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We have developed a novel, single-step, isothermal, signal-amplified, and sequence-specific RNA quantification method (L-assay). The L-assay consists of nicking endonuclease, a dual-labeled fluorescent DNA probe (DL-probe), and conformation-interchangeable oligo-DNA (L-DNA). This signal-amplified assay can quantify target RNA concentration in a sequence-specific manner with a coefficient of variation (Cv) of 5% and a lower limit of detection of 0.1 nM. Moreover, this assay allows quantification of target RNA even in the presence of a several thousandfold excess by weight of cellular RNA. In addition, this assay can be used to measure the changes in RNA concentration in real-time and to quantify short RNAs (<30 nucleotides). The L-assay requires only incubation under isothermal conditions, is inexpensive, and is expected to be useful for basic research requiring high-accuracy, easy-to-use RNA quantification, and real-time quantification.

Gluten peptides are prepared using limited hydrolysis of wheat gluten, resulting in improved solubility and physico-chemical properties. In general, the hydrolysis is performed using a batch-wise process. In this study, we developed a bioreactor system that can continuously produce gluten peptides with foaming properties. Two kinds of acid protease, pepsin and rapidase, were immobilized on porous chitosan beads. The partial deamidation of gluten in advance increased initial velocity of hydrolysis by immobilizing protease and also worked to enhance foaming properties. A packed-bed reactor filled with immobilized protease was designed, and operating parameters were optimized. The optimized conditions were as follows: pH, 3.0; temperature, 40 °C; substrate concentration, 40 mg/ml; and space velocity, 2.0 h −1. Based on these results, a bench-scale reactor was manufactured to determine the stability of continuous operation. The half-life of the reactor was approximately 45 days, and both productivity and quality were stable and excellent.

A directed evolution with phage-displayed random polypeptides of about 140 amino acid residues was followed until the sixth generation under a selection based on affinity to a transition state analog for an esterase reaction. The experimental design deliberately limits the observation to only 10 clones per generation. The first generation consists of three soluble random polypeptides and seven arbitrarily chosen clones from a previously constructed library. The clone showing the highest affinity in a generation was selected and subjected to random mutagenesis to generate variants for the next generation. Even within only 10 arbitrarily chosen polypeptides in each of the generations, there are enough variants in accord to capacity of binding affinity. In addition, the binding capacity of the selected polypeptides showed a gradual continuous increase over the generation. Furthermore, the purified selected random polypeptides exhibited a gradual but significant increase in esterase activity. The ease of the functional development within a small sequence variety implies that enzyme evolution is prompted even within a small population of random polypeptides.

The liposome consisting of eggPC, cholesterol, and DSPE-PEG5000 with a molar ratio of 1.5:1:0.08 was used to entrap cell-free protein synthesis reaction mixture. The synthesis of a mutant green fluorescent protein in the liposome was confirmed by the fluorescence emitted from the liposome on flow cytometry analysis and fluorescence microscopy. The protein synthesized in the liposome is hence functional.

The mathematical model presented here aims to elucidate the essential mechanisms of coexistence of species, especially those of closely related forms, as a result of competition in the same environment. It describes a system where the fate of the competitors or mutants is observed at the initial stage of evolution. The model encompasses both the external variables and the internal state of the competitors, which differ only in one of the metabolic rate constants. Results of simulations, even with the simplified form of the model, show that stable coexistence of closely related forms in a uniform environment is possible. In addition, the model allows the analysis of the limitations on the level of differences and similarities among the competitors for achieving a state of coexistence. The essential mechanisms for the coexistence of closely related competitors are proposed to be the involvement of the metabolic network in allowing the same growth rate of competitors which have different internal states, and the interplay between the internal states of the competitors and the external variables of their environment.

A simple system was constructed and used in the experimental elucidation of the fate of a mutant emerging in a population. Three Escherichia colistrains having the same genetic background except for their glutamine synthetase gene were used as model competitors. The difference in the enzyme gene were introduced by random mutation. Competition between these bacterial strains was carried out and observed in a continuous liquid culture. In most cases, the competitors stably coexist either in a steady state or in an oscillating state. In addition, the competition between the strains was found to be a deterministic process and not a stochastic one. These results showed that an emerging mutant in a population, be it a closely related one to the original members, can attain a state of stable coexistence even in a homogeneous environment. The ability of each of the emerging mutants to maintain its stable coexistence with the original population gives rise to the accumulation of various mutants in a population. Therefore, evolution starts from gradual accumulation of various mutants in the population, which in turn leads to the diversification of the population. As our experimental system is a minimum model for the various competitions in the natural ecosystem, the observed competitive coexistence is proposed to be a general phenomenon in nature.

A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino acids was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coliare expected to be soluble. The soluble random proteins RP3–42 and RP3–45 and insoluble RP3–70 were purified. The solubility of the purified form is the same as that in the cell lysate.

