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1
Allouch, Julie [Liberté Lumière]. "Etude structurale et fonctionnelle de deux béta-agarases de la famille 16 des glycoside hydrolases." Aix-Marseille 2, 2003. http://www.theses.fr/2003AIX22066.
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Hehemann, Jan-Hendrik. "Structural and functional organisation of the agarolytic enzyme system of the marine flavobacterium Zobellia galactanivorans." Paris 6, 2009. http://hal.upmc.fr/tel-01110381v1.
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Zobellia galactanivorans est une Flavobacterie marine capable de dégrader des polysaccharides complexes tels que l’agar qui est largement utilisé en biotechnologie et agroalimentaire. Dans l’écosystème marin les organismes photosynthétiques tels que les algues et les cyanobactéries sont les principaux producteurs de carbone organique. Cette source de carbone est utilisée par les bactéries marines qui sécrètent des glycosides hydrolases afin d’attaquer les algues ou d’hydrolyser la paroi des algues en décomposition. Pour comprendre les mécanismes de recyclage du carbone à partir de ces zones de nutrition préférentielles nous avons décidé d’analyser le système agarolytique de Z. Galactanivorans. Le génome bactérien complet a mis en évidence la présence de neuf agarases de la famille GH 16, confirmant que Z. Galactanivorans possède un système agarolytique complexe pour dégrader cette ressource naturel qu’est l’agar. Sept séquences originales ont été surexprimées grâce à une stratégie de clonage à moyen débit. Une nouvelle ß-agarase (AgaD) a été purifiée à l’homogénéité, puis cristallisée et les données de diffraction ont été collectées à une résolution de 1. 5 Å. La structure a été résolue par remplacement moléculaire et la caractérisation biochimique a été réalisée. De plus, les deux premières ?-porphyranases PorA et PorB ont été identifiées, cristallisées et caractérisées biochimiquement. La diversité d’enzymes agarolytiques reflète les adaptations biologiques dont ont fait preuve certaines bactéries pour s’accommoder à la grande variabilité de l’agarose de la paroi des algues rouges.
3
Ekborg, Nathan Alexander. "The agarase system of saccharophagus degradans strain 2-40 analysis of the agarase system and protein localization /." College Park, Md. : University of Maryland, 2005. http://hdl.handle.net/1903/3188.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2005.Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
4
BERNARD, ISABELLE. "Production de l'alpha-agarase par alteromonas agarlyticus." Compiègne, 1996. http://www.theses.fr/1995COMP872S.
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Les enzymes qui hydrolysene l'agarose, un polysaccharide compose d'une alternance de molecules de d-galactose et de 3,6-anhydro-l-galactose reliees entre elles par des liaisons beta1-4 et alpha1-3, sont appelees les beta-agarases et les alpha-agarases suivant la liaison hydrolysee. Seule l'agarase produite par alteromonas agarlyticus, une bacterie marine, a ete clairement definie comme etant une alpha-agarase. Notre etude a concerne l'analyse de sa production afin de pouvoir l'optimiser. L'activite enzymatique a ainsi ete multipliee par 3,6 (un maximum de production de 7,7 u. E par ml lors d'essais en fermenteur), ceci grace a une hydrolyse partielle de l'agar avant son utilisation. Une hydrolyse menagee a l'acide et a chaud produit une degradation partielle de l'agar au niveau des liaisons alpha liberant ainsi des agaro-oligosides, inducteurs de la production de l'alpha-agarase. L'existence d'une repression catabolique a aussi ete demontree. Cette derniere s'effectue vraisemblablement par l'intermediaire d'un produit libere par l'action de l'alpha-agarase sur l'agarose: l'agarotetraose. La production de cette enzyme est donc soumise au mecanisme d'induction-repression. Durant des essais de cultures en volume plus importants (20 et 100 litres) ayant donne lieu a une optimisation partielle des parametres de fermentation, des problemes (en microfiltration et ultrafiltration) lors de la recolte du surnageant ont ete mis en evidence. Des mutations aleatoires (par u. V. ) pour obtenir une synthese de l'agarase constitutive ont ete entreprises ; car une synthese constitutive de cette derniere pourrait permettre d'utiliser un milieu de culture depourvu d'agar, une perspective particulierement interessante economiquement et pratiquement. C'est pourquoi outre la poursuite de l'etude de la production en fermenteur de l'alpha-agarase (culture en continu par exemple), un remaniement genetique de cette enzyme peut aussi etre une perspective au travail deja realise
5
ALISTE, ANTONIO J. "Efeito da radiacao na viscosidade de carragenanas, agaranas e alginatos utilizados na industria alimenticia." reponame:Repositório Institucional do IPEN, 1999. http://repositorio.ipen.br:8080/xmlui/handle/123456789/9281.
