• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.576-581
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• 1999

Effects of signal sequences, protein sizes and dissolved oxygen on the secretion of human lysozyme from a recombinant yeast were experimentally characterized. The systems consisted of Saccharomyces cerevisiae host SEY2102 that was transformed with two different plasmids. These plasmids were identical with an exception to the plasmid pMC614, which contained the native yeast MFα1 sequence and the plasmid pMC632 with the non-native rat α-amylase signal sequence. The expression of human lysozyme was controlled by the ADHI promoter. The native yeast MFαl signal sequence was more efficient than the non-native rat α-amylase signal sequence in directing the secretion of human lysozyme. Lysozyme secreted with the α-amylase signal was retained inside the cells and released to the medium very slowly, thereby causing a lower cell growth rate and a decreased product secretion rate. Lysozyme was secreted more efficiently than invertase, which is an order of magnitude bigger in molecular size compared to lysozyme, which was under the direction of the MFαl signal sequence, suggesting that protein sizes may affect the secretion efficiency. When expressed in anaerobic conditions in the medium where the ADHI promoter was derepressed, the amount of lysozyme secreted was about twice higher than that of the aerobic culture. However, the secretion rates were identical. This result showed that the dissolved oxygen level may affect the efficiency of protein secretion only, and not the secretion rate of the product protein.

• 한국안광학회지
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• 제2권1호
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• pp.85-90
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• 1997

안구의 각막과 결막은 외계에 노출되어 있어서 손상을 받기 쉽고, 손상부위에 병균이 감염되면 염증이 유발되어 심한 시력장애를 초래하기도 하는데, 누액 속에 포함되어 있는 lysozyme은 이러한 병균에 대해 항균작용을 가지므로 평소에는 안구가 노출되어 있어도 각 결막염이 발생하지 않는다. 본 연구는 lysozyme의 항균효과를 조사하고자 각 결막염의 원인균 중 5가지를 선별하여 배양하였고, 그 배양액에 lysozyme을 농도별로 첨가하면서 생육저해 정도를 측정하였다. 그 결과, 배지 중의 lysozyme 농도가 증가할수록 공시균의 생육은 저해되었다. 1mM 농도의 lysozyme에 의해 Pseudomonas는 43%, Neisseria는 41%, KlebsielIa는 35%, Staphylococus는 22% 생육이 저해되었고, 그람음성균은 그람양성균인 Staphylococus보다 lysozyme에 대한 감수성이 약 1.5~2.0배 큰 것으로 나타났다. 그러나 lysozyme은 진균인 Fusarium의 생육에는 매우 미약한 저해양상을 나타내었다.

• 한국미생물·생명공학회지
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• 제23권2호
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• pp.138-144
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• 1995

The cDNA, encoding human lysozyme (HLY) which was isolated from a human placenta cDNA library, has been well characterized (Yoshimura et al., 1988). Based on the communication, we have prepared an artificial HLY gene from chemically synthesized 38-oligomer with high codon usage in Saccharomyces cerevisiae. For directing the synthesis and secretion of HLY in S. cerevisiae, an expression vector, pHKl was constructed by inserting the HLY gene, containing a synthetic HLY secretion signal sequence, between the yeast GAP promoter and PH05 terminator. From a lysoplate assay, we have confirmed an yeast transformant harboring a pHK1 which makes a clearing zone on the overlayed Micrococcus luteus. This result means a chemically synthesized HLY gene which was normally expressed and secreted in yeast.

• BMB Reports
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• 제31권4호
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• pp.413-417
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• 1998

Transgenic mice containing a bovine ${\beta}-Casein/Human$ lysozyme fusion gene (pBZ) were generated in order to produce human lysozyme in their milk. The expression vector was a quadripartite fusion consisting of a 2 kb upstream DNA of the bovine ${\beta}-casein$ gene, human lysozyme gene, intron II of the rabbit ${\beta}-globin$ gene, and the polyadenylation/termination signals of SV40 DNA. Fertilized mouse zygotes were microinjected with pBZ, then transferred into the oviduct of foster mothers. Out of 20 mice born, 11 survived until postweaning and three were identified as positivetransgenic by Southern blot analysis (one male and two females). The founder mice were mated to BCFl mice to produce transgenic progeny. It was confirmed by RT-PCR and Northern blot analyses that the transgene was specifically expressed in the mammary gland of the founder mice. Furthermore, the artificial introns within the transgenic RNA was proven to be correctly spliced out as judged by RT-PCR analysis. These results indicated that transgenic mice generated in this study properly expressed the human lysozyme RNA in their mammary gland.

