D-Allulose 3-epimerase catalyzes C-3 epimerization between D-fructose and D-allulose was found in Arthrobacter globiformis strain M30. The enzyme gene was cloned, and its recombinant enzyme and the mutant variants were expressed in E. coli. Using the information of the sequence and model structure, we succeed in the improvement of melting temperature for the enzyme without significant loss of the enzyme activity by protein engineering method. The melting temperatures were increased by 2.7, 2.1, 3.7, 5.1, and 8.0 °C for the mutants Glu75Pro, Arg137Lys, Ala200Lys, Ala270Lys, and Val237Ile, respectively. Each effect of the mutation was independent and additive. By integrating the above mutations, we constructed a thermostable mutant that exhibits a melting temperature 12 °C higher than wild type, and remains stable at 65 °C for 2 h. These highly stable properties suggest that the thermostable enzymes represent an ideal enzyme candidate for the industrial production of D-allulose.

Enzymatic hydrolysis of cellulosic biomass is a complex process involving many factors, including multiple enzymes, heterogeneous substrates, and multi-step enzyme reactions. Cellulase researchers have conventionally used a double-exponential equation to fit the experimental time course of product formation, but no theoretical basis for this has been established. Here we present a mechanism-based equation that fits well the progress curves of cellulase reaction, incorporating the concepts of non-productive and productive binding on the cellulose surface and processivity. The derived equation is double exponential. Our findings indicate that the reaction mechanism of cellulase itself can account for the double-exponential nature of the progress curve independently of other factors that may contribute, such as substrate heterogeneity and involvement of other enzymes.

The branched structure of amylose was probed using concanavalin A (ConA) lectin, which forms precipitable aggregates with highly branched glucans, such as glycogen and amylopectin. Rice (japonica cultivar) amylose was fractionated from de-fatted, gelatinized starch by precipitation with 1-butanol (BuOH) and purified by ultracentrifugation and repeated crystallization. The purified amylose still has short side chains, whose chain-length (CL) distribution resembles that of amylopectin. More than 96 wt% of the amylose was not precipitated with ConA and remained in the resultant supernatant. The amylose recovered from the supernatant exhibited essentially the same size distributions of molecules and the CL distributions of main and side chains as those of amylose without ConA precipitation. The molar % of branched molecules was slightly decreased by ConA precipitation (-ConA, 11.6; +ConA, 8.1). These results suggest that the side chains detected in BuOH-precipitable amylose preparation are essentially attributable to amylose itself. Also, the non-precipitable nature of the branched molecules of amylose by ConA supports our previous proposal that the organization of the short side chains on amylose molecules is quite different from that found in amylopectin, in which the short side chains are arranged in a cluster fashion, and the branched glucan interacts with ConA to form precipitable aggregates. A tiny amount of ConA-precipitable glucan was detected, but its CL distribution was inconsistent with the size distribution of the branched molecules. Even if the precipitable glucans were fragments of amylopectin, their contribution to the branches detected in amylose should be minor.

The application of flour is determined by the composition of its starch and storage proteins. Previously isolated diploid waxy wheat is known to be amylose-free and possesses the same amylopectin structure as the wild-type. To reveal its characteristics, starch, protein, lipid, fiber, gluten, and allergen contents and rheological properties were analyzed and compared to its parental wild-type diploid wheat and commercially available hexaploid wheats. The results showed that the starch content of diploid waxy wheat was similar, but its protein, lipid, and fiber contents were higher than that of the wild-type. In addition, diploid waxy wheat produced high levels of gluten unlike its wild-type while its allergen level was similar to its wild-type. The storage modulus of diploid waxy wheat was significantly lower than that of other wheat lines at high temperatures. These results suggest that diploid waxy wheat holds different characteristics from hexaploid wheats for food processing.

β-Glucose 1-phosphate (βGlc1P) is a donor substrate in the synthesis of various α-glucosides by glycoside phosphorylases belonging to the glycoside hydrolase family 65. This study presents an efficient synthesis of βGlc1P combining enzymatic phosphorolysis of inexpensive maltose and baker's yeast fermentation to bias the equilibrium toward maltose phosphorolysis by removing released glucose. Mass production of βGlc1P was obtained in a 2 L reaction mixture initially containing 500 mM maltose and inorganic phosphate, with a yield of 76 %. βGlc1P was isolated from the reaction mixture by crystallization after electrodialysis to obtain 181 g of βGlc1P as a bis(cyclohexylammonium) salt.