• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.277-288
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• 2017

Rhizomucor miehei NRRL 5282 and Rhizopus oryzae NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified R. miehei and Rh. oryzae enzymes, respectively. p-Nitrophenyl palmitate (pNPP) hydrolysis was maximal at $40^{\circ}C$ and pH 7.0 for the R. miehei lipase, and at $30^{\circ}C$ and pH 5.2 for the Rh. oryzae enzyme. The enzymes showed almost equal affinity to pNPP, but the $V_{max}$ of the Rh. oryzae lipase was about 1.13 times higher than that determined for R. miehei using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM $Hg^{2+}$, $Zn^{2+}$, or $Mn^{2+}$, 10 mM N-bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of n-hexane, cyclohexane, n-heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the pNPP hydrolyzing activity of R. miehei lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed p-nitrophenyl (pNP) esters with C8-C16 acids, exhibiting maximum activity towards pNP-caprylate (R. miehei) and pNP-dodecanoate (Rh. oryzae). The purified lipases are promising candidates for various biotechnological applications.

• 한국응용과학기술학회지
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• 제18권4호
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• pp.306-315
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• 2001

Our study is aimed at proposal of systematic verification method of molecular structure using measuring method of selective ionic determination and spectrometry on 34 kinds of surfactants such as sodium dodecyl sulfate(SDS) which are most widely used today. In the IR spectrum, unsaturated fatty acids reveal themselves by HC= at $3000{\sim}3020cm^{-1}$, and intensity of $720cm^{-1}$ depends on carbon length of alkyl group. Also ethylene oxide(EO) adducts exhibit weak characteristic bands by $-CH_{2}-CH_{2}-O$ at 1350, 1100 and $950cm^{-1}$. Isethionate can be distinguished from diester succinate by intensity ratio of 1740 and $1200cm^{-1}$ spectrums, the ratio of latter is close to 1 due to 2 carboxylate radical in diester succinate. Quaternary ammonium salts exhibit characteristic band of $C_{4}N^{+}$ at $1000-900㎝^{-1}$. In the case of dialkyl dimethyl ammonium salts in quaternary ammonium surfactants, the spectrum of $3000cm^{-1}$ by $N-CH_{3}$ collapses to a very weak band at $3020cm^{-1}$. In ammonium heterocyclic derivatives, pyridinium salts show characteristic bands at 1640 and $1460cm^{-1}$, while imidazolinium salts exhibit characteristic band at $1620-1610cm^{-1}$. In the characteristic spectrum at $1080-1050cm^{-1}$ on OH radicals of the alkyl esters, primary alcohol appears as weak band and the 2 bands show in almost same intensity when primary and secondary alcohols exist together in one molecule. Also, alkyl ester of polyhydric alcohols appears as various broad band.

• 한국식품영양과학회지
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• 제18권3호
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• pp.287-299
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• 1989

폭렬종 옥수수를 가열가공 처리하여 가열 처리과정중에 일어나는 단백질 및 지질의 열안정성을 검토한 결과 옥수수 가열 가공 처리에 따른 영양학적 평가의 기초자료를 얻을 수 있었다. 폭렬종 옥수수의 중성지지질은 93.5%에서 95.5%로 가열 처리 후 당지질 및 인지질의 상대적 함량이 감소하는 반면, 중성지질은 증가하였다. 전지질과 중성지질의 구성지방산은 가열처리 후 불포화 지방산이 상당히 증가하였고, 당지질과 인지질의 구성지방산은 가열처리 후 당지질의 경우 margaric acid가, 인지질의 경우 linoleic acid의 함량이 각각 증가하였다. 가열처리후 albumin분획은 lysine이 다소 증가하고, aspartic acid, arginine등은 급격히 감소하였으며, globuline분획도 이와 비슷하였다. zein분획은 glutamic acid, proline, leucine, alanine등을 적게 함유하였으며, 가열처리에 의해 함량변화가 거의 없었다. 한편, glutelin분획은 zein분획과 비교하여 lysine, histidine등을 많이 함유하는 반면, glutamic acid는 적었고, 가열처리에 의한 변화도 거의 없었다.