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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
6
Schroeder, Declan Cosmo. "Isolation and characterization of a β(1-4) agarase of an epiphytic bacterial pathogen, Pseudoalteromonas gracilis B9, of the red alga, Gracilaria gracilis." Doctoral thesis, University of Cape Town, 2001. http://hdl.handle.net/11427/4328.
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Aliste, Antonio João. "Uso de substâncias antioxidantes na resposta à radiação dos hidrocolóides carragenanas, agaranas e alginatos utilizados na indústria alimentícia." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-15052012-111215/.
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Carragenanas, agaranas e alginatos são hidrocolóides largamente utilizados em todo tipo de produtos alimentícios como aditivos espessantes. Eles não são absorvidos pelo organismo e, portanto não introduzem calorias extras na dieta. A irradiação se apresenta com grande potencial como um método alternativo na preservação de alimentos pois não induz aumento da temperatura, e é, portanto, de grande eficácia na descontaminação de ingredientes alimentícios sensíveis ao calor. Neste trabalho, soluções dos hidrocolóides agararana, carragenana e aiginato de sódio, foram irradiadas com diferentes doses (0-10 kGy) de radiação gama de Co-60 na presença de antioxidantes também utilizados na indústria alimentícia: ácido ascórbico, extrato vegetal de rosela (Híbiscus sabdariffa L.) e isofiavona de soja. As soluções dos polissacarídeos comestíveis agarana, carragenana e alginato de sódio mostraram ser bons sistemas para avaliar o efeito da radiação ionizante por apresentarem radiossensibilidade característica medida pelas mudanças na viscosidade. Os resultados obtidos mostram que esses antioxidantes apresentam, no geral, ação radioprotetora o que pode ser de grande valia nas aplicações futuras da irradiação de alimentos em escala comercial.Carrageenan, agaran e alginates are hydrocolloids largely employed in every kind of food products as stabilizing agent and viscosity builder. The human body does not absorb them, so they do not introduce extra calories in the diet. Irradiation is presented as an important alternative method in food preservation because do not induce temperature increase being of good efficiency in cold food ingredients decontamination. In this work aqueous solutions of carrageenan, agar e sodium alginate were gamma irradiated (0-10 kGy) in presence of ascorbic acid, roselle {Hibiscus sabdariffa L.) extract and soy isoflavone. Edible polysaccharide solutions showed to be suitable systems for the evaluation of ionizing radiation effects as they presented a singular radiosensitivity through viscosity changes. The results obtained showed that in general the antioxidants employed had a radioprotective action that can be of importance in the future commercial applications of food irradiation.
8
ALISTE, ANTONIO J. "Uso de substancias antioxidantes na resposta a radiacao dos hidrocoloides carragenanas, agaranas e alginatos utilizados na industria alimenticia." reponame:Repositório Institucional do IPEN, 2006. http://repositorio.ipen.br:8080/xmlui/handle/123456789/11386.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
9
Vidal, Gabarró Marcel·la. "Estudi de l'expressió i secreció de proteïnes recombinants (agarasa i lacasa) en una soca SipY- de Streptomyces lividans." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/285079.
