Hidekatsu Maeda

Constructing an efficient trans‐acting genomic HDV ribozyme

We have engineered a genomic HDV ribozyme to construct several trans‐acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88‐Trans had cleavage activity on the 13‐nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88‐Trans ribozyme (kclv = 0.022 min−1), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88‐Trans, HDV88‐Trans‐4 yielded kclv = 1.7 min−1. HDV88‐Trans‐4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37°C with 1 mM of MgCl2).

Properties of NAD (P) H-linked aldose reductase from Crytococcus lactativorus

Abstract A yeast growing at 48°C was isolated from soil and the strain was identified as Cryptococcus lactativorus . The aldose reductase which the strain produced was purified 114-fold with an overall recovery of 36%. The stability of the enzyme was higher than that of other aldose reductases. The half life of the enzyme was 800 h and 14 h at 30°C and 50°C, respectively. The enzyme showed the best activity with d -xylose. l -Sorbose and d -fructose were also reduced by the enzyme. The enzyme was active with both NADPH and NADH as a conenzyme, and the activity with NADH was 1.25 times higher than that with NADPH. The K m app value for d -xylose was 8.6 mM and the V max app was 20.8 units/mg NADH was used as a coenzyme. The K m app values for NADPH and NADH were 6μM and 170 μM, respectively, when d -glucose was used as a substrate.

Construction of a bioreactor for production of (R)-(−)-mandelate, a typical specialty chemical

This review describes our research on a new type of bioreactor system for the production of (R)-(-) mandelate, a useful chiral synthon for the chain modifier of semisynthetic penicillins and cephalosporines, and chemical intermediate of ephedrine.

The separation of solids from the liquefied mash of cassava tuber and continuous saccharification by immobilized glucoamylase

SummaryThe solid material in liquefied mash of cassava tuber was very efficiently separated by a mixture of Trichoderma cellulase and Aspergillus niger pectinase. The solid content of the residue after the treatment and centrifugation decreased from 29.5% to 7.0%. The transparent digested solution from cassava tuber after centrifugation was continuously saccharified by glucoamylase immobilized in a gel which was prepared using polyvinylpyrrolidone and γ-ray irradiation. The addition of 50 ppm of sulfite ion completely prevented microbial contamination during the 18 days of operation. The final DE (dextrose equivalent), glucose content and disaccharide content in the hydrolyzate were 98, 94.4 and 3.3%, respectively.

The Construction of a Bioreactor for the Production of an Optically Active Compound

A droplet gel-entrapping method used for enzyme immobilization was improved to simplify the procedure and to increase the enzyme stability. This immobilization technique is suitable for coupled enzyme reactions requiring cofactors. Leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) were freeze-dried with bovine serum albumin, dextrin and stabilizers. The freeze-dried enzyme powder was suspended in a methylcellosolve solution containing poly- ethyleneglycol (# 4000) diacrylate, N,N’-methylenebisacrylamide and 2-hydroxyethylacrylate, and the suspension was gelled with initiators. The gel was cut up and the pieces were washed in a buffer to remove the methylcellosolve and the dextrin inside. The maximum conversion ratio for a LeuDH-FDH gel column was determined to be 99.8% by means of the recycling reaction. On long-term operation at 30 °C for leucine production, the initial conversion ratio (7.2%) gradually decreased to 6.6% over the first 10 days. However, the conversion ratio remained almost constant after the 10th day. The effects of flow rate, temperature, pH, and the concentrations of formate, α-ketoisocaproate, ammonium and NAD on the leucine productivity with the gel column were also investigated.