• BMB Reports
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• v.44 no.4
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• pp.279-284
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• 2011

The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1 : 200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.

• Mass Spectrometry Letters
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• v.8 no.3
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• pp.53-58
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• 2017

Glycated hemoglobin ($HbA_{1c}$) has been commonly used to screen and diagnose for patients with diabetes mellitus. Here the accelerated procedure of microwave-assisted sample treatment from acid hydrolysis to enzyme digestion followed by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was optimized and applied to measure $HbA_{1c}$ in an effort to speed up analysis time. First, two signature peptides of $HbA_{1c}$ and hemoglobin $A_0$ were certified with amino acid analysis by setting optimized acid hydrolysis conditions to $150^{\circ}C$, 1.5 h and $10{\mu}M$ sample concentration in 8 M hydrochloric acid. Consequently, the accurate certified peptides above were used as calibration standards to implement the proteolytic procedure with endoproteinase Glu-C at $37^{\circ}C$, 700 W for 6 h. Compared to the traditional method, the microwave heating not only shortened dramatically sample preparation time, but also afforded comparable recovery yields. The optimized protocol and analytical conditions in this study are suitable for a primary reference method of $HbA_{1c}$ quantification with full SI-traceability and other similar proteins in complex biological samples.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.151-156
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• 1985

To investigate the new Yoghurt starter, Lactobacillus helveticus YM-1, which was selected among 14 Lactobacillus strains, and Streptococcus thermophilus CH-1 were inoculated together in reconstituted non-fat skim milk medium and their growth characteristics and cultural conditions for the mixed culture were examined. The main results of this study were obtained as follows. The typical symbiotic growth was shown between the two strains and pH and temperature for optimal growth were 6.5 and 4$0^{\circ}C$, respectively. Heat treatment of milk was most effective at 10$0^{\circ}C$ for 30 min. The cell-free filtrate of Lactobacillus helveticus YM-1 had stimulatory effect on Streptococcus thermophilus CH-1 but the reverse case was slightly observed. Significant difference was observed in the proteolytic activities between Lactobacillus helveticus YM-1 and Streptococcus thermophilus CH-1. The former liberated 135$\mu\textrm{g}$ free amino acid per $m\ell$ of cultured milk, the latter 35$\mu\textrm{g}$ per $m\ell$.

• The Korean Journal of Food And Nutrition
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• v.23 no.3
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• pp.399-404
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• 2010

This study was conducted to determine the influence of culture conditions such as temperature, time, water content, koji-thickness, and agitation on the production of protease by Rhizopus sp. ZB9, isolated from Korean Nuruk, during the preparation of rice koji, which is used in brewing the Korean rice wines, Takju and Yakju. Rice koji was made under different culture conditions, and the proteolytic activity of each koji was tested. The temperature range suitable for the production of protease was $28~32^{\circ}C$. Based on the protease and color, 60 hours of cultivation at $28^{\circ}C$ was shown to produce optimum results. The production of protease increased in proportion to the increase in water content of steamed rice from 25% to 35%. An increase in koji-thickness induced no adverse effects on the production of protease, and agitation during cultivation showed beneficial effects.

• BMB Reports
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• v.35 no.2
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• pp.143-154
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• 2002

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the $\alpha+\beta$ class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0-2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly $\beta$-sheet conformation and shows a strong binding to 8-anilino-1-napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1133-1140
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• 2006

Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.

• International Journal of Industrial Entomology and Biomaterials
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• v.33 no.2
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• pp.78-84
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• 2016

The antioxidant activity of silkworm powder treated by proteolytic enzyme was investigated. Total protein content of silkworm power was assayed using BCA, Bradford assays and SDS-polyacrylamide gel electrophoresis (PAGE) with alkaline protease treatment conditions including temperature and pH. The optimum condition of alkaline protease treatment for silkworm powder was found to be $60^{\circ}C$ and pH 7. The alkaline protease treatment resulted in increased contents of free amino acids, total polyphenol and total flavonoid compared to control group. The silkworm hydrolysates showed excellent antioxidant activities in various in vitro models such as 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2 - azino-bis(3-ethylbenzthiazoline-6)-sulfonic acid (ABTS) radical scavenging activity. These results provide useful information for using silkworm powder as an ingredient in functional foods and for exploiting alkaline protease treatment to improve the extractability and bioactivity of a raw material.

