사람, 집박쥐 및 마우스 파네트세포의 Lysozyme 면역반응성

정진웅,차정호,오수자,전정수 대한해부학회 1991 Anatomy & Cell Biology Vol.24 No.2

호알칼리성, 고온성 Bacillus sp. TA-11에 대한 오골계 난백 Lysozyme의 용균특성

이성훈,조창호,안용근,이종수 한국식품영양학회 1996 韓國食品營養學會誌 Vol.9 No.4

교분석하였다. 오골계 난백 lysozyme의 용균활성은 Bacillus sp. TA-11를 50℃에서 18시간 정치배양한 대수기 후기의 세포에 대하여 가장 높았고, lysozyme의 농도는 0.25%가 최적이었다. 또한 lysozyme의 최적반은 pH와 온도는 각각 4.5와 35℃였다. 일반 난백 lysozyme의 용균활성은 시험균주를 24시간 배양한 정지기의 세포에 대하여 가장 높았고 lysozyme의 최적 농도는 0.5%였으며 반응 최적 pH와 온도는 각각 5.5과 40℃이었다. Lytic activities of the egg white lysozyme from Korea-native Ogol fowl and a fowl against the alkalophilic and thermophilic Bacillus sp. TA-11 were investigated and compared. Lytic actinity of the Ogol fowl lysozyme for Bacillus sp. TA-11 was the highest for the cell of post-logatithm phase and optimum concentration of the lysozyme was 0.25%. Optimum reaction pH and temperature were 4.5 and 35℃, respectively. Lytic activity of egg white lysozyme from fowl for Bacillus sp. TA-11 was the highest for the cell of stationary phase and optimum concentraion of the lysozyme was 0.5%. Optimum reaction pH and temperature were 5.5 and 40℃, respectively. Bacillus sp TA-11에 대한 오골계 난백 lysozyme과 일반 난백 lysozyme의 용균성을 비

Effects of heavy metals and albumin on lysozyme activity

( Eun Ko ),( Seul-i Ku ),( Dae-yoon Kim ),( Sooim Shin ),( Moonsung Choi ) 한국응용생명화학회(구 한국농화학회) 2018 Journal of Applied Biological Chemistry (J. Appl. Vol.61 No.4

Lysozyme is an antibacterial enzyme that is found in most of body fluids. Lysozyme in tears plays a primary role in protecting eye from harmful environments; if lysozyme is degraded or inhibited, eyes are likely to be more vulnerable to bacterial infection. In this study, lysozyme activity was evaluated according to varying concentrations of heavy metals, copper, zinc, cobalt and manganese and light metal, calcium that are frequently found in airborne particulate matters and was assayed using a dyequenching lysozyme substrate, Micrococcus lysodeikticus. Less fluorescence intensity was observed with increasing amounts of copper, zinc, manganese and cobalt but not with calcium suggesting that these metals have some affinity with lysozyme and inhibit lysozyme activity. When albumin, the second most common protein in tears, was added on the reaction of lysozyme and metals, lysozyme activity was partially restored. This finding suggests that the albumin might protect damage caused by metals on lysozyme. To identify whether the decrease in enzymatic activity was related to structural changes of lysozyme, SDS-PAGE was conducted and only with copper did lysozyme show marked smearing bands on the SDS-gel, meaning that copper degraded lysozyme consistent with the sharpest activity decrease.

오골계 난백 lysozyme 이 몇가지 동물성 식품의 보존에 미치는 영향

이종수,최영준,김나미,안용근,오홍록,Lee, Jong-Su,Choe, Yeong-Jun,Kim, Na-Mi,An, Yong-Geun,O, Hong-Rok 배재대학교 자연과학연구소 1995 自然科學論文集 Vol.7 No.-

