• 한국식품과학회지
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• 제27권5호
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• pp.794-798
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• 1995

식품단백질의 효율적인 이용을 위하여 neutrase에 의한 glutamine과 asparagine 잔기의 탈아미드화에 관한 연구를 행하였다. Neutrase에 의한 BSA의 최적반응 조건은 $20^{\circ}C$, pH 10에서 3시간 반응시켰을 때로, 이때 BSA는 24%가 탈아미드화되었고. 동시에 2.9%가 가수분해되었다. 또한 pronase, bromelain, ficin 보다는 neutrase에 의한 BSA의 탈아미드화율이 높은 것으로 나타났다. 최적 탈아미드화 조건에서 neutrase에 의한 egg albumin, 분리콩단백 및 casein의 경우 탈아미드화에 수반하여 펩티드결합의 분해가 비교적 높게 관찰되었다.

• 한국식품영양과학회지
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• 제24권5호
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• pp.811-815
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• 1995

BSA, egg albumin (EA), 그리고 분리대두단백질(SPI)의 Neutrase에 의한 탈아미드화가 용해도에 미치는 영향을 조사하여 보았다. BSA는 탈아미드화로 pH 4~8 사이의 물에 대한 용해도가 천연 BSA에 비하여 98~83%로 감소하였으며, pH 6 부근에서 가장 낮은 용해도를 보았다. 천연 BSA와 탈아미드화된 BSA 모두 0.2M NaCl 용액에서는 100%의 용해도를 보였으나, 산성 1.0M NaCl용액에서는 용해도가 모두 크게 떨어졌다. EA의 용해도는 탈아미드화로 pH3 이하와 6 이상의 수용액에서는 증가하였으나, 산성 NaCl용액에서는 크게 감소하였다. SPI는 탈아미드화로 수용액에서의 용해도가 모든 pH 범위에서 크게 증가하였으나, NaCl용액에 대한 용해도는 pH 6 이상에서는 증가하였고, pH 5 이하에서는 변화가 없었다.

• Mass Spectrometry Letters
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• 제14권2호
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• pp.48-55
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• 2023

This study compares the efficiency of weak acid hydrolysis (WAH) using formic acid (FA) and hydrochloric acid (HCl) in the analysis of myoglobin peptides. WAH using 2% and 5% formic acid resulted in the identification of 32 peptides, with varying degrees of cleavage at the C-terminus of aspartic acid residues. HCl WAH with different concentrations demonstrated an increase in the total number of identified peptides but a decrease in fully cleaved peptides as the HCl concentration increased. Notably, deamidation was observed during HCl WAH but not in FA WAH. The addition of HCl WAH after FA WAH provided a similar pattern to HCl WAH, with slightly higher levels of hydrolysis. These findings highlight distinct cleavage patterns and deamidation effects between FA and HCl in the context of WAH.

• Molecules and Cells
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• 제37권11호
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• pp.819-826
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• 2014

Protein modifications of recombinant pharmaceuticals have been observed both in vitro and in vivo. These modifications may result in lower efficacy, as well as bioavailability changes and antigenicity among the protein pharmaceuticals. Therefore, the contents of modification should be monitored for the quality and efficacy of protein pharmaceuticals. The interface of EPO and its receptor was visualized, and potential amino acids interacting on the interface were also listed. Two different types of modifications on the interface were identified in the preparation of rHu-EPO BRP. A UPLC/Q-TOF MS method was used to evaluate the modification at those variants. The modification of the oxidized variant was localized on the Met54 and the deamidated variants were localized on the Asn47 and Asn147. The extent of oxidation at Met54 was 3.0% and those of deamidation at Asn47 and Asn147 were 2.9% and 4.8%, respectively.

• Bulletin of the Korean Chemical Society
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• 제10권6호
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• pp.600-604
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• 1989

In order to find the rational methods for improving the thermal stability of subtilisin Carlsberg, the mechanisms of irreversible thermoinactivation of the enzyme were studied at $90^{\circ}C.$ At pH 4, the main process was hydrolysis of peptide bond. This process followed first order kinetics, yielding a rate constant of $1.26\;{\times}\;10^{-1}h^{-1}$. Hydrolysis of peptide bond of PMS-subtilisin occurred at various sites, which produced new distinct fragments of molecular weights of 27.2 KD, 25.9 KD, 25.0 KD, 22.3 KD, 19.0 KD, 17.6 KD, 16.5 KD, 15.7 KD, 15.0 KD, 13.7 KD, and 12.7 KD. Most of the new fragments originated from the acidic hydrolysis at the C-side of aspartic acid residues. However 25.0 KD, 15.7 KD, and 13.7 KD which could not be removed in purification steps stemmed from the autolytic cleavage of subtilisin. The minor process at pH 4 was deamidation at asparagine and/or glutamine residues and some extend of aggregation was also observed. However, the aggregation was main process at pH 7 with a first order kinetic constant of $16 h^{-1}.$ At pH 9, the main process seemed to be combination of deamidation and cleavage of peptide bond.