• 한국식품영양과학회지
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• 제38권6호
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• pp.801-806
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• 2009

본 연구는 transglutaminase 생산능이 우수한 토양유래방선균 strain YK-2를 TGase 최적생산배지에서 $28^{\circ}C$, 5일간 배양하여 얻은 배양여액으로 본 효소의 정제 및 정제된 효소의 효소화학적 특성에 관하여 검토한 것이다. 본 효소의 정제는 50% methanol precipitation, DEAE-Sephadex column chromatography의 정제 절차를 거쳐 56.5%의 수율로 정제되었고, 정제된 효소의 순도는 12.5% SDS-PAGE에서 단일 밴드를 나타내어, 서브유닛트의 분자량이 약 45,000 dalton으로 추정되는 호모형 효소인 것을 알 수 있었다. 정제된 TGase의 생화학적 제 특성을 검토한 결과, 등전점은 pH $6.0{\sim}7.0$ 부근에 있는 것으로 나타났으며, 본 효소의 기질인 CBZ-L-Gln-Gly 농도에 대한 Km치는 18.5 mM으로 추산되었다. 또한 금속이온 및 저해제의 영향으로는 $Hg^{++}$에 의해서 본 효소의 활성이 강하게 저해되었으나, DTT 및 mercaptoethanol에 의해 각각 293% 및 219% 활성이 증가하였다.

• 농업과학연구
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• 제45권4호
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• pp.635-643
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• 2018

Bovine milk is widely consumed by humans and is a primary ingredient of dairy foods. Proteomic approaches have the potential to elucidate complex milk proteins and have been used to study milk of various species. Here, we performed a proteomic analysis using 2-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF MS) to identify whey proteins in bovine milk obtained soon after parturition (bovine early milk). The major casein proteins were removed, and the whey proteins were analyzed with 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The whey proteins (2 mg) were separated by pI and molecular weight across pH ranges of 3.0 - 10.0 and 4.0 - 7.0. The 2-DE gels held about 300 to 700 detectable protein spots. We randomly picked 12 and nine spots that were consistently expressed in the pH 3.0 - 10.0 and pH 4.0 - 7.0 ranges, respectively. Following MALDI-TOF MS analysis, the 21 randomly selected proteins included proteins known to be present in bovine milk, such as albumin, lactoferrin, serum albumin precursor, T cell receptor, polymeric immunoglobulin receptor, pancreatic trypsin inhibitor, aldehyde oxidase and microglobulin. These proteins have major functions in immune responses, metabolism and protein binding. In summary, we herein identified both known and novel whey proteins present in bovine early milk, and our sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed their expression pattern.

• Fisheries and Aquatic Sciences
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• 제24권12호
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• pp.406-414
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• 2021

The objective of this study was to investigate the yield and characteristics of collagen protein extracted from the muscle of four different species of mantis shrimp: Miyakella nepa, Harpiosquilla harpax, Erugosquilla woodmasoni, and Odontodactylus cultrifer. Mantis shrimp muscle was extracted by using a pepsin-solubilization technique, with 0.5 M acetic acid and 5% pepsin enzyme. The highest collagen yield was from M. nepa muscle (0.478 ± 0.06%), which was significantly greater (p < 0.05) than that from H. harpax, O. cultrifer, and E. woodmasoni (0.313 ± 0.03%, 0.123 ± 0.02%, and 0.015 ± 0.00%, respectively). The freeze-dried collagen appeared as thin fibers, and formed an opaque film. The pepsin-soluble collagen (PSC) from four mantis shrimp species was analyzed by gel electrophoresis. The results showed that all species of mantis shrimp contained type I collagen, consisting of β, α1, and α2 subunits with average molecular weights of 250, 145, and 118 kDa, respectively. The study of the solubility of collagen showed that, for NaCl, collagen had the highest relative solubility in 2% NaCl (80.20 ± 4.95%). In contrast, the solubility decreased at higher NaCl concentrations. However, in terms of pH, collagen had the highest relative solubility at pH 3 (91.32 ± 5.14%), and its solubility decreased at higher pH. FT-IR spectroscopy was used to compare the collagen with a model compound. Five wavenumbers in the spectrum for model collagen were identified: Amide A (3,406-3,421 cm^-1), amide B (2,916-2,940 cm^-1), amide I (1,639-1,640 cm^-1), amide II (1,539-1,570 cm^-1), and amide III (1,234-1,250 cm^-1).

