• Title, Summary, Keyword: alliinase

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Identification of an Essential Tryptophan Residue Residue in Alliinase from Garlic (Allium sativum) by Chemical Modification

  • Jin, Yeong Nam;Choe, Yong Hun;Yang, Cheol Hak
    • Bulletin of the Korean Chemical Society
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    • v.22 no.1
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    • pp.68-76
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    • 2001
  • We have employed chemical modification to identify amino acids essential for the catalytic activity of alliinase (EC 4.4.1.4) from garlic (Allium sativum). Alliinase degrades S-alkyl-L cysteine sulfoxides, causing the characteristic odor of garlic. The activity of alliinase was rapidly and completely inactivated by N-bromosuccinimide(NBS) and slightly decreased by succinic anhydride and N-acetylimidazole. These results indicate that tryptophanyl, lysyl, and tyrosyl residues play an important role in enzyme catalysis. The reaction of alliinase with NBA yielded a characteristic decrease in both the absorbance at 280 nm and the intrinsic fluorescence at 332 nm with increasing reagent concentration of NBS, consistent with the oxidation of tryptophan residues. Kinetic analysis, fluorometric titration of tryptophans and correlation to residual alliinase activity showed that modification of only one residue present on alliinase led to complete inhibition of alliinase activity. To identify this essential tryptophan residue, we employed chemical modification by NBS in the presence and absence of the protecting substrate analogue, S-ethyl-L-cysteine (SEC) and N-terminal sequence analysis of peptide fragment isolated by reverse phase-HPLC. A fragment containing residues 179-188 was isolated. We conclude that Trp182 is essential for alliinase activity.

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High Pressure Inactivation of Alliinase and Its Effects on Flavor of Garlic (고압처리에 의한 Alliinase의 불활성화가 마늘의 풍미에 미치는 영향)

  • Sohn, Kyung-Hyun;Lim, Jae-Kag;Kong, Un-Young;Park, Ji-Yong
    • Korean Journal of Food Science and Technology
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    • v.28 no.3
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    • pp.593-599
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    • 1996
  • The effects of high pressure on alliinase and on flavor of garlic (Alliiium sativum L.) were investigated. After pressurized at 150 MPa, 300 MPa, and 500 MPa for 10 min, the activities of purified alliinase were reduced approximately 30%, 80%, and 100%, respectively, while the enzyme activities of pressurized garlic cloves were reduced 0%, 7%, and 100%, respectively. This indicated that the intact garlic has a protective effect against pressure-inactivation of alliinase. Alliinase was more effectively inactivated when high pressure treatment was carried out at high ($>40^{\circ}C$) or low temperature ($>10^{\circ}C$) than ambient temperature. Pressure treated garlic at 500 MPa had little pungency and sulfuryl odor compared to raw garlic indicating that high-pressure processing can be used to produce garlic without pungent flavor.

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Studies on the Changes in the Alliinase Activity during the Aging of Pickled Garlic (마늘장아찌 숙성 중 Alliinase 활성 변화에 관한연구)

  • 채수규
    • The Korean Journal of Food And Nutrition
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    • v.12 no.1
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    • pp.55-62
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    • 1999
  • Changes in the alliinase activity during the aging of pickled garlic samples prepared by the several methods were investigated. The activity of alliinase in raw garlic was 8,37 units/mg protein. The ac-tivity in the garlic pickled with swoy sauce was reduced to 4.57 units/mg with 52% remaining by 1st week of pickling and to 1.05 units/mg protein with 12% remaining by 2nd week of pickling. The ac-tivity of alliinase in the garlic pickled with vinegar was 2.79 units/mg protein with 32% remaining by 1st week of pickling and was 0.26 units/mg protein only with 3% remaining by 2nd week of pickling. The activity of alliinase in the garlic pickled with 10% salt solution was 5.06 units/mg protein with 58% remaining by 1st week of pickling. After one week pickling the juice of pickled garlic was removed. Then garlics were pickled again with vinegar. The allinase acting in was reduced to 0.85 units/mg pro-tein with 10% remaining by 2nd week of pickling. The activity of alliinase in the garlic pickled with vin-egar was 2.79 units/mg protein with 32% remaining by 1st week of pickling. The juice of pickled garlic was removed after one week. Then the galics were again pickled with saysauce. The allinase activity in the garlic the garlic pickled again with soy sauce was reduced to 0.43 units/mg protein with 5% remain-ing by 2 week of pickling.

