A novel protein-deamidating enzyme, which has potential for industrial applications, was purified from the culture supernatant of Chryseobacterium proteolyticum strain 9670 T isolated from rice field soil in Tsukuba, Japan. The deamidating activities on carboxybenzoxy (Cbz)-Gln-Gly and caseins and protease activity were produced synchronously by the isolate. Both deamidating activities were eluted as identical peaks separated from several proteases by phenyl-Sepharose chromatography of the culture supernatant. The enzyme catalyzed the deamidation of native caseins with no protease and transglutaminase activities. Phenotypic characterization and DNA analyses of the isolate were performed to determine its taxonomy. Physiological and biochemical characteristics, 16S rRNA gene sequence analysis, and DNA-DNA relatedness data indicated that the isolate should be placed as a new species belonging to the genus Chryseobacterium. The isolate showed no growth on MacConkey agar and produced acid from sucrose. The levels of DNA-DNA relatedness between the isolate and other related strains were less than 17%. The name Chryseobacterium proteolyticum is proposed for the new species; strain 9670 is the type strain (‫؍‬FERM P-17664).An enzyme catalyzing deamidation of proteins has a great potential for industrial applications. Deamidation of proteins can improve protein functionalities such as solubility, emulsification, and foaming and gelation properties, which are desired properties in some food proteins. Most plant proteins have poor solubility and functionality under mild acidic conditions, which are the pH ranges of most food systems, resulting in their limited use in foods. Because the contents of glutamine residue in plant proteins are generally high, deamidation of such proteins is one of the most promising ways to expand their uses and to improve their functionalities. Many studies of the chemical (mild acid or alkaline treatment) or physical (dry heat treatment) deamidation of food proteins had reported and demonstrated the effectiveness of deamidation for improvement of protein functionalities (see reference 25 for review). To avoid unfavorable side effects brought about by nonenzymatic treatments-for example, concomitant peptide bond cleavage, off-flavor formation, and amino acid racemization-enzymatic deamidations of proteins have been examined (see reference 10 for review). Protease (16), transglutaminase (23), and peptidoglutaminase (9) were used for this purpose. None of their primary reactions were deamidations, or the enzymic substrates were peptides rather than proteins. Besides the improvement in protein functionalities, protein-deamidating enzymes could be used for many applications, including protein structure analysis.In 1971, Kikuchi et al. (17) found an enzyme, peptidoglutaminase, from Bacillus circulans that deamidates the peptidebound glutamines. This enzyme was not active on high-molecular-weight peptides, i.e., proteins such as caseins, unless the proteins were hydrolyzed to short peptides (17). In p...

Protein-glutaminase (PG) purified from Chryseobacterium proteolyticum was used to investigate its deamidation effects on wheat gluten. Water-insoluble gluten was able to be deamidated to the extent of deamidation degree (DD) 72% in 200 mM sodium phosphate buffer (pH 7) at 40 degrees C for 30 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited an upper shift of gluten bands with only deamidation for 1.5-2.0 h (DD 35-45%) compared to the bands of nondeamidated gluten. Results of Fourier transform infrared analysis revealed alterations in secondary structure of gluten by PG deamidation. The assignment within amide I region showed decreases in both inter- (around 1695 cm(-1)) and intramolecular beta-sheets (around 1680 cm(-1)) by deamidation suggesting the deterioration of the aggregation ability of gluten molecules. Solubility and emulsification properties of gluten at pH 7 were improved by deamidation, while both properties at pH 3 were deteriorated by deamidation. Enzyme-linked immunosorbent assay identified that allergenicity of deamidated gluten as compared to the nondeamidated cohorts was decreased remarkably as the deamidation time was prolonged.

A novel protein-deamidating enzyme was purified to homogeneity from Chryseobacterium proteolyticum and the gene encoding it was cloned. The enzyme is a monomer with a pI of 10.0, a measured M r of < 20 000 and a calculated M r of 19 860. Extensive comparison with Streptoverticillium transglutaminase showed that the protein-deamidating enzyme lacked transglutaminase activity in terms of hydroxamateformation between benzyloxycarbonyl-Gln-Gly and hydroxylamine, or monodansylcadaverine incorporation into casein. The enzyme deamidated the two glutaminyl residues in the oxidized insulin A chain and deamidated both casein and the oxidized insulin B chain with higher catalytic efficiencies (k cat /K m ) than with short peptides. The enzyme was active against several proteins, including insoluble wheat gluten, but did not deamidate asparaginyl residues in peptides, free glutamine or other amides. The enzyme was therefore named protein-glutaminase (EC 3.5.1). The gene encoding the protein was cloned and, when expressed in Escherichia coli, the protein product had proteinglutaminase activity and cross-reacted with antiserum raised against the purified enzyme. The protein-glutaminase was shown to be expressed as a prepro-protein with a putative signal peptide of 21 amino acids and a prosequence of 114 amino acids. The amino-acid sequence had no obvious homology to any published sequence and is therefore a novel protein-glutaminase.

The performance of novel protein-glutaminase (PG) purified from Chryseobacterium proteolyticumon alpha-zein was investigated. Highly insoluble alpha-zein was able to be deamidated to the extent of deamidation degree 62% by using 50 mM potassium phosphate (pH 8) containing 11.7% ethanol, at 40 degrees C for 137 h. Analysis by sodium dodecyl sulfate polyacrylamide-gel electrophoresis showed that deamidated and non-deamidated zeins have different mobilities. Results of circular dichroism spectra revealed the decline in alpha-helix contents of alpha-zein by deamidation. Besides, Fourier transform infrared spectroscopy analysis demonstrated alterations in the secondary structure of alpha-zein by deamidation. The assignment of the amide I region showed a remarkable decrease in antiparallel intermolecular beta-sheets (around 1690 cm(-1)) as an indication of the weakening aggregation ability of the deamidated molecules. Solubility and emulsification properties of alpha-zein, particularly at pH 7, were remarkably improved after the deamidation by PG. Gas chromatography and peroxide value studies pointed out that deamidated alpha-zein in powder form exhibited an inferior antioxidative property as compared with the non-deamidated one.