• BMB Reports
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• v.30 no.2
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• pp.125-131
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• 1997

This work reports studies of the catalytic and structural properties of pyridoxal kinase (ATP: pyridoxal 5' -phosphotransferase, EC. 2.7.1.35), Pyridoxal kinase catalyzes the phosphorylation of vitamin $B_6$ (pyridoxal, pyridoxamine, pyridoxine) using ATP-Zn as a phosphoryl donor. The enzyme purified from brain tissues is made up of two identical subunits of 40 kDa each. Native enzyme was inhibited by a substrate analogue, pyridoxal-oxime. Limited chymotrypsin digestion of pyridoxal kinase yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were isolated by DEAE ion exchange chromatography and used for binding studies with fluorescent ATP and pyridoxal analogues. The spectroscopic properties of both fluorescent pyridoxal analogue and Anthraniloyl ATP (Ant-ATP) bound to the 24 kDa fragment are indistinguishable from those of both pyridoxal analogue and Ant-ATP bound to the native pyridoxal kinase, respectively. The small 16 kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the substrate analogues. Binding characteristics of Ant-ATP were extensively studied by measuring the changes in fluorescence spectra at various conditions. From the results presented herein, it is postulated that the structural domain associated with catalytic activity comprises approximately one-half of the molecular mass of pyridoxal kinase (24 kDa). whereas the remaining portion (16 kDa) of the enzyme contains a regulatory binding domain.

• Microbiology and Biotechnology Letters
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• v.2 no.2
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• pp.89-93
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• 1974

The various conditions of substituting wheat gluten for the bean, one of the raw materials for soy sauce manufacture, was studied by measuring the activities of the amylase and proteolytic enzyme of koji. It was found that substitute wheat gluten for up to 60％ of bean content (30％ of the total bran and wheat content) yielded good quality of soy sauce. By using more than 30％ of wheat gluten the availability of nitrogen of raw materials was decreased. This was attributed to the low enzyme activity in koji containing more than 30％ wheat gluten.

• Journal of Life Science
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• v.11 no.1
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• pp.76-82
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• 2001

Fifteen species of microorganisms were isolate form the intestines of fishes, fish feed, and ferment. Eleven microorganisms except HY4, HY8, HY12, and HY13 were Gram-positive, and HY1, HY2, HY3, HY5, HY6, and HY7 produced lactic acid. The species of HY1, HY2, HY3, HY4, HY5, HY6, HY13, and HY14 showed some growth in the medium containing 1% of NaCl. Except HY6, HY7, HY8, HY12 and HY5, 10 isolates had proteolytic activity, whereas only HY13 and HY14 had lipase activity. From all the results four isolates (HY3, HY4, HY13 and HY14) were chosen for the degradation of fish wastes. There was no mutual inhibition among the microorganisms, and the optimum temperature and pH for the growth of the mixed culture were found to be 3 2$\^{C}$ and 7, respectively. Under the optimum growth conditions the maximum optical density and the maximum specific growth rate were estimated to be 2.35 and {TEX}$0.46h^{-1}${/TEX}, respectively. Major microorganisms in the mixed culture at the log-phase were HY3 and HY4, which occupied 70%. The degrading efficiency of fish waste by the mixed microorganisms was 2.3 times higher, compared to control. The total amount of free amino acids in the degraded products from fish wastes was 39g/100g protein and little odor was produced by the mixed microorganisms after 48 hours.