• International Journal of Industrial Entomology and Biomaterials
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• 제45권2호
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• pp.93-98
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• 2022

To assess the feasibility of silk sericin for non-textile application, the storage stability and biological safety of sericin were examined. It was extracted at 37℃, 70℃, 100℃, and 121℃ for 1, 3, and 5 h to elucidate the effect of extraction condition on the stability and safety of silk sericin. The solubility was increased till approximately 26% with extraction temperature of 121℃ for 1 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight distribution depended on the extraction conditions. Extracted sericin displayed typical UV absorption bands upon spectrometric analysis. To examine the reproducibility of its obtained conformation, sericin was extracted thrice and its circular dichroism (CD) spectra was measured each time. Most CD spectra showed reproducibility regardless of temperature and time except under 100℃ extraction condition. The diversity of CD spectrum showed gradual reduction and was finally coincident with extraction time from 1 to 5 h. Notably, sericin has a negative peak of approximately 200 nm attributed to random coil conformation, regardless of extraction condition. However, at the 100℃ extraction condition, sericin showed both bands to be negative bands of approximately 200 and 220 nm, respectively. Sericin was centrifuged to determine the stability of storage conditions. The sericin extracted at 100℃ and 121℃ for 1 h was found to form gel rapidly within 1 h, but at 121℃ condition, the gel fraction was approximately 20% within 1 h which retained its phase regardless of storage time. The gel fraction of sericin extracted at 100℃ for 5 h increased with time, however at the 121℃ for 5 h condition, the gel fraction was measured to be less than 10% regardless of increase in storage time. Petriflim^TM AC plates test showed that sericin was safe from aerobic bacteria activity by extraction under high temperature.

• 한국축산식품학회지
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• 제28권2호
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• pp.154-159
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• 2008

근육단백질과 카제인염 단백질간의 상호작용의 촉매제로서 TGase의 배양시간과 온도에 따른 물성효과를 측정하기 위하여 본 연구를 실시하였다. 돈육 등심부위의 근원섬유단백질을 추출하였고 배양온도는 $4^{\circ}C$, $37^{\circ}C$로, 배양시간은 0, 0.5, 2, 4시간으로 단백질의 열량분석, 점도, 겔 강도, 전기영동상 패턴의 변화를 측정하였다. 단백질열량변화는 각 단백질 별로 열량변화 패턴이 상이하게 나타났으며 근원섬유와 카제인염의 혼합액은 각각의 단백질 피크와 유사하게 나타났고 배양시간과 온도에 따라 차이를 보여 $4^{\circ}C$에 비하여 $37^{\circ}C$에서 열량변화의 차이가 크게 나타났다. 점도의 경우 배양하지 않은 것과 비교했을 때 $37^{\circ}C$에서 2시간 배양했을 때부터 유의적인 차이를 보이며 증가하였다. 근원섬유단백질을 4.5%의 농도로 가열에 의한 겔의 강도를 측정한 결과, 배양시간이나 온도에 따른 뚜렷한 차이를 보이지 않았다. 전기영동의 경우에도 $4^{\circ}C$ 와 $37^{\circ}C$의 배양의 경우 myosin heavy chain과 카제인 염 단백질 분획이 배양시간이 경과함에 따라 점차 감소하였고, 특히 $37^{\circ}C$에서 30분까지는 큰 변화를 나타내지 않았으나 2시간부터 32-34 kDa 분자량을 갖는 카제인 염단백질의 저분자의 밴드가 사라지고 고분자의 biopolymer를 형성하였다. 이상의 결과를 종합하면 $4^{\circ}C$보다 $37^{\circ}C$에서 단백질 분자간의 상호작용에 의한 TGase의 효과가 뚜렷하였으며 $37^{\circ}C$에서 2시간 이상 배양시 TGase에 의한 현저한 물성의 차이를 보인 것으로 평가된다.