• 한국축산식품학회지
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• 제34권2호
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• pp.207-213
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• 2014

This study investigated the effects of microbial transglutaminase (MTGase) and pH-shift processing on the functional properties of porcine myofibrillar proteins (MP). The pH-shift processing was carried out by decreasing the pH of MP suspension to 3.0, followed by re-adjustment to pH 6.2. The native (CM) and pH-shifted MP (PM) was reacted with and without MTGase, and the gelling and emulsion characteristics were compared. To compare the pH-shifted MTGase-treated MP (PT), deamidation (DM) was conducted by reacting MTGase with MP at pH 3.0. Rigid thermal gel was produced by MTGase-treated native MP (CT) and PT. PM and DM showed the lowest storage modulus (G') at the end of thermal scanning. Both MTGase and pH-shifting produced harder MP gel, and the highest gel strength was obtained in PT. All treatments yielded lower than CM, and CT showed significantly higher yield than PM and DM treatments. For emulsion characteristics, pH-shifting improved the emulsifying ability of MP-stabilized emulsion, while the treatments had lower emulsion stability. PM-stabilized emulsion exhibited the lowest creaming stability among all treatments. The emulsion stability could be improved by the usage of MTGase. The results indicated that pH-shifting combined with MTGase had a potential application to modify or improve functional properties of MP in manufacturing of meat products.

• Mass Spectrometry Letters
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• 제5권4호
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• pp.95-103
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• 2014

Characterization and studies of proteome are challenging because biological samples are complex, with a wide dynamic range of abundance. At present the proteins are identified by digestion into peptides, with subsequent identification of the peptides by mass spectrometry (MS). MS is a powerful technique for the purpose, but it cannot identify every peptide in such complex mixtures simultaneously. For accurate analysis and quantification it is important to separate the peptides first by chromatography into fractions of a size that MS can handle. With these less complex fractions, the probability is increased of identifying peptides of low abundance that would otherwise experience ion suppression effects due to the presence of peptides of high abundance. Enrichment for peptides with certain post-translational modifications helps to increase their detection rates as well. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is a mixed-mode chromatographic technique which combines the use of electrostatic repulsion and hydrophilic interaction. This review provides an overview of ERLIC and its various proteomics applications. ERLIC has been demonstrated to have good orthogonality to reverse phase liquid chromatography (RPLC), making it useful as a first dimension in multidimensional liquid chromatography (MDLC) and fractionation of digests in general. Peptides elute in order of their isoelectric points and polarity. ERLIC has also been successfully utilized for the enrichment for phosphopeptides and glycopeptides, facilitating their identification. In addition, it is promising for the study of peptide deamidation. ERLIC performs comparably well or better than established methods for these various applications, and serves as a viable and efficient workflow alternative.

• KSBB Journal
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• 제8권4호
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• pp.405-408
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• 1993

Absidia zychae NRIC 1199 produced two forms of carboxypeptidase(CPZ-1 and CPZ-2) which were distinguished in their isoelectric points but had almost identical properties(1). The amino acid sequences for the N-terminal of both enzymes were the same (Tyr-Thr-Ser-Pro-Lys-Leu-Xaa-Asp-Pro-Asp-Val) and any significant difference was not observed between amino acid compositions of the two enzymes. The ouchterlony double diffusion technique using antibody raised against the CPZ-2 protein demonstrated a good cross-reaction between CPZ-1 and CPZ-2 Genomic Southern analysis showed only one gene encoding CPZ in the genome of Absidia zychae. However, a significant difference between two enzymes was observed on peptide map using Staphylococcus aureus V8 protease, distinguishable only one band, indicating that multiple forms of CPZ are caused by post-translational modification, such as deamidation.

• Journal of Microbiology and Biotechnology
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• 제21권6호
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• pp.617-626
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• 2011

Transglutaminase from Streptomyces mobaraensis is an enzyme of unknown function that cross-links proteins to high molecular weight aggregates. Previously, we characterized two intrinsic transglutaminase substrates with inactivating activities against subtilisin and dispase. This report now describes a novel substrate that inhibits papain, bromelain, and trypsin. Papain was the most sensitive protease; thus, the protein was designated Streptomyces papain inhibitor (SPI). To avoid transglutaminase-mediated glutamine deamidation during culture, SPI was produced by Streptomyces mobaraensis at various growth temperatures. The best results were achieved by culturing for 30-50 h at $42^{\circ}C$, which yielded high SPI concentrations and negligibly small amounts of mature transglutaminase. Transglutaminasespecific biotinylation displayed largely unmodified glutamine and lysine residues. In contrast, purified SPI from the $28^{\circ}C$ culture lost the potential to be cross-linked, but exhibited higher inhibitory activity as indicated by a significantly lower $K_i$ (60 nM vs. 140 nM). Despite similarities in molecular mass (12 kDa) and high thermostability, SPI exhibits clear differences in comparison with all members of the wellknown family of Streptomyces subtilisin inhibitors. The neutral protein (pI of 7.3) shares sequence homology with a putative protein from Streptomyces lavendulae, whose conformation is most likely stabilized by two disulfide bridges. However, cysteine residues are not localized in the typical regions of subtilisin inhibitors. SPI and the formerly characterized dispase-inactivating substrate are unique proteins of distinct Streptomycetes such as Streptomyces mobaraensis. Along with the subtilisin inhibitory protein, they could play a crucial role in the defense of vulnerable protein layers that are solidified by transglutaminase.