Hyun Woo
HW
Lee
Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon, 57922, Republic of Korea.
Won Kyeong
WK
Jung
Department of Agricultural Chemistry, Sunchon National University, Suncheon, 57922, Republic of Korea.
Hyo-Seung
HS
Gang
Department of Biological Sciences, Konkuk University, Gwangjin-gu, Seoul, 05029, Republic of Korea. lkang@konkuk.ac.kr.
Lin-Woo
LW
Kang
Department of Pharmacy, Sunchon National University, Suncheon, 57922, Republic of Korea. hoon@sunchon.ac.kr.|Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon, 57922, Republic of Korea. hoon@sunchon.ac.kr.
Hoon
http://orcid.org/0000-0002-7203-3712
H
Kim
D002384:Catalysis; D003001:Cloning, Molecular; D003460:Crystallization; D017931:DNA Primers; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature; D047071:beta-Glucans / Q000378:metabolism
D017931:DNA Primers; D047071:beta-Glucans; D006026:Glycoside Hydrolases; C028020:licheninase
Activity improvement; Crystallography; Error-prone PCR; Truncation; β-1,3-1,4-Glucanase (BGlc8H)