• 한국식품영양과학회지
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• 제33권10호
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• pp.1668-1675
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• 2004

근원섬유단백질과 알칼리 용액으로 처리하여 회수한 단백질의 가열 중 ANS 소수성의 변화, IR스펙트럼의 변화, SH기의 변화, 전기영동상의 변화 및 엔탈피의 변화로 비교하여 회수 단백질의 가열 변성기구를 조사하였다. 갈고등어와 냉동 꼬마민어의 근원섬유단백질은 가열 온도의 상승과 더불어 소수성 잔기가 외부로 노출되고 소수성 상호작용은 6$0^{\circ}C$ 부근에서 최대로 일어나지만, 근형질 단백질을 포함하는 회수단백질은 근원섬유 단백질과 ANS 소수성에서 차이를 보였다. pH가 증가함에 따라 1636 $cm^{-1}$ /에 해당하는 peak가 증가하였다. 반응성 SH기와 총 SH기 수의 차이는 pH 7.0과 pH 10에서 비교적 큰 것으로 나타났다. SDS-PAGE 상에서 pH가 상승함에 따라 myosin heavy chain의 중합체가 확인되었다. 알칼리 공정으로 제조한 회수 단백질을 시차주사열량계로 분석한 결과, 회수 단백질은 33.1,44.3 및 65.5$^{\circ}C$에서 전이 온도가 나타나며, myosin의 변성 에 해당하는 온도인 51.7$^{\circ}C$의 peak는 보이지 않았다. 회수 단백질의 가열 젤은 극단적 인 pH 처리에 의한 $\alpha$-helix 구조의 $\beta$-sheet 구조 전환, 가열에 의한 S-S 결합의 형성과, myosin heavy chain 중합체 형성에 의한 것으로 보인다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권4호
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• pp.534-541
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• 2009

The aim of this study was to evaluate the effects of gamma irradiation (${\gamma}$-irradiation) at doses of 15, 30 and 45 kGy on chemical composition, anti-nutritional factors, ruminal dry matter (DM) and crude protein (CP) degradibility, in vitro CP digestibility and to monitor the fate of true proteins of full-fat soybean (SB) in the rumen. Nylon bags of untreated or ${\gamma}$-irradiated SB were suspended in the rumens of three ruminally-fistulated bulls for up to 48 h and resulting data were fitted to a nonlinear degradation model to calculate degradation parameters of DM and CP. Proteins of untreated and treated SB bag residues were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Digestibility of rumen undegraded CP was estimated using the three-step in vitro procedure. The chemical composition of raw and irradiated soybeans was similar. Results showed that phytic acid in ${\gamma}$-irradiated SB at dose of 30 kGy was eliminated completely. The trypsin inhibitor activity of 15, 30 and 45 kGy ${\gamma}$-irradiated SB was decreased (p<0.01) by 18.4, 55.5 and 63.5%, respectively. From in sacco results, ${\gamma}$-irradiation decreased (p<0.05) the washout fractions of DM and CP at doses of 30 and 45 kGy, but increased (p<0.05) the potentially degradable fractions. Gamma irradiation at doses of 15, 30 and 45 kGy decreased (p<0.05) effective degradability of CP at a rumen outflow rate of 0.05 $h^{-1}$ by 4.4, 14.4 and 26.5%, respectively. On the contrary, digestibility of ruminally undegraded CP of irradiated SB at doses of 30 and 45 kGy was improved (p<0.05) by 12 and 28%, respectively. Electrophoretic analysis of untreated soybean proteins incubated in the rumen revealed that ${\beta}$-conglycinin subunits had disappeared at 2 h of incubation time, whereas the subunits of glycinin were more resistant to degradation until 16 h of incubation. From the SDS-PAGE patterns, acidic subunits of 15, 30 and 45 kGy ${\gamma}$-irradiated SB disappeared after 8, 8 and 16 h of incubation, respectively, while the basic subunits of glycinin were not degraded completely until 24, 48 and 48 h of incubation, respectively. It was concluded that ${\gamma}$-irradiated soybean proteins at doses higher than 15 kGy could be effectively protected from ruminal degradation.