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Byline: Hirofumi Motoi (1), Shinichi Fukudome (2), Itaru Urabe (3) Keywords: Gluten peptides; Immobilized enzyme; Bioreactor; Foaming properties; Deamidation Abstract: 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 degC substrate concentration, 40 mg/ml and space velocity, 2.0 h.sup.-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. Author Affiliation: (1) Research Center for Basic Science, Research and Development, Quality Assurance Division, Nisshin Seifun Group Inc., 5-3-1 Tsurugaoka, Oi-Machi, Iruma-Gun, 356-8511 , Saitama, Japan (2) Food Research Department, Nisshin Foods Inc., 19-12, Koami-cho, Nihonbashi, Chuo-ku, 103-8544 , Tokyo, Japan (3) Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871, Osaka, Japan Article History: Registration Date: 09/07/2004 Received Date: 23/03/2004 Online Date: 29/07/2004

A self-encoding system designed to have strict 'compartition' of the molecules, i.e., to contain only a single molecule of DNA in each compartment, was established, and its evolutionary fate was analyzed. The system comprised the Thermus thermophilus DNA polymerase gene as the informational molecule and its protein product replicating the gene as the functional molecule. Imposing strict compartition allows the self-encoding system to last up to at least the tenth generation, whereas the system ceased to work after the third generation when loose compartition initiated with 100 molecules was imposed. These results provide experimental evidence on the importance of compartition for the maintenance of a self-encoding system. In addition, the extent of diversity in self-replication activity of the compartments was found to be another vital difference in the evolutionary dynamics between the strict and loose compartitions. Although the system with strict compartition provides widely diversified activity of the compartments at each generation, the values of the activity diverge only within a small range in the system with loose compartition. When the variety in the activity of a compartment is small, functional selection becomes weak, and to conform Darwinian evolution may become unfeasible. Therefore, strict compartition is essential for the evolvability of a self-encoding system.

The amino acid sequence of the Flavobacterium sp. K1723T1 plasmid pOAD2, which forms nylon degrading enzymes, is given. The plasmid consists of 45519 bp and contains 876 termination codons in the clockwise direction and 778 termination codons in the anticlockwise direction. The G+C content of the plasmid is high. The structure of the plasmid is similar to substances such as oligopeptide permeases and isopenicillin N-acyltransferase. The position of the genes forming the degradation enzymes on the genome map is given.

The nucleotide sequence I reveals that the five constituent regions possess identical 880 bp sequences that resemble the IS6100 insertion sequence of the IS6 family. The nylon oligomer-degrading plasmid pNAD2, seen in Pseudomonas sp. strain NK87, also exhibits sequences homologous to IS6100 of the Mycobacterium fortuitum.

The 6-aminohexanoate-dimer hydrolase (P-EII) from Pseudomonas sp. was characterizedand its base sequence described. The DNA sequence contained 396 amino acids, was initiated by ATG and terminated by TAG. The F-nylB and P-nylB genes had 35%similarity to amino acid sequences in the whole coding region. The F-EII and P-EII enzymes showed similar DFP inhibition profiles. No significant sequence similarities were observed for F-E1 and F-EII enzymes. Therefore, the nylA and nylB families could have evolved independently.

A cDNA isolated encoding the 27-kilodalton diphtheria toxin receptor-associated protein (DRAP27) in monkey Vero cells was isolated and sequenced. The deduced peptide primary structure showed that DRAP27 is the homologue of human CD9 antigen. Transfection experiments using a human-mouse hybrid cell line and a mouse cell line showed that DRAP27 does not bind diphteria toxin (DT), but it enhances the binding of DT by increasing the number of DT binding sites if the cell expresses the DT receptor. DRAP27 thus enhances the DT sensitivity of cells that express the DT receptor.

Bacillus megaterium cells have more than one type of glucose dehydrogenase (GlcDH) genes which are regulated differently. To understand the expression mechanisms and physiological role of each isozyme, the genes coding for GlcDH were isolated. Two new genes were identified and were designated as gdhIII and gdhIV, bring to four the total number of gdh genes in B. megaterium IAM1030. Characterization of the four different isozymes showed that they differ in electrophoretic mobility, immunoreactivity, salt stability and coenzyme preference.

The nucleotide sequences of the genes for nylon oligomer degradation were examined. Flavobacterium sp. KI72 and Pseudomonas sp. NK87 have the same two enzymes, EI and EII, encoded by the genes, nylA and nylB. It was shown that these nylB genes have a long stretch of sequence without stop codons, defined as a nonstop frame (NSF), on their antisense strands. These NSFs seem to be a basic mechanism for the occurrence of new enzymes.

When Pseudomonas aeruginosa PAO is grown in a culture containing 6-aminohexanoate (Ahx) which is a by-product of nylon-6, it acquires the ability to obtain carbon and nitrogen from the Ahx cyclic dimer (Acd). This ability of the bacteria to use Ahx is due to a genetic change and two new enzymes are produced which can hydrolyse the cyclic dimer of Ahx. This metabolic adaptation of Pseudomonas aeruginosa PAO is useful in the biodegradation of nylon-6.