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La producció de proteïnes homòlogues i heteròlogues a escala industrial requereix sistemes
d’expressió variats i eficients. Els bacteris grampositius, com Streptomyces, poden ser una
alternativa per a la producció de proteïnes recombinants ja que aquestes són produïdes i
secretades al medi. La seqüenciació del genoma d’algunes espècies de Streptomyces i el seu
anàlisi post-genòmic ha permès determinar i caracteritzar la seva maquinària de secreció. Així,
se sap que els precursors de les proteïnes que s’han de secretar són exportats majoritàriament
per una via principal de secreció (via Sec) o bé per una via secundària (via Tat). Aquests
precursors contenen un pèptid senyal de tipus I que permet la seva correcta translocació a través
de la membrana plasmàtica, la seqüència del qual determina per quina de les dues vies es
dirigeix. Un cop translocades, les peptidases senyal de tipus I (SPases) processen la majoria
d’aquestes proteïnes secretades i les alliberen al medi extracel·lular en la seva forma madura.
Estudis previs en la maquinària de secreció han determinat que Streptomyces lividans té
quatre peptidases senyal Sip (SipW, SipX, SipY i SipZ) de les quals, SipY és la que desenvolupa el
paper majoritari. La soca deficient en aquesta proteïna SipY té la capacitat de secreció
notablement afectada però, paradoxalment, és un bon hoste per a la sobreproducció de
proteïnes secretades, ja que presenta una activitat proteàsica considerablement disminuïda.
En aquest treball s’ha estudiat la producció d’agarasa i de lacasa en la soca SipY- (S. lividans
ΔsipY) a escala de bioreactor per avaluar si aquesta soca pot ser considerada com a possible
hoste per a la producció de proteïnes recombinants per la indústria. Aquestes dues proteïnes se
secreten majoritàriament per la via Tat. En el cas de l’enzim agarasa, el gen expressat prové de
S. coelicolor A3(2) i s’ha produït l’enzim en discontinu amb la soca salvatge (S. lividans
TK21pAGAs5) i la soca mutada (S. lividans ΔsipYpAGAs5). La soca S. lividans ΔsipYpAGAs5 ha
presentat una millor producció i s’ha seleccionat el medi més adequat per tal de produir aquesta
proteïna estudiant el seu comportament operant en continu. També s’han estudiat diferents
estratègies en discontinu alimentat, entre les quals la major producció s’ha obtingut amb una
addició de mannitol i evitant que aquesta font de carboni s’esgoti en el medi.
En el cas de la lacasa, s’han construït tres soques en les quals el gen de la pròpia lacasa de S.
lividans és regulat per tres promotors diferents: el promotor propi de la lacasa, el promotor del
gen de resistència a eritromicina i el promotor del gen agarasa de S. coelicolor. La soca en el qual
el gen és regulat pel promotor del gen agarasa (S. lividans ΔsipYpFD-DagA-laccase) ha estat
seleccionada i s’ha estudiat la producció de l’enzim utilitzant mannitol o glucosa com a font de
carboni en discontinu i discontinu alimentat. En l’operació en discontinu alimentat van ser
necessàries dues addicions de glucosa per assolir els mateixos nivells de producció que amb una
addició de mannitol.Recombinant protein production on industrial scale requires varied and efficient expression systems. Gram-positive bacteria such as Streptomyces, may be an alternative since proteins are secreted through the medium. The sequence of the genome of several Streptomyces species and their post-genomic analysis has allowed to determine and characterize the secretion machinery. Two secretion pathways are mainly use in Streptomyces: Sec pathway and Tat pathway. Protein precursors contain type I signal peptide that allows a proper translocation though membrane and whose sequence determines which pathway must be used in order to export each protein. During its translocation, type I signal peptidases (SPases) cleave the signal peptide leading the secretion of the mature protein through the medium. Previous studies on the secretory machinery have described four signal peptidases in Streptomyces lividans (SipW, SipX, SipY and Sip Z) and they have postulate SipY as the one which develops the main role in protein secretion. SipY deficient strain has a secretion ability significantly affected but, paradoxically, it is a good host for the protein overproduction since it has a considerably reduced protease