• 농업과학연구
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• 제21권2호
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• pp.122-132
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• 1994

본(本) 연구(硏究)는 초유(初乳) 중(中)에 다량(多量) 존재하면서, 국소면역(局所免疫)을 담당하고 있는 것으로 알려진 Secretory immumoglobulin A (SIgA)를 분리(分離)하여 사람에게 급성위장병 및 집단 식중독을 일으키는 장독소형(腸毒素型) Salmonella속(屬) (S. enteritidis, S. typhimurium)의 성장억제효과(成長抑制效果)를 시험하였다. 우유(牛乳) 초유(初乳) 중(中)에서 분리한 SIgA를 각각 $0.1{\sim}5.0mg/m{\ell}$의 수준이 되도록 stock solution을 제조하여 성장억제효과(成長抑制效果)를 비교 검토하였으며, 한편 항균성물질(抗菌性物質로)로 알려진 egg lysozyme을 $0.5mg/m{\ell}$의 수준으로 단독(單獨) 첨가(添加) 및 SIgA와 혼합(混合) 첨가(添加)하면서, 그에 따른 성장억제효과(成長抑制效果)를 검토(檢討)하였다. 1. 우유(牛乳) 초유(初乳) 중(中)에 존재(存在)하는 SIgA는 gel filtration 및 ion exchange chromatography 방법으로 순수하게 분리(分離)할 수 없었으며, 또한 SIgA rich fraction 중(中)에는 dimeric-$IgG_1$이 상당히 혼재(混在)되어 있는 것이 확인되었다. 2. Human SIgA는 $0.5{\sim}1.0mg/m{\ell}$의 농도(濃度)에서 효과적인 성장억제효과(成長抑制效果)를 보였으며, 그 이상의 농도(濃度)에서는 억제효과(抑制效果)의 증가추세가 둔화되었다. Bovine SIgA는 농도가 증가함에 따라 점진적인 성장억제효과(成長抑制效果)를 보였으며, 특히 $2.0mg/m{\ell}$이상의 농도(濃度)에서는 뛰어난 억제효과(抑制效果)를 보여 주었다. Bovine IgG는 Salmonella속 (屬) 2균주(菌株)에 대하여 성장억제효과(成長抑制效果)를 보이지 않았다. Egg lysozyme의 성장억제효과(成長抑制效果)는 bovine SIgA와 비슷한 경향을 나타냈으며 농도(濃度)를 증가시킬수록 억제효과(抑制效果)가 증가(增加)하는 경향을 보여 주었다. 3. Human SIgA와 egg lysozyme을 단독(單獨) 또는 혼합첨가(混合添加)하면서 시간 경과에 따른 성장억제효과(成長抑制效果)를 시험한 결과, 2균주(菌株) 모두에서 egg lysozyme은 대조구(對照區)에 비하여 월등한 성장억제효과(成長抑制效果)를 나타내는 반면 human SIgA 단독(單獨) 및 egg lysozyme과 혼합처리구(混合處理區)에는 경미한 억제효과(抑制效果)를 나타내었다. 4. Bovine SIgA와 egg lysozyme을 단독(單獨) 또는 혼합(混合) 첨가(添加)하면서 시간경과에 따른 성장억제효과(成長抑制效果)를 시험한 결과, S. enteritidis의 경우 세처리구 모두 월등한 성장억제(成長抑制)를 보였으며, S. typhimurium의 경우 egg lysozyme 단독처리구(單獨處理區)만 뛰어난 성장억제효과(成長抑制效果)를 보여 주었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권1호
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• pp.52-55
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• 2002

Flow cytometric techniques were used to investigate cell size, protein content and cell cycle behavior of recombinant Saccharomyces cerevisiae strains producing human lysozyme (HLZ). Two different signal sequences, the native yeast $MF\alpha1$ signal sequence and the rat $\alpha-amylase$ signal sequence, were used for secretion of HLZ. The strain containing the rat $\alpha-amylase$ signal sequence showed a higher level of internal lysozyme and lower specific growth rates. Flow cytometric analysis of the total protein content and cell size showed the strain harboring the native yeast signal sequence had a higher total protein content than the strain containing the rat $\alpha-amylase$ signal sequence. Cell cycle analysis indicated that the two lysozyme producing recombinant strains had an increased number of cells in the $G_2+M$ phase of the yeast cell cycle compared with the host strain SEY2102.