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Characteristics of Thiosulfinates and Volatile Sulfur Compounds from Blanched Garlic Reacted with Alliinase (Alliinase 첨가에 의한 열처리 마늘로부터 생성된 함황 화합물의 특성)

  • Choi, Yoon-Hee;Shim, You-Sin;Kim, Cheong-Tae;Lee, Chan;Shin, Dong-Bin
    • Korean Journal of Food Science and Technology
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    • v.39 no.6
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    • pp.600-607
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    • 2007
  • In this study, attempts were made to develop a method for controlling the volatile sulfur compounds in garlic. Crude alliinase extracted from fresh garlic was applied to garlic pulp blanched far 5 min at $100^{\circ}C$, and the changes in the thiosulfinates and volatile compounds of the blanched garlic pulp reacted with the enzyme were investigated. Eight kinds of thiosulfinates from garlic were separated by HPLC, and identified by LC/MS/MS. When the alliinase was added to the blanched garlic pulp at 100, 200, 300, or 400 units, and reacted for 15 min, respectively, thiosulfinates were generated in the amounts of 37, 68, 77, and 80% of the fresh garlic content (control). Under the same conditions, we analyzed the volatile compounds, where 28 peaks were identified by GC/MSD. Of the 28 peaks, 23 were volatile sulfur compounds. The results of the analysis showed that all the volatile compounds were generated at amounts of 25, 36, 66, and 76% of the content of the control, respectively. These results indicate that the sulfur compound content of garlic can be regulated, depending upon the reaction conditions of allinase.

A Study on the Changes in the Alliinase Activity during the Vacuum Freeze Drying of Onions(Allium cepa L.) (양파의 진공 동결 건조 과정 중 Alliinase 활성 변화에 관한 연구)

  • Chae, Soo-Kyu;Yun, Mi-Suk
    • Culinary science and hospitality research
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    • v.14 no.1
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    • pp.144-151
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    • 2008
  • This study investigated the changes in the contents of allicin and diallyl disulfide and in the alliinase activity during the vacuum freeze drying of onion samples treated as the whole, sliced and crushed forms. The contents of allicin and diallyl disulfide in raw onions were 26.40ppm and 2.78ppm respectively. The contents of allicin and diallyl disulfide of onion samples treated as the whole, sliced and crushed forms increased with the progress of vacuum freeze drying. The degree of increase was different in each onion sample form prepared by vacuum freeze drying(p<0.05). The specific activity of alliinase in raw onions was 7.536 units/mg protein. The activity in onion samples treated as the whole, sliced and crushed forms decreased with the progress of vacuum freeze drying. The activity in the whole onion prepared by the vacuum freeze drying for 8 hrs reduced to 5.516 units/mg protein with 73.2% remaining and to 3.304 units/mg protein with 43.8% remaining for 36 hrs. The activity in the sliced onion prepared by the vacuum freeze drying for 36 hrs reduced to 2.366 units/mg protein with 31.4% remaining and the activity in the crushed onion prepared by the vacuum freeze drying for 36 hrs reduced to 2.232 units/mg protein with 29.6% remaining. The alliinase in onion sample treated as the whole form showed the highest remaining activity during the vacuum freeze drying.

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Studies on the Changes in the Alliinase Activity during the Drying of Garlic (마늘의 건조과정 중 Alliinase 활성 변화에 관한 연구)

  • Chae, Soo-Kyu
    • Journal of environmental and Sanitary engineering
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    • v.22 no.1
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    • pp.57-66
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    • 2007
  • Changes in the alliinase activity during the hot air drying and vacuum freeze drying of garlic samples treated as the whole, sliced and crushed state were investigated. The specific activity of alliinase in raw garlic was 8.52 units/mg protein. The activity in the whole garlic prepared by the hot air drying for 8 hrs was reduced remarkably to 5.22 units/mg protein with 61% remaining and to 4.25 units/mg protein with 50% remaining for 36 hrs. The activity in the sliced garlic prepared by the hot air drying for 36 hrs was reduced to 3.55 units/mg protein with 42% remaining and the activity in the crushed garlic prepared by the hot air drying for 36 hrs was reduced to 3.12 units/mg protein with 37% remaining. The garlic sample sliced or crushed was higher than the whole state in the efficiency of drying but was lower in the remaining activity of alliinase. The activity in the whole garlic prepared by the vacuum freeze drying for 8 hrs was reduced to 7.21 units/mg protein with 85% remaining and to 5.53 units/mg protein with 65% remaining for 36 hrs. The activity in the sliced garlic prepared by the vacuum freeze drying for 36 hrs was reduced to 4.55 units/mg protein with 53% remaining and the activity in the crushed garlic prepared by the vacuum freeze drying for 36 hrs was reduced to 4.16 units/mg protein with 49% remaining. The remaining activity of alliinase in the garlic samples prepared by the vacuum freeze drying was higher than the remaining activity in the garlic samples prepared by the hot air drying.