activity. In this work, we have studied the production of agarase and laccase in SipY- strain (S. lividans ΔsipY) in a bioreactor scale to assess whether this strain can be considered as a host for the production of recombinant proteins in the industry. These two proteins are mainly secreted by Tat pathway. Firstly, agarase from S. coelicolor A3(2) was cloned in a multicopy plasmid in S. lividans wild type strain and SipY- mutant strain (S. lividans TK21pAGAs5 and S. lividans ΔsipYpAGAs5). Batch cultures have been carried out and S. lividans ΔsipYpAGAs5 of which has presented better agarase production and it has been selected. Appropriated medium has been also selected operating in continuous. Secondly, focusing on laccase production, three strains have been constructed in which the homologous gene of S. lividans is regulated by three different promoters: its own promoter, the promoter of erythromycin resistance gene and the S. coelicolor agarase promoter. The strain in which the laccase gene is regulated by agarase promoter (S. lividans ΔsipYpFD-DagAplaccase) has been selected and studied in batch and fed-batch mode using mannitol or glucoses as carbon source. In fed-batch cultures, two glucose addition have been necessary to achieve the same production as unic mannitol addition.
10
Gildenhuys, Carin. "Investigation of the role of the extracellular β-agarase, produced by the bacterial epiphyte Pseudoalteromonas sp. LS2i, in the virulence response towards the agarophyte Gracilaria gracilis." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/4266.
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Includes abstractIncludes bibliographical references
Gracilaria gracilis that grows naturally at Saldanha Bay, South Africa is economically important as a source of agar. The Gracilaria yields from natural beds at Saldanha Bay are however unreliable, and consequently the South African Gracilaria industry has experienced a number of setbacks over the years. The only way a consistent supply can be assured is by mariculture to supplement the natural harvests. In 1993 the Seaweed Research Institute (SRU) found that mariculture of G. gracilis in Saldanha Bay is feasible but that there is potential to improve yields by technical research and development (Anderson et al.1996a). Jaffray and Coyne (1996) developed a pathogenicity assay that demonstrated that agarolytic bacteria isolated from Saldanha Bay Gracilaria induced disease symptoms such as thallus bleaching, while non-agarolytic isolates did not. It is thought that unfavorable environmental conditions such as elevated water temperature and nutrient depletion, which occur during the summer months in the surface layers of the water column in Saldanha Bay, induce the onset of agarase production or result in changes in the bacterial community structure in which agarase-producers become more dominant. By using the pathogenicity assay, Jaffray and Coyne (1996) identified the highly agarolytic Gracilaria gracilis pathogen, Pseudoalteromonas sp. LS2i. The aim of this study was to characterize the bacterial pathogen, Pseudoalteromonas sp. LS2i to further our understanding of virulence regulation and specifically, the role of the agarase enzymes in the process of seaweed-pathogen interaction. Two agarolytic clones, pEB1 and pJB1, were obtained after constructing and screening a Pseudoalteromonas sp. LS2i genomic library in Esherichia coli. Restriction enzyme mapping suggested that both clones contain the same agarase gene. Southern hybridization studies confirmed the origin of the cloned DNA and sequencing studies revealed the 1062 bp ORF, putative promoter region, putative ribosome binding site and putative transcriptional start point of the cloned agarase gene. The ORF showed sequence identity to several other β-agarases and was identified as a member of the GH-16 family of glycoside hydrolases. The agarase was purified from the E. coli JM109 (pEB3) transformant. The molecular weight was estimated to be 39 kDa by SDS-PAGE. Zymogram analysis confirmed that the purified protein is agarolytic and TLC analysis revealed that the predominant end-products of agar hydrolysis are neoagarohexaose and neoagarobiose, which indicates the same mode of action as that observed for the agarase produced extracellularly by Pseudoalteromonas sp. LS2i.
