• Journal of Microbiology and Biotechnology
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• v.9 no.5
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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.

• Journal of Korean Ophthalmic Optics Society
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• v.2 no.1
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• pp.85-90
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• 1997

The cornea and conjunctiva of the human eye are exposed to external environment and thus are damageable. If the damaged part is infected with some pathogenic microorganisms. serious visual loss may be occured by inflammation. Keratitis or conjunctivitis does not always occur if the eyes are routinely exposed to pathogenic factors because lysozyme in human tears has antimicrobial activity against the microorganisms. 10 this study we have selected 5 strains causing keratitis and/or conjunctivitis. and cultured them in the optimum media. And then we have estimated the growth inhibition of the strains with the addition of various concentration of lysozyme to media to investigate the antimicrobial activity of lysozyme. The results are as follows. The growth of the strains were decreased according to the increase of lysozyme concentration. The growth of Pseudomonas. Neisseria. Klebsiella and Staphylococcus were inhibited 43%, 41%, 35% and 22% respectively by 1 mM concentration of lysozyme. The susceptibility of the gram-negative bacteria to lysozyme is 1.5~2 times higher than the Staphylococcus which is gram-positive bacteria in 1 mM concentration of lysozyme. But lysozyme inhibited the growth of Fusarium which is fungi slightly.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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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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• v.31 no.4
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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.

• Korean Journal of Agricultural Science
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• v.21 no.2
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• pp.122-132
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• 1994

Bacteriostatic activity of secretory immumoglobulin A (SIgA) in human and bovine colostrums on enterotoxigenic type Salmonella was tested in the tissue culture medium. SIgA was used in $0.1{\sim}5.0mg/m{\ell}$ concentration with or without the addition of egg lysozyme tested for theirs bacteriostatic activites. 1. Bovine SIgA rich fraction with a large amount of $IgG_1$-dimer could be prepared from bovine colostrum of Holstein cows by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200 and Sepharose 6B. 2. Human SIgA appeared to be the most bacteriostatic effect for all varieties of Salmonella in a range of $0.5{\sim}1.0mg/m{\ell}$ Bovine SIgA showed a marked bacteriostatic effect increased by increasing the concentration. Bovine IgG had not show bacteriostatic effect against both enterotoxigenic type Salmonella. Egg lysozyme as well as bovine SIgA also showed a marked bacteriostatic effect increased by increasing the concentration. 3. When the growth inhibition of human SIgA was tested by adding egg lysozyme with time interval, egg lysozyme showed bacteriostatic effect as compared with control. But human SIgA and adding with lysozyme showed slight the bacteriostatic effect. 4. When the growth inhibition of bovine SIgA was tested by adding egg lysozyme with time interval, all treatment against S. enteritidis showed bacteriostatic effect as compared with control In the case of S. typhinwrium, egg lysozyme showed a marked bacteriostatic effect as compared with control.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.1
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.2
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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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• v.39 no.3
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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.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.34-39
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• 1998

We have already prepared a human lysozyme (HLY) structural gene from chemically synthesized 38 oligomers with high codon usage in Saccharomyces cerevisiae. For directing the synthesis and secretion of HLY in S. cerevisiae, two types of expression vectors, a YCp centromere-based vector, pHK101 and a YEp 2-$\mu\textrm{m}$ circle-based vector, pHK501 were constructed. With the resulting plasmids, we have confirmed that yeast transformant harboring pHK501 has more secreted HLY than pHK101-transformant by using a lysoplate and a turbidimetric assay. In flask cultivation, pHK501-transformant produced active HLY about 8 times (55 units/$m\ell$) higher than pHK101-transformant. From batch cultivation, the HLY productivity was obtained with 1.12 units/$m\ell$/h, corresponding to a 1.8-fold increase compared with flask fermentation. These results indicate that yeast transformant with pHK501 vector overexpressed and secreted HLY than that of YCp type vector.

• Journal of Microbiology
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• v.34 no.1
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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.