Improved Thermostability of Maltooligosyltrehalose Synthase from Arthrobacter ramosus by Directed Evolution and Site-Directed Mutagenesis

Abstract

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in trehalose production. MTSase from Arthrobacter ramosus has poor thermostability, limiting its industrial use. In this study, mutant G415P was obtained by directed evolution and S361R/S444E was subsequently generated based on a structure analysis of the region around G415. The t 1/2 of G415P and S361R/S444E at 60 °C increased by 3.0-and 3.2-fold, respectively, compared with the wild-type enzyme. A triple mutant (G415P/S361R/S444E) was obtained through a combination of the above mutants, and its t 1/2 significantly increased by 19.7fold. Kinetic and thermodynamic stability results showed that the T 50 and T m values of the triple mutant increased by 7.1 and 7.3 °C, respectively, compared with those of the wild-type enzyme. When the triple mutant was used in trehalose production, the yield reached 71.6%, higher than the 70.3% achieved with the wild-type. Thus, the mutant has a potential application for industrial trehalose production.

Enhanced Thermostability of Pseudomonas nitroreducens Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis

“…The modeled structure of IEM showed that K83, K95, and L273 were all located on the protein surface (Figure 5a), which was consistent with the opinion that irreversible thermal denaturation of a protein usually contained an unfolding step and such process primarily involved surface-located regions [40]. Intramolecular interactions including hydrogen bonds, salt bridges, and disulfide bonds had pivotal roles in enhancing the thermal stability of proteins [25,26,28]. When the distance between two polar non-hydrogen atoms was less than 3.5 Å, a hydrogen bond which contributed 0.6 kcal/mol energy to protein stability could be formed [41].…”

“…Directed evolution is a useful protein engineering method [23], and numerous researches enhanced enzymes' performances at elevated temperatures by employing errorprone PCR [24,25]. However, it is very time-consuming to screen large mutant pools, especially for those enzymes without efficient high-throughput screening approaches.…”

“…The purpose of this work was to enhance the thermostability of IEM from P. nitroreducens Jin1 by combining the strategies of surface residue replacement and consensus mutagenesis for the first time. The GETAREA (http://curie.utmb.edu/getarea.html) [38] and Directed evolution is a useful protein engineering method [23], and numerous researches enhanced enzymes' performances at elevated temperatures by employing errorprone PCR [24,25]. However, it is very time-consuming to screen large mutant pools, especially for those enzymes without efficient high-throughput screening approaches.…”

Enhancing Activities of Salt-Tolerant Proteases Secreted by Aspergillus oryzae Using Atmospheric and Room-Temperature Plasma Mutagenesis

“…Although the directed mutagenesis breeding technology has been developed rapidly in the past decade, the random chemical and physical mutagenesis breeding technologies still play an important role in obtaining organisms with high enzymic activities or ideal properties. − Recently, ARTP, a newly developed mutation system, has shown great potential in microbe breeding. Its core component is a helium radio-frequency atmospheric-pressure glow discharge (RF APGD) plasma generator .…”

Enzymatic Preparation of Non‐Reducing Oligosaccharides from Maltodextrins and Nigerooligosaccharides

“…Q36 MTSase is similar to those of the MTSases from A. ramosus (Ar-MTSase; PDB entry 6LCU), Sulfolobus acidocaldarius (Sa-MTSase; PDB entry liv8), Sulfolobus shibatae DSM5389 (5389-GTSase; PDB entry 5zcr) and Sulfolobus tokodaii (St-MTSase; PDB entry 3hje). [27][28][29][30][31] The MTSase first split the C1-O bond of the glucoside linkage at the reducing end of the substrate. [32] The glycosyl-enzyme intermediate was formed after glycosyl residue bound to the enzyme, and the glycosyl reside was transferred to the glucose in the cleft by the -1,1-linkage.…”