우유와 쏘세지 등 몇가지 동물성 식품의 보존에 미치는 오골계 난백 lysozyme의 효과를 검토하기 위하여 이들을 0.05% lysozyme 으로 처리한 후 $20^{\circ}C$, $30^{\circ}C$ 및 $37^{\circ}C$에 저장하면서 경시적으로 이들의 pH와 휘발성 염기질소 함량 및 생균수의 변화를 조사 하였다. 생우유를 lysozyme 으로 처리하여 $20^{\circ}C$에서 48시간 저장 하였을 때 휘발성 염기질소 함량이 63mg%로 제일 낮아 보존효과가 있었고 $30^{\circ}C$에서는 lysozyme 과 glycine의 혼용처리시 그 보존효과가 상승하였다. 또한 시판우유와 어묵에서도 lysozyme처리에 의한 보존효과가 있었으며 특히 솔빈산을 첨가하지 않은 쏘세지에 lysozyme 을 처리하여 $5^{\circ}C$에 저장 하였을 때 휘발성 염기질소 함량이 약 150 mg%로 비교적 낮았다. To elucidate the effect of egg white lysozyme from Ogol fowl on the preservation of milks, fishcurd and sausage, changes of pH, volatile base nitrogen content and viable cell count were investigated during the storage periods at $20^{\circ}C$, $30^{\circ}C$ and $37^{\circ}C$ after the addition of lysozyme in each foods. Volatile base nitrogen count of raw milk as marker of spoilage was lowest(63 mg%) in 0.05% lysozyme addition lot which was stored at $20^{\circ}C$ for 2 days, and its preservation effect by lysozyme at $30^{\circ}C$ was enhanced with addition of glycine(0.1%). Preservation effect by lysozyme in commercial milk at $37^{\circ}C$ and in fishcurd at $5^{\circ}C$ and $20^{\circ}C$ were also good, and when sausage was stored at $5^{\circ}C$ after treatment of lysozyme instead of sorbic acid, its preservation effect was acceptable.

한외여과 공정을 이용한 고순도 향균 Lysozyme 의 분리 및 막 침착 특성

이은영,우건조,Lee, Eun-Young,Woo, Gun-Jo 한국식품과학회 1999 한국식품과학회지 Vol.31 No.2

난백 lysozyme은 박테리아 세포벽을 선택적으로 분해하므로 식품 가공 및 저장용 천연 식품보존제로서의 이용 가치가 높다. 농도(0.25, 0.5, 1.0%, w/v)를 달리한 난백 용액을 예비여과하여 얻은 여액(PFS)과 PFS를 $35^{\circ}C$에서 한외여과하여 얻은 여액(PMS)의 단백질 농도와 lysozyme 농도를 측정하고 비활성도와 purification factor를 계산하여 최적 분리 농도를 결정하였다. 난백 용액의 각 단계별 lysozyme의 분리 정제도를 겔 크로마토그래피와 전기영동으로 확인하였으며, PM30 막에 대한 난백 단백질의 막 침착도는 SEM으로 관찰하였다. 예비여과와 한외여과 단계를 거치면서 lysozyme 이외의 비효소성 단백질은 99% 이상이 제거되었다. 비활성도는 0.25% PMS>0.50% PMS>1.0% PMS 순이었으며 PFS에 비해 PMS의 비활성도는 한외여과 단계를 거치면서 최저 18배에서 최고 31배까지 증가하였으므로, 비활성도와 purification factor가 가장 높은 0.25% 난백 용액을 최적 농도로 결정하였다. 난백 용액(0.25%, w/v)의 겔투과 크로마토그래피 결과 비효소 획분은 대부분 ovalbumin으로 판명되었으며, 전기영동 결과 PMS내에는 고순도의 lysozyme이 존재하였다. 분자량이 큰 단백질들이 $0.9{\mu}m$의 두께로 한외여과 막 표면에 침착되었으나, SEM 관찰 결과 이들 침착 단백질의 대부분은 0.1 N NaOH로 세척시 제거되는 것으로 나타났다. 따라서, polysulfone계의 PM30 막을 사용한 한외여과 공정이 고순도의 lysozyme만을 선택적으로 분리하는데 매우 효과적이었다. The value of lysozyme as a natural food preservative is continuously increased due to its unique antimicrobial activity. To determine the optimum separation concentration among the various hen egg white protein (HEWP) concentrations (0.25, 0.5, 1.0, w/v), protein concentrations, lysozyme concentrations, specific activities (SA), and purification factors of prefiltered solution (PFS) and PM30 permeate solution (PMS) were compared. The purity of lysozyme separated at each step was analyzed and confirmed by gel permeation chromatography and electrophoresis. The fouling deposits on membrane were observed by SEM. The non-enzymatic proteins were removed over 99% by ultrafiltration (UF). The increased feed concentration did not contribute to the increase of SA. SA of PMS was 18 to 31 times higher than that of PFS. The optimum feed concentration was decided as 0.25% based on SA and purification factor. The non-enzymatic region of gel chromatogram was proved to be ovalbumin. The thickness of deposit on the UF membrane was approximately $0.9{\mu}m$ and removed by cleaning with 0.1 N NaOH. Therefore, UF using PM30 membrane was very effective to separate the antimicrobial lysozyme from various HEWPs.