Purification and Characterization of Alliinase from Garlic of Korean Origin (한국산 마늘 alliinase의 분리 및 특성)

  • Kim, Mee-Ree;Song, Moung-Jhu;Jhee, Ok-Hwa;Ahn, Seung-Yo
    • Korean journal of food and cookery science
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    • v.10 no.4
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    • pp.376-380
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    • 1994
  • 마늘의 alliinase를 ammonium sulfate 분획과 hydroxylapatite chromatography, concanavalin A-Sepharose affinity chromatography에 의해 정제하여, 23% 의 회수율과 7.6배 정제도를 나타내었다(specific activity 116.6 units/mg). SDS-polyacrylamide gel electrophoresis에서 단일 band를 나타내므로 순수한 aliinase 로 추측되며 이 효소의 분자량은 42K 로 추정된다. 기질로서 S-ethyl-L-cysteine sulfoxide를 사용한 이 효소의 $V_max$값은 2.27${\mu}$monl/mg.min이고 $K_m$은 1O mM이다 . 정제효소의 optimum pH는 6.5 phosphate buffer이며, 40$^{\circ}C$에서 최대활성을 나타내었다. Activation energy value($E^*$)는 4.6Kcal/mole로 추정된다.

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The Chemical Basis of Green Pigment Formation ('Greening') in Crushed Garlic (Allium sativum L.) Cloves

  • Lee, Eun-Jin;Cho, Jung-Eun;Lee, Seung-Koo
    • Food Science and Biotechnology
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    • v.15 no.6
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    • pp.838-843
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    • 2006
  • The chemical processes involved in the formation of green pigment in crushed garlic cloves were investigated based on the principle of pink pigmentation in macerated onions. Intact greening and non-greening garlic cloves were either left untreated or heated at $90^{\circ}C$ for 3 min to inactivate enzyme activities. First, a colorless ether soluble compound referred to as color developer reacted with glycine (among all free amino acids) in garlic to form a second compound insoluble in ether. The latter compound then reacted with formaldehyde to yield the green colored pigment. Alliinase activity was necessary for the production of color developer and for the development of green pigment. In greening garlic that had been heat treated, green pigmentation did not proceed due to the heat-inactivation of alliinase, but the addition of alliinase solution into the garlic homogenates restored the pigmentation. However, this phenomenon was not observed in non-greening garlic with or without heat treatment. Finally, the mechanism of green pigment formation in crushed garlicis similar to that of pink pigment formation in macerated onions.

Comparative Investigation of Glutathione S-Transferases, Glyoxalase-I and Alliinase Activities in Different Vegetable Crops

  • Hossain, Md Daud;Rohman, Md Motiar;Fujita, Masayuki
    • Journal of Crop Science and Biotechnology
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    • v.10 no.1
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    • pp.19-26
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    • 2007
  • Glutathione S-transferases(GSTs, EC 2.5.1.18), glyoxalase-I(EC 4.4.1.5) and alliin lyase(alliinase, EC 4.4.1.4) are important enzyme systems in plant bodies. The first two are mainly detoxifying enzymes that utilize glutathione(GSH) in the defense mechanism, and the last one is mainly involved in secondary metabolism and relevant to sulfur compounds derived from GSH. The activities of the three enzymes have been investigated in soluble extracts of vegetable crops, including pumpkin, cabbage, broccoli, radish, carrot, potato, sweet potato, mungbean, and onion. GST activities were detected in all of the vegetables, and the extract of onion bulb exhibited the highest specific activity(648 nmol/min/mgP). The putative GSTs of most of the vegetables were found to be induced by ethanol. The activities of GSTs in onion bulb were found to be markedly inhibited by S-hexyl glutathione and were also inhibited by S-butyl glutathione and S-propyl glutathione. The anti-CmGSTF1 antiserum recognized a thick band for putative onion GST. The estimated glyoxalase-I activity level was also high in onion bulb(4540 nmol/min/mgP), indicating that the thick band detected by Western blot analysis might result from partial recognition of glyoxalase-I by the antiserum. The specific activities for glyoxalase-I were moderate in radish and carrot, and the extracts of other vegetables had rather low levels of activities. The extract of onion also showed the highest specific activity level for alliinase(2069nmol pyruvate/mgP). The extracts of other vegetables also had alliinase activities, although the estimated values were much lower than that of onion.