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Toussaint, Grégoire. "Apport des écoulements secondaires et pulsés et de la vélocimétrie IRM à la filtration de fluides biologiques." Compiègne, 2000. http://www.theses.fr/2000COMP1310.
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Dans le but d'améliorer les performances de la filtration membranaire par des moyens hydrodynamiques, notre travail était d'explorer l'apport à la filtration de tourbillons de Dean générés par la courbure de l'écoulement dans des modules de filtration spécialement construits à cet effet. Nous avons effectué des essais sur trois modules de géométrie différente en testant la superposition de pulsations de débit qui provoquent une inversion du sens de rotation des tourbillons de Dean, Le besoin de mieux connaître et vérifier les profils de vitesse et les taux de cisaillement générés par ces écoulements complexes et tridimensionnels présents dans nos modules nous a amenés à expérimenter une nouvelle technique de vélocimétrie basée sur l'imagerie par résonance magnétique ( IRM). Les techniques de filtration que nous avons utilisées étant par leur complexité plus adaptées à la séparation de molécules à haute valeur ajoutée nous avons choisi comme exemple d'application l'extraction d'une enzyme, lagarase à partir d'un moût de fermentation. Toutefois, du fait des faibles volumes de moûts disponibles nous avons été amenés à effectuer les essais comparatifs sur les divers modules avec des suspensions de levures de bière considérées comme fluide modèle d'une suspension bactérienne.
12
蔡品嫻. "Study on Sequential Expression of Pseudomonas vesicularis MA103 Agarases and the Profiles of Hydrolytic Oligosaccharides Produced by Individual Agarase." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/21638022318434914829.
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Yan, Jia-Yi, and 顏嘉怡. "Characterization of an Agarolytic Thermophilic Bacterium from Marine and Its agarases." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/01721482419305735288.
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碩士東吳大學
微生物學系
90
英文摘要 In a previous work of 1995, an agar-degrading bacterial strain S3PY was isolated from a hot spring in the interidal zone of Lutao, Taiwan. In the present study, cells of the isolate were found to be Gram-negative cocci with a dimension of 1μm and moving with flagella. The G+Ccontent of this isolate was 64.5 mol %. By sequence analysis of 16 S rDNA sequence of 16S rDNA sequence of strain S3PY was found to be 100 % similar to that of Alterococcus agarolyticus ADT3, apreviously isolated strain obtained to by Shieh and Jean in 1998. Many other physiological properties of strain S3PY were similar to those of strain ADT3. In the analysis of the composition of fatty acid of strains S3PY and ADT3, anteiso-C15:0 was found to be the highest one in quantity , and both the ratios of branch chain/straight chain fattyacids of these two strains were much higher than that of E.coli. k12. Accocding to these data, strain S3PY was identified as A. agarolyticus. The optimal temperature of agar-degrading activity of crude agarase of strain S3PY was between 50-55℃, and the optimal pH was between 6.0-6.5. Besides, the 0-5 % NaCl had no significant effect on agarase activity. In the respect of the thermostability of crude agarase, after incubating at 60℃ for 180 min, the crude agarase still remained 70-80 % of activity, but only 30 % of activity was remained after incubated at 65℃ for 30 min. The crude agarase could not degrade -and -Carrageenan. The performance of crude agarase of strain S3PY in the degradation of high-melting point agarose、 agar and Gracilaria spp. extract were better than that in low-melting point agarose and Porphyra spp. extract. Detecting reducing sugar released with β-galactosidase, the existence of α-agarase andβ-agarase was found. At least eight kinds of agarase were found in the crude agarase preparation of strain S3PY means of SDS-PAGE and activity stain. These argarases were named argarase-I~VIII, and their molecular weight were found to be 175, 89, 82, 70, 58, 50, 46, 40 KDa. The activities of agarase-I and agarase-II were weaker than others, and their activities could be increased after heating at 65、70、75℃. Purification of enzymes was performed with 75 % ammonium sulfate precipitation, ion-exchange (DEAE-cellulose), gel filtration(Sephacryl S-300 HR, Sephacryl S-100 HR), affinity column( Sepharose CL-6B). The specific activities of partially purified agarases were increased but no any single agarase was found to be purified after the analysis with electrophoresis and active staining. Hydrolytic products of 1.5 % agar digested with crude agarase or partially purified agarase-A1、B、C of strain S3PY were analyzed by TLC, The products of agarase-A1 which contained tetraose, hexaose and, other oligosaccharides were the same as those of crude agarases. The hydrolytic products of agarase - B contained tetraose, hexaose and, little oligosaccharides. The hydrolytic products of agarase- C contained biose, tetraose, hexaose and, little oligosaccharides.