Cloning and Prokaryotic Expression of C-type Lysozyme Gene from Agrius convolvuli

김종완,여성문 한국통합생물학회 2008 Animal cells and systems Vol.12 No.3

We have isolated and characterized Agrius convolvuli cDNA encoding a c-type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino-terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21 (DE3) pLysS cells for pGEX 4T-1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T-1 vector, which contained the glutathione Stransferase (GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS-PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was purified by glutathion-Sepharose 4B affinity chromatography. Western blot analysis of this protein revealed an immunoreactivity with the anti-Agrius lysozyme. We have isolated and characterized Agrius convolvuli cDNA encoding a c-type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino-terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21 (DE3) pLysS cells for pGEX 4T-1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T-1 vector, which contained the glutathione Stransferase (GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS-PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was purified by glutathion-Sepharose 4B affinity chromatography. Western blot analysis of this protein revealed an immunoreactivity with the anti-Agrius lysozyme.

Use of Lysozyme as a Feed Additive on In vitro Rumen Fermentation and Methane Emission

Biswas, Ashraf A.,Lee, Sung Sill,Mamuad, Lovelia L.,Kim, Seon-Ho,Choi, Yeon-Jae,Bae, Gui-Seck,Lee, Kichoon,Sung, Ha-Guyn,Lee, Sang-Suk Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.11

This study was conducted to determine the effect of lysozyme addition on in vitro rumen fermentation and to identify the lysozyme inclusion rate for abating methane ($CH_4$) production. An in vitro ruminal fermentation technique was done using a commercial concentrate to rice straw ratio of 8:2 as substrate. The following treatments were applied wherein lysozyme was added into 1 mg dry matter substrate at different levels of inclusion: Without lysozyme, 2,000, 4,000, and 8,000 U lysozyme. Results revealed that, lysozyme addition had a significant effect on pH after 24 h of incubation, with the highest pH (p<0.01) observed in 8,000 U lysozyme, followed by the 4,000 U, 2,000 U, and without lysozyme. The highest amounts of acetic acid, propionic acid (p<0.01) and total volatile fatty acid (TVFA) (p<0.05) were found in 8,000 U after 24 h of incubation. The $CH_4$ concentration was the lowest in the 8,000 U and the highest in the without lysozyme addition after 24 h of incubation. There was no significant differences in general bacteria, methanogen, or protozoan DNA copy number. So far, addition of lysozyme increased the acetate, propionate, TVFA, and decreased $CH_4$ concentration. These results suggest that lysozyme supplementation may improve in vitro rumen fermentation and reduce $CH_4$ emission.

Isolation and Characterization of the C-type Lysozyme Gene from the Common Cutworm Spodoptera litura

김종완,Sung Moon Yoe 한국통합생물학회 2009 Animal cells and systems Vol.13 No.3

We have isolated and characterized a new insect chicken type (c-type) lysozyme gene from the common cutworm, Spodoptera litura. The full-length cDNA of Spodoptera lysozyme is cloned by rapid amplification of cDNA ends PCR (RACE-PCR). The isolated cDNA consists of 1039 bp including the coding region for a 142-amino acid residue polypeptide, which included a signal peptide of 21-amino acid residue and a mature protein of 121-amino acid residue. The predicted molecular weight of mature lysozyme and its theoretical isoelectric point from amino acid composition is 13964.8 Da and 9.05, respectively. The deduced amino acid sequence of Spodoptera lysozyme gene shows the highest similarity (96.7%) to Spodoptera exigua lysozyme among other lepidopteran species. Amino acid sequence comparison with other the c-type lysozymes, Spodoptera lysozyme has the completely conserved Glu32 and Asp50 of the active site and eight Cys residues are completely conserved in the same position as that of other lepidopteran lysozymes. We have isolated and characterized a new insect chicken type (c-type) lysozyme gene from the common cutworm, Spodoptera litura. The full-length cDNA of Spodoptera lysozyme is cloned by rapid amplification of cDNA ends PCR (RACE-PCR). The isolated cDNA consists of 1039 bp including the coding region for a 142-amino acid residue polypeptide, which included a signal peptide of 21-amino acid residue and a mature protein of 121-amino acid residue. The predicted molecular weight of mature lysozyme and its theoretical isoelectric point from amino acid composition is 13964.8 Da and 9.05, respectively. The deduced amino acid sequence of Spodoptera lysozyme gene shows the highest similarity (96.7%) to Spodoptera exigua lysozyme among other lepidopteran species. Amino acid sequence comparison with other the c-type lysozymes, Spodoptera lysozyme has the completely conserved Glu32 and Asp50 of the active site and eight Cys residues are completely conserved in the same position as that of other lepidopteran lysozymes.

