Noriko Miwa

Kinetic modeling and sensitivity analysis of xylose metabolism in Lactococcus lactis IO-1

We proposed a kinetic simulation model of xylose metabolism in Lactococcus lactis IO-1 that describes the dynamic behavior of metabolites using the simulator WinBEST-KIT. This model was developed by comparing the experimental time-course data of metabolites in batch cultures grown in media with initial xylose concentrations of 20.3-57.8 g/l with corresponding calculated data. By introducing the terms of substrate activation, substrate inhibition, and product inhibition, the revised model showed a squared correlation coefficient (r2) of 0.929 between the experimental time-course of metabolites and the calculated data. Thus, the revised model is assumed to be one of the best candidates for kinetic simulation describing the dynamic behavior of metabolites. Sensitivity analysis revealed that pyruvate flux distribution is important for higher lactate production. To confirm the validity of our kinetic model, the results of the sensitivity analysis were compared with enzyme activities observed during increasing lactate production by adding natural rubber serum powder to the xylose medium. The experimental results on pyruvate flux distribution were consistent with the prediction by sensitivity analysis.

Effect of Enzymatic Deamidation on the Heat-induced Conformational Changes in Whey Protein Isolate and Its Relation to Gel Properties

The effect of protein-glutaminase (PG) on the heat-induced conformational changes in whey protein isolate (WPI) and its relation to gel properties was investigated. The structural properties of WPI treated with PG were examined by several analytical methods. The analysis of the fluorescence spectrum and the binding capacity of a fluorescent probe demonstrated that deamidation prevented the increase in the fluorescence intensity caused by subsequent heat treatment. Measurements of the molecular weight distribution of WPI showed that PG-treated WPI was not likely to polymerize even after heating. This is thought to be due to an increase in electrostatic repulsion between carboxylic acid groups and a decrease in the formation of disulfide bonds, which results in the decrease in heat-induced aggregation. The properties of heat-induced WPI gels were modified by deamidation. PG-treated WPI gels had a soft texture and a high water-holding capacity in the presence of salts.

The NMR structure of protein-glutaminase from Chryseobacterium proteolyticum

Protein deamidation, the hydrolysis of side chain amido groups of protein-bound glutaminyl or asparaginyl residues to release ammonia, has received focused attention especially in food industries since protein deamidation is regarded as a promising method to improve functional properties of food proteins. Deamidation generally decreases an isoelectric point of proteins due to increase in number of negatively charged carboxyl groups and enhances protein solubility. In addition, deamidation leads to alteration of the tertiary structures of proteins with an improved amphiphilic character that is useful as an emulsifier or a foaming agent. Therefore, deamidation of food proteins have been investigated by various methods including mild acid treatment, anion-catalyzed deamidation, dry heating under mild alkaline conditions, and thermal treatment. Although deamidation by these treatments improved protein functionalities, there were undesired side-effects, such as concomitant peptide bond cleavages, that were unavoidably brought about by the chemical/ physical treatments. Therefore, enzymatic methods have advantages due to their selectivity and mild treatments. The possibilities of the use of transglutaminases, peptidoglutaminases, and proteases have been explored for this purpose. These enzymes, however, are not suitable because the primary catalytic reactions of transglutaminases and proteases are not deamidation itself, and primary substrates of peptidoglutaminases are not proteins. Protein-glutaminase (PG) is an enzyme produced from the microorganism Chryseobacterium proteolyticum strain 9670 (Yamaguchi et al. 2001). PG catalyzes only the deamidation of the side chain amido group of protein-bound glutaminyl residues to release ammonia without catalyzing the transglutamination and hydrolysis of asparaginyl residues or producing other undesirable changes in protein structures. PG is a monomeric single polypeptide with pI = 10.0 and a molecular weight of 19,860 and is synthesized as a prepro-form, containing a 21-amino-acid signal polypeptide, a 114-amino acid pro-region, and a sequence for the mature enzyme. PG with the pro-region (pro-PG) has no enzymatic activities, but when pro-PG is removed by an extracellular protease, an active enzyme is produced in C. proteolyticum. However, the amount of PG produced by C. proteolyticum is too small to be used for industrial application that limits the application of PG to deamidation of food proteins. Recently, we have constructed the high expression system of PG with Corynebacterium glutamicum, which enables us to prepare an amount of stable-isotope labeled PG for NMR experiments (Kikuchi et al. 2009). Here, we report the solution structure of mature PG determined by NMR and discuss the catalytic mechanism of PG on the structural basis.

Incorporation of 15N-Labeled Ammonia into Glutamine Amide Groups by Protein-Glutaminase and Analysis of the Reactivity for α-Lactalbumin

Protein-glutaminase (PG) is an enzyme that catalyzes the deamidation of protein-bound glutamine residues. We found that an enzyme labeling technique (ELT), which is a stable isotope labeling method based on transglutaminase (TGase) reaction, is applicable for PG. PG catalyzed incorporation of (15)N-labeled ammonium ions into reactive glutamine amide groups in α-lactalbumin similarly to TGase and deamidated the most reactive glutamine amide group once labeled with (15)N. Furthermore, we investigated the effect of ammonium ions on the PG activity by peptide mapping, and more reactive glutamine residues were detected than were detected by the ELT in the presence of ammonium ions. This is probably because ammonium ions are competitive inhibitors, causing decreased reactivity for glutamine residues. We propose the reaction scheme of PG in the presence of the (15)N-labeled ammonium ions and show that the ELT method with PG is useful for evaluating the activity of PG.

Screening of microorganisms producing a novel protein-asparaginase and characterization of the enzyme derived from Luteimicrobium album

A screening system using enrichment culture has been established with the aim of obtaining a novel enzyme for protein modification that has not been previously reported. This enzyme catalyzes deamidation of the side-chain amide group of asparagine in proteins. Enrichment culture of 390 soil samples was carried out with Z-Asn-Gly as the sole source of nitrogen, and the reaction product, Z-Asp-Gly, was detected in the culture supernatant of 102 strains. Strains with particularly high activity were Leifsonia sp., Luteimicrobium sp., Microbacterium sp., and Agromyces sp., all belonging to the class Actinobacteria. Of these, a protein-asparaginase (PA) was obtained from the culture supernatant of Luteimicrobium album 333B-h1, and its reactivity with different substrates and its basic enzymatic characteristics were investigated. Addition of the enzyme solution resulted in specific deamidation of only the asparagine residue in insulin chain B. The enzyme showed no reactivity with free asparagine or asparagine in low molecular weight peptides. It was demonstrated that the enzyme reacts with various protein substrates. In particular, proteins that have open structures, such as casein or gelatin, were good substrates. The activity and stability of PA at different temperatures and pH values were investigated. It was found that a temperature of 37°C and a roughly neutral pH are optimal conditions for the enzyme.