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SU, CHIA YAN, and 蘇家彥. "Characterization of agarases from Alterococcus agarolytics S3PY and Cellvibrio sp. AG1W." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/52680755298610280201.
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碩士東吳大學
微生物學系
93
We isolated the agar-degrading bacterial strain AG1W from the Waishuangshi. After biochemical test, we found AG1W was bacillus, gram-negative, size is about 0.5 × 3.0 μm, movable, aerobe, able to hydrolyze starch and lipid, catalase、oxidase are positive reaction, doesn't have tryptophanase. AG1W after compare with 16S rDNA sequence, we found out that AG1W with Cellvibrio sp. R4079 has 98 %, with Cellvibrio mixtus has 97 %, with Cellvibrio ostraviensis has 97 %, and Cellvibrio fulvus has 97 % similarity. Therefore we defined the agar-degrading bacterial strain AG1W was belong to Cellvibrio sp. To compare agarases from the agar-degrading bacterial strain S3PY, E. coli 3A which possess agarase gene and can represent agar-degrading activity, the agar-degrading bacterial strain AG1W, found that crude agarases of strain S3PY have the best stability to temperature, we made it at 60 degree, after 30 minutes, it still have over 90 % residual activity. We also found S3PY crude agarases stability are the best at pH and chemical. S3PY crude agarases at pH value 3, 10, after 30 minutes, still owned over 70 % residual activity; S3PY crude agarases at 4 M Urea, 9.5 % ethanol, 2.5 % SDS, after 1 hour, it still have over 50 % residual activity. We analyze S3PY and E. coli 3A hydrolysis products with TLC, which the main products was neoagarotetraose and neoagarohexarose; hydrolysis products of AG1W analyze with TLC, found that the main products was neoagarobiose. The agarase G, agarase B ( which was not completely purified ), agarase E which were purified from the S3PY culture supernatant by ammonium sulfate precipitation, anion exchange and gel giltration column chromatographic methods. Estimation of the molecular mass of agarase G, agarase B, agarase E by SDS-PAGE gave values of 39, 128, 55 KDa, respectively. The KM of agarase G, agarase B, agarase E gave values of 19.4 、11.6 、6.8 g/L, respectively. To analyze S3PY crude agarases, agarase G, agarase B ( which was not completely purified ), agarase E, hydrosis products of hydrolyze agarose by thin-layer chromatogram, we find hydrolysis products of agarase G, agarase B, agarase E are all different from hydrolysis products of S3PY crude agarases.To analyze agarase G, agarase B ( which was not completely purified ), agarase E hydrolysis products of hydrolyze neoagarobiose, neoagarotetraose, neoagarohexaose, neoagarooctaose, neoagarodecaose, neoagarododecaose by thin-layer chromatogram, find that hydrolysis products of three kinds agarases has different.