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마늘의 저장 및 효소처리 조건에 따른 함황화합물의 변화

  • Sin, Dong-Bin
    • Food preservation and processing industry
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    • v.7 no.1
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    • pp.33-44
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    • 2008
  • 마늘의 저장 중 생리활성과 향미에 중요한 영향을 미치는 성분의 변화를 발표된 논문을 중심으로 정리하여 살펴보았다. 또한 마늘의 냄새를 최소화하고 생리활성을 극대화할 수 있는 기능성식품의 소재개발 타당성을 살펴보기 위하여 열처리(blanching)로 마늘 중의 효소를 모두 불활성화 시킨 후, 이에 마늘로부터 추출한 alliinase를 가하여 반응조건에 따른 alk(en)yl thiosulfinates 생성 및 이들의 분해산물인 휘발성 황함유화합물의 함량변화를 측정하였다. 수확한 마늘의 최종 저장물질로 알려진 -glutamyl-S-alk(en)yl-L-cysteines는 마늘중에 존재하는 -glutamyl-transpeptidase 및 oxidase의 작용에 의하여 감소한 반면 S-alk(en)yl cysteine sulfoxide는 감소한 만큼 증가하였으며, 이는 $-3^{\circ}C$ 및 실온($23^{\circ}C$)에서 보다도 냉장온도($4^{\circ}C$)에서 가장 많이 변화하는 것으로 나타났다. 이러한 감소 및 증가현상은 -glutamyl-S-(2-popenyl)-L-cysteine이 -glutamyl-S-(trans-1-propenyl)-L- cysteine이나 -glutamyl-S-methyl-L-cysteine보다 더 컸다. -glutamyl-S-(2-propen yl)-L-cysteine은 $4^{\circ}C$에서 저장 60일 만에 66%가 감소한 반면 이로부터 생성된 S-(2- popenyl)-L-cysteine sulfoxide는 그 만큼 증가하였다. -glutamyl-S-(trans-1-propenyl)-L-cysteine 및 -glutamyl-S-methyl-L-cysteine도 $4^{\circ}C$에서 150일간 저장한 경우 각각 81% 및 39%가 감소하고, 이들로부터 각각 생성된 S-(trans-1- propenyl)-L-cysteine sulfoxides 및 S-methyl-L-cysteine sulfoxide는 증가하였다. 한편 열처리 마늘에 alliinase를 가하여 함황화합물을 재생성 시킨 결과 8종의 S-alk(en)yl cysteine sulfoxides를 확인할 수 있었다. S-(2-propenyl)-L-cysteine sulfoxide은 전체 thiosulfinates함량의 약 60%를 차지하는 것으로 나타났다. 100, 200, 300 및 400 unit의 alliinase를 첨가하여 15분간 반응시킨 결과 총 thiosulfinates는 생마늘(대조구)에 비하여 각각 37, 68, 77 및 80%가 생성되는 것으로 나타났다. GC/MSD를 이용하여 대조구 및 효소를 첨가하여 반응시킨 시료의 휘발성 향기성분을 분석한 결과 alliinase를 100, 200, 300 및 400 unit 첨가하여 15분간 반응시키면 각각 마늘의 휘발성 향기성분이 25, 36, 66및 76% 씩 재 생성되는 것으로 나타났다. 이상의 결과를 종합해 볼 때 마늘을 이용한 제품개발이나 연구를 할 경우 마늘의 저장조건에 따른 생리활성물질의 분석결과를 근거로 하여 이루어져야 하며, 또한 효소를 이용하여 적절히 반응시키면 마늘 냄새를 $30{\sim}80%$ 범위 내에서 조절이 가능한 것으로 나타났다.

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