Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase

Abstract

The thermostability of potato type L ␣-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations-Phe393Leu (F39L), Asn1353Ser (N135S), and Thr7063Ile (T706I)-by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.␣-Glucan phosphorylase (EC 2.4.1.1) catalyzes the reversible phosphorolysis of ␣-1,4 glucan and is widely distributed in microorganisms, plants, and animals. All known ␣-glucan phosphorylases require pyridoxal 5Ј-phosphate (PLP) for activity and seem to share a similar catalytic mechanism (17). Although all ␣-glucan phosphorylases belong to a large highly homologous group that includes glycogen phosphorylases from bacteria, yeast, and animals, starch phosphorylase from plants, and maltodextrin phosphorylases of bacteria, these enzymes from distinct origins are known to differ in their substrate preference and their mode of regulation (14,22).The wide distribution of ␣-glucan phosphorylase suggests that this enzyme plays an important role in the cellular metabolism of reserve polysaccharides: starch and glycogen. ␣-glucan phosphorylase is also potentially useful for the production of glucose 1-phosphate (G-1-P) and for the synthesis of linear amylose and other glucose polymers with various structures (6,23). To obtain ␣-glucan phosphorylase suitable for industrial applications, we previously reported the properties and primary structures of thermostable ␣-glucan phosphorylases from Bacillus stearothermophilus (19) and Thermus aquaticus (18). In these studies, we found that bacterial ␣-glucan phosphorylases appeared to have higher K m values of malto-oligosaccharides such as maltotetraose as the primer for glucan synthesis compared to plant ␣-glucan phosphorylases. To engineer useful ␣-glucan phosphorylase for various applications, the potato type L ␣-glucan phosphorylase gene was subjected to random mutagenesis by error-prone PCR, and thermostable variant enzymes were screened. Based on the results of random mutagenesis, we focused on specific amino acid replacements and obtained double and triple mutants by site-directed mutagenesis. We describe here the enhanced thermostability of potato type L ␣-glucan phosphorylase. Possible antiproteolytic degradation of the mutant enzymes is also described. MATERIALS AND METHODS Materials.Bacto tryptone and Bacto yeast extract ...

Preparation of inclusion complexes composed of amylose and biodegradable poly(glycolic acid-co-ɛ-caprolactone) by vine-twining polymerization and their lipase-catalyzed hydrolysis behavior

“…36 Lipase from the fungus Phycomyces nitens (B150 U mg À1 ) was purchased from Wako Pure Chemical Industries (Osaka, Japan). P(GA-co-CL)s were prepared by copolymerization of GA and CL in a small amount of water with nitrogen flowing at 200 1C for 4 h according to a previously described procedure.…”

Enzymatic transformation of nonfood biomass to starch

“…Wild-type PGP has an estimated TTN value of ∼6 × 10 4 at 30°C. Via directed evolution, a PGP mutant has a nearly two orders of magnitude enhancement in TTN values, being ∼1.7 ×10 6 (43). By considering the stability of immobilized glucose isomerase (29) and other immobilized thermophilic enzymes (44), further stability improvement of CBP and PGP is expected to be achieved by more rounds of mutagenesis plus enzyme immobilization soon.…”

Enhancing the Thermal Stability of Sucrose Phosphorylase from Streptococcus mutans by Random Mutagenesis

“…Most of such thermostable enzymes are derived from the thermophilic archea or bacteria, 14 17) although some were produced by protein engineering. 11,18) In the current study, we attempted to obtain a highly thermostable mutant of S. mutans SPase by random mutagenesis using error prone PCR. The screening method we employed is based on the detection of amylose synthesized from sucrose by the actions of SPase and glucan phosphorylase.…”

“…It is widely known that the thermal stability of an enzyme can be elevated by combining each mutation that contributes to the enhancement of the thermal stability. 11,12) To evaluate the effect of the combination of the eight amino acid substitutions on thermal stability, those mutations were combined and their thermal stabilities were then examined. The enzyme with two mutations, T47S and D249G, retained more than 90% of its initial activity after heating at 55 C for 20 min (Fig.…”