A library of artificial proteins of 141 amino acid residues, of which 95 are random and which include 20 kinds of amino acids, was prepared. As the properties of the artificial random proteins are free from the evolutionary constraint, they can be used as a standard to discriminate the specialized properties of natural proteins. Out of the 25 identified random proteins, 5 are soluble in the cell lysate, indicating that about 20 of the random proteins expressed in Escherichia coliare expected to be soluble. Therefore, as natural soluble or insoluble proteins can arise from the line of soluble or insoluble ancestry, respectively, solubility does not seem a specialized property of natural proteins. The soluble random proteins RP3-42 and RP3-45 were purified and their properties were investigated.

Closely related competitors comprising of Escherichia colistrains having the same metabolic system and differing only with a few bases on the glutamine synthetase gene in the plasmid pKGN were previously shown to coexist in a chemostat. The differences among these closely related competitors can be considered large enough to allow coexistence as the level of enzyme activity is different. To bring the difference among competitors to the slightest possible, the mutation was introduced on the noncoding region of the plasmid pKGN harbored in the wild-type strain (strain W). The new strain, strain W’, carries the plasmid pKGN’ with a 4-base insertion at the HindIII site in the polycloning site of pKGN. As the noncoding region is a nucleotide segment that is not translated into amino acids, the relatedness between strains W and W’ is the closest possible from the genetic point of view. Interestingly, though both strains are almost identical, they can coexist stably in a chemostat irrespective of the initial population size. These experimental results suggest that in the natural ecosystem, no matter how akin competitors are, coexistence is not impossible.

Four stability-increasing mutants of glucose dehydrogenase from Bacillus megateriunwere purified together with the wild-type enzyme; each has a single amino acid change of Glu-96 to Gly, Glu-96 to Ala, Gln-252 to Leu, or Tyr-253 to Cys. These mutant enzymes are more heat resistant at pH 6.5 than the wild-type enzyme; the replacement of Glu-96 by Ala increases the thermostability by about 20°C. The mutant enzymes are also resistant to inactivation in alkaline solutions. The replacement of Glu-96 by Gly or Ala protects the enzyme from alkaline inactivation almost completely. The kinetic constants for the activities of these mutant enzymes do not differ significantly from those of the wild-type enzyme.

Four heat-resistant mutants of xylanase (N56, N102, N104 and F1) were obtained by random mutagenesis. The mutant genes had the following amino acid changes: N56, Ser-26 to Trp, Gly-38 to Asp and Thr-126 to Ser; N102, Gly-38 to Asp; N104, Gly-38 to Ser and Arg-48 to Lys; F1, Ser-12 to Cys. Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.

The structural and catalytic properties of two soluble random proteins, RP3‐42 and RP3‐45, of 141 amino acid residues were investigated. Although no marked secondary structure was detected by CD spectrum, sedimentation equilibrium and small‐angle X‐ray scattering studies showed that they form an oligomeric structure and are as compact as the molten globule. The random proteins have low but distinct esterase activity; the values of the second‐order rate constant for the hydrolysis of p‐nitrophenol were 0.78 and 1.39 M−1s−1for RP3‐42 and RP3‐45, respectively. The differences in the properties of the random and the native proteins are discussed from the evolutionary point of view.

The structural and catalytic properties of two soluble random proteins, RP3-42 and RP3-45, of 141 amino acid residues were investigated. Although no marked secondary structure was detected by CD spectrum, sedimentation equilibrium and small-angle X-ray scattering studies showed that they form an oligomeric structure and are as compact as the molten globule. The random proteins have low but distinct esterase activity; the values of the second-order rate constant for the hydrolysis of p-nitrophenol were 0.78 and 1.39 M −1s −1for RP3-42 and RP3-45, respectively. The differences in the properties of the random and the native proteins are discussed from the evolutionary point of view.

Four stability‐increasing mutants of glucose dehydrogenase from Bacillus megateriunwere purified together with the wild‐type enzyme; each has a single amino acid change of Glu‐96 to Gly, Glu‐96 to Ala, Gln‐252 to Leu, or Tyr‐253 to Cys. These mutant enzymes are more heat resistant at pH 6.5 than the wild‐type enzyme; the replacement of Glu‐96 by Ala increases the thermostability by about 20°C. The mutant enzymes are also resistant to inactivation in alkaline solutions. The replacement of Glu‐96 by Gly or Ala protects the enzyme from alkaline inactivation almost completely. The kinetic constants for the activities of these mutant enzymes do not differ significantly from those of the wild‐type enzyme.

Four heat‐resistant mutants of xylanase (N56, N102, N104 and F1) were obtained by random mutagenesis. The mutant genes had the following amino acid changes: N56, Ser‐26 to Trp, Gly‐38 to Asp and Thr‐126 to Ser; N102, Gly‐38 to Asp; N104, Gly‐38 to Ser and Arg‐48 to Lys; F1, Ser‐12 to Cys. Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.