• 한국식품영양과학회지
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• 제28권2호
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• pp.348-352
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• 1999

We have intended to accelerate the secretion of human lysozyme(HLY) with permeabilizing agents from the cultivated cells of the recombinant Saccharomyces cerevisiae. The five agents CaCl2, Tween 80, ethanol, Triton X 100, and cetyltrimethylammonium bromide(CTAB) were used as permeabilizing agents. Treatments of the yeast cell with CaCl2, Tween 80, and ethanol were effective to increase the secretion from the yeast cells. Especially, treatment of 10% ethanol increased the extracellular HLY activity by 38.6% at 30oC for 48 h in culture broth. But Triton X 100 and CTAB unexpectedly didn't play a role in increase of HLY secretion. Recovery of a foreign protein by permeabilizing agents is easier than by osmotic shock, and is less expensive than enzymatic digestion.

• Journal of Oral Medicine and Pain
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• 제39권3호
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• pp.90-95
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• 2014

Purpose: To investigate viscosity and wettability of hyaluronic acid (HA) solutions according to supplementation of lysozyme and/or peroxidase, and different ionic strength and pH conditions. Methods: Solutions containing HA were prepared using distilled deionized water (DDW) and simulated salivary buffer (SSB) in different conditions. Different concentrations of hen egg-white lysozyme and bovine lactoperoxidase was added into HA solutions. HA solutions with antimicrobials in different ionic strength and pH conditions were prepared. Viscosity was measured using cone-and-plate digital viscometer at six different shear rates and wettability on acrylic resin and Co-Cr alloy was determined by contact angle. Results: The viscosity values of HA dissolved in DDW were decreased in order of HA, HA containing lysozyme, HA containing peroxidase, and HA containing lysozyme and peroxidase. The viscosity values for HA in DDW were decreased as the concentration of lysozyme and/or peroxidase increased. However, the viscosity values for HA in SSB showed no significant changes according to the concentration of lysozyme and/or peroxidase. The viscosity values of HA solutions were inversely proportional to ionic strength and pH. The contact angle of HA solutions showed no significant differences according to tested surface materials, addition of lysozyme and/or peroxidase, and different ionic strength and pH conditions. Contact angles on acrylic resin by HA solutions in all tested conditions were much higher than those by human saliva. Conclusions: The rheological properties of HA supplemented with lysozyme and/or peroxidase in different ionic strength and pH conditions were objectively confirmed, indicating the possibility of HA with lysozyme and/or peroxidase as main components in the development of effective saliva substitutes.

• 한국미생물·생명공학회지
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• 제26권1호
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• pp.34-39
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• 1998

본 연구에서는 안정성이 보장된 효모를 숙주로 하여 이미 lysozyme 생산능이 확인된 저 copy수의 YCp type인 pHK101의 생산능을 높이기 위해 고발현 벡터인 다 copy 수의 YEp type인 pHK501을 구축하였다. pHK501과 pHK101형질전환체의 M. luteus를 기질로 한 lysoplate assay 비교에서 확실한 생산량의 증가를 생육저지환을 통해 확인하였다. 또한 E. coli에서 peptidoglycan만을 추출하여 기질로 사용한 lysoplate assay에서도 pHK501형질전환체의 배양액 중에는 정상적 인 HLY의 생산을 직접 확인할 수 있었다. 플라스크 배양에서 배양시간에 따른 HLY의 최대 생산량은 81시간만에 pHK501형질전환체가 55 units/$m\ell$에 도달됨으로써 pHK101(7 units/$m\ell$)에 비해 약 8배 증가됐다. 발효조규모에서의 HLY 생산은 24시간만에 26.8 units/$m\ell$(1.12 units/$m\ell$/h)에 도달하였고 전체 생산성은 플라스크배양(0.625 units/$m\ell$/h)에 비해 약 1.8배 정도 증가되었다.

• Journal of Microbiology
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• 제34권1호
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• pp.95-100
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• 1996

The effect of the extracellular protease of Salmonella schottmulleri on human serum constituents such as immunoglobulins, hemoglobin and lysozyme and tissue constituents such as fibronectin and collagens was investigated. This protease degraded collagens (type I and III), fibronectin and serum proteins such as human hemoglobin and lysozyme. Bovine serum albumin was degraded slightly. Thus, the present study suggested the possibility that this protease is not only played an important role in invasion of S. schottmulleri by degrading the constituent proteins such as collagens and fibronectin but also induced complications observed in septicemia and chronic infections by degrading the serum proteins. This protease is also capable of degrading defence-oriented humoral proteins, immunoglobulins (IgG and IgM). Furthermore, it is toxic to HEp-2 cells. These findings clarified the possible role of Salmonella protease as a virulence factor in the pathogenesis of Salmonella infections.