Cloning and expression analysis of c-type and g-type lysozymes in yellow catfish (Pelteobagrus fulvidraco)

Fei Ke,Yun Wang,XiaoYao Cao,GaoWei Pan,Rui Geng 한국유전학회 2016 Genes & Genomics Vol.38 No.8

Lysozymes have important roles in innate immune system. Here, a c-type and a g-type lysozyme were identified from yellow catfish (Pelteobagrus fulvidraco). The deduced amino acid sequences of both lysozymes were conserved in catalytic sites and structural features as compared to their counterparts from other species. It was interesting that the g-type lysozyme possessed a signal peptide. The c-type and g-type lysozymes had the highest identity 89.4 and 76.2 % with that from channel catfish respectively. Phylogenetic analysis showed that the two lysozymes had a closely relationship with that from channel catfish and Astyanax mexicanus. Lysozymes from one order could form more than one clade in the phylogenetic tree, which indicated the gene duplications in evolution. Expression analysis with real time quantitative PCR revealed that the two lysozyme genes were constitutively expressed in all the tested tissues. The highest expression of c-type lysozyme was observed in liver, followed by spleen, head kidney, and trunk kidney, while the g-type lysozyme had highest expression in intestine, followed by spleen, head kidney, and trunk kidney. The mRNA levels of both genes were all up-regulated after challenging with Aeromonas hydrophila. However, there were differences in tissues and time points when the mRNA levels reached its peak between the two lysozymes. It indicated the diversity in regulation mechanisms and detailed functions among lysozymes. Taking together, these results will benefit the understanding of yellow catfish lysozymes.

이온교환 칼럼 크로마토그래피를 이용한 난백에서 Lysozyme의 연속추출

박성준,김현석,김현위,안태회,박기문,최춘언,Park, Seong-Joon,Kim, Hyeon-Seok,Kim, Hyean-Wee,Ahn, Tae-Hoe,Park, Ki-Moon,Choi, Chun-Un 한국식품과학회 1990 한국식품과학회지 Vol.22 No.6

식품 및 의약품 원료로 이용되고 있는 Iysozyme을 난백으로부터 이온교환 크로마토그래프를 이용하여 연속추출하였다. 사용된 수지는 지금까지 수율, 순도, 작동용이성에서 가장 효과적이라고 밝혀져 있는 Duolite C-464 양이온교환수지를 선택하여 $Na^+$형으로 $pH\;7.9{\pm}0.1$로 평형화하여 사용하였다. 조제용 자동 Liquid Chromatography(LC) system(column size ; i.d. 50 mm, bed volumn ; 1020 ml)에서 수지 평형화, 난백접촉, rinse, lysozyme용출 순으로 cycle을 연속적으로 반복하면서 시행하고 그 재현성을 관찰하기 위해 각 cycle별 rinse끝점과 용출끝점의 UV level을 비교하였다. 그 결과 rinse끝점은 19 cycle까지 30% 이하를 유지하였다. 용출 끝점은 17 cycle까지는 30% 이하에서 비교적 안정하였으나 18 cycle 이후 부터는 50% 이상으로 용출능력이 현저하게 감소하였다. 또한, 회수율 비교에서 17cycle까지는 90% 이상을 유지하다가 18cycle에서는 72%, 19 cycle에서는 65%로 격감하였다. 본 추출 정제 lysozyme은 전기영동상 단일 band로 나타났고, densitometer로 측정한 순도는 99% 이상이었다. Continuous column chromatographic separation of lysozyme from egg white was investigated. A weak acid type cation exchange resin, Duolite C-464, was used because of high lysozyme recovery and ease of column operation in this experiment. The resin was equilibrated at $pH\;7.9{\pm}0.1$ in Na+form. Continuous lysozyme separation was processed by repeating cycles(one cycle : resin equilibration, flow egg white, rinse, lysozyme elution) in automated preparative Liquid Chromatography(LC) system(column size ; i.d. 50 mm, resin bed volumn ; 1020 ml). At comparison of UV levels in rinse end point and elution end point of every cycle, the UV levels of rinse end point are maintained below 30% for 19 cycles and that of elution end point are also maintained below 30% for 17 cycles, stably, but was increased above 50% after 18 cycle. That indicated the eluting ability of lysozyme was reduced conspicuously after 18 cycle in continuous cycling process. The recovery of lysozyme was maintained above 90% from one to 17 cycle, but was decreased to 72% and 65% in 18 cycle and 19 cycle, respectively.