Catalase I from Bacillus stearothermophilushas the interesting property of increasing its enzyme activity on heating. It was confirmed that after heating at 70 °C for 10 min or 65 °C for 20 min, almost all the enzyme molecules were converted irreversibly to the activated form. The increase in kcatfrom 1400 to 3930 s−1and the decrease in Kmfor H2O2from 4.4 to 2.7 mmby heat activation indicate changes in the kinetic property of the enzyme molecule. Therefore, it follows that catalase I has two active forms, a high-activity form and a low-activity form. The heat activation process followed the first-order kinetics with an activation enthalpy (ΔH*) of 191 kJ/mol while the heat denaturation process had a ΔH* of 545 kJ/mol. The CD spectra of the two enzyme forms had small but marked differences. The conversion of the low-activity form to the high-activity form was an endothermic process with aTmof 56 °C, which is much lower than that of the heat denaturation (Tm= 76 °C), and the enthalpy change for the transition was only 5% of that for the denaturation. It has to be noted that the high-activity form of the enzyme was converted back to a low-activity form through the process of denaturation, refolding, and reconstitution with heme. In addition, the newly obtained low-activity form was brought to a high-activity form by heating. These results suggest that the native state of catalase I has two active conformations that are roughly the same but not identical and are separated by a high energy barrier.

The bacterial DNA sequence in GenBank database were divided into coding and noncoding regions and examined for the base-trimer distribution in every triplet frame on the sense and antisense strands. The results revealed that for the noncoding region, both strands have very similar base-trimer distributions and have no frame specificity; that is, DNA is symmetric in the noncoding region. For the coding region, on the other hand, the symmetry is broken only in the triplet framework, and we found a special triplet-frame-specific symmetry which appears when the two complementary strands of the coding region are read from their 5' ends. In addition, the following frame specificity was also observed in the distribution of stop codons on the antisense strand of the coding region. When the antisense sequences of the open reading frames (ORFs) in the database are read in the three reading frames, the same reading frame as the corresponding ORF contains a significantly larger amount of long open frames without stop codons (i.e., nonstop frames [NSFs]) than expected, while the number of NSFs in the other two reading frames is similar to that of the expected one. That is, NSFs as well as ORFs are maintained in a frame-specific manner, and in this sense, DNA becomes symmetrical even in the coding region. These two kinds of frame-specific symmetries indicate that only an ORF and its complementary triplets are specifically recognized and maintained in DNA. We suppose that the antisense strands as well as the sense strands in the coding region may be transcribed, thereby producing various kinds of proteins corresponding to NSFs, though their amount may not be large. The presence of these proteins should have some benefits for living organisms, and therefore we propose that these proteins are upcoming enzymes having novel functions.

The steady-state velocity equation is derived for the general reaction scheme containingnkinds of enzyme species connected by a network of reversible reaction steps. The general equation is represented by the net rate constants as well as the true rate constants of the individual reaction steps. Using a general equation, equations for simpler schemes can easily be derived. Furthermore, the velocity equation expressed by net rate constants is useful for understanding the dynamic state of any reaction be it simple or complex. The generality of the presented equation is tested in a complex reaction involvingBacillus stearothermophiluscatalase I. In addition, the applicability of the general equation in analysing the reaction specificity and dynamic state of the reaction is shown.

Escherichia coliJM109 (pGDA2) overexpressing the glucose dehydrogenase (GDH) gene from Bacillus megateriumIWG3 was examined for use as a cofactor regenerator. In the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by E. coliJM109 (pKAR) which is an aldehyde reductase-overproducing transformant, E. coliJM109 (pGDA2) can act as an NADPH regenerator with NADP+and glucose, similarly to commercially available GDH.

The cloned peroxidase gene from Bacillus stearothermophilus was highly expressed in Escherichia coli. Using the high copy number plasmid which is temperature-sensitive and its own strong promoter, this thermostable peroxidase was produced at 28% of the total cell proteins when the cells were grown at 42°C. The enzyme could be easily purified from E. coli by heat treatment and single-column Sephadex G-200 chromatography. From a 200 ml culture, 30 mg of purified enzyme was obtained. The peroxidase produced by E. coli showed a thermostability, haem type and content identical with those of the peroxidase produced by B. stearothermophilus.

Catalase I (wild type) from Bacillus stearothermophilus, which was found to have catalase activity, catalyzed dioxygen-inserted indole-ring opening reaction of methyl N-acetyl L-tryptophanate as tryptophan 2,3-dioxygenase (TDO) model in the presence of Na2S2O4. Heme-reconstruction with protoporphyrin IX manganese(III) chloride (MnClPP) viaannealing was examined and the reconstituted MnClPP-catalase I also revealed dioxygenolytic behaviour with higher TDO activity and higher selectivity than the catalase I (wild type).

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