15
Chen, Tze-Yuan, and 陳姿元. "Studies on Purification and Properties of Agarases from Aeromonas salmonicida MAEF108." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/00465691231785546381.
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碩士國立臺灣海洋大學
食品科學系
94
Aeromonas salmonicida MAEF108 is a marine bacterium, which has the ability to degrade agar. Strain MAEF 108 was grown at 20oC for 24 hr in MMB-MAEF108 broth with shaking speed of 240 rpm. Collected crude enzymes after removing the cells then to proceed separation and purification of the agarases. First, after concentrated and dialysed the specific activity, purification fold, and yield of the resulted solutions were 2225.10 AU/mg, 2.32 fold, and 24.66%, respectively. Chromatographed with DE-52 gel achieved the separation of three agarases in the concentrated and dialysised broth, they are named MAEF108-agarase Ι, ΙΙ, and ΙΙΙ, by their agarase activities in each group of eluted fractions. The following properties of these Ι, ΙΙ, and ΙΙΙ agarases were: specific activity, 855.35, 174.47, and 1069.46 AU/mg; purification fold, 0.89, 0.18, and 1.11 fold; and yield, 4.97%, 0.27%, and 13.32%, respectively. MAEF108-agarase Ι was purified further by second DE-52 and then Bio-Gel P-100 chromatographs. The final purified MAEF108-agarase Ι showed its specific activity, purification fold, and yield were 233.49 AU/mg, 0.24 fold, and 0.40%, respectively. The optimum pH for MAEF108-agarase Ι was found to be 6.0 or 7.5-8.0. The optimum temperature for MAEF108-agarase Ι was 45oC, but approximately 50% of the enzyme activity was inactivated in 60 minutes under 40oC. The optimum pH for MAEF108-agarase ΙΙ was found to be 5.0 or 7.5. The optimum temperature for MAEF108-agarase ΙΙ was 45oC, but almost inactivated in 20 minutes under 50oC and more than 50% of the enzyme activity was inactivated after exposure to the rest tested temperature. The optimum pH for MAEF108-agarase ΙΙΙ was found to be 6.0 or 7.5. The optimum temperature for MAEF108-agarase ΙΙΙ was 45oC, but more than 60% of the enzyme activity was inactivated while stand 60 minutes under the rest tested temperature. The best substrate specificity activities of MAEF108-agarase Ι, ΙΙ, and ΙΙΙ toward high-melting-point agarose were produced equivalent 3.76, 2.17, and 9.46 mg D-galactose, respectively. MAEF108-agarase Ι, ΙΙ, and ΙΙΙ did not have the capability to degrade �晻d-carrageenan and �菇e-carrageenan. Hydrolytic products of agarose digested by MAEF108-agarases, MAEF108-agarase Ι, ΙΙ, or ΙΙΙ were analyzed by HPLC, and the results showed that they could be monosaccharides, which molecular weight were smaller than galactose, and disaccharides, which molecular weight were between galactose and lactose. Hydrolytic products of neoagarohexaose digested by MAEF108-agarase Ι, ΙΙ, or ΙΙΙ were analyzed by HPLC. The products examined could be monosaccharides, which molecular weight were smaller than galactose, and disaccharides, which molecular weight were between galactose and lactose.
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Tao, Yuan Ching, and 袁景道. "The Studies on the Effect of Fermenter Production Conditions on Pseudomonas vesicularis MA103 Agarases and on the Application of algal Polysaccharides and Its Oligosaccharides via Agarases Hydrolysis." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/59625431032593577036.
Full textAbstract:
碩士國立海洋大學
食品科學系
89
Pseudomonas vesicularis MA103 is a marine bacterium, which has the ability to degrade agar. As P. vesicularis MA103 was incubated in MM-P broth, containing 0.15% agar, 0.1% yeast extract, 1.0 mM CaCl2, 1% b-glycerophosphate and 0.6% NaCl, at 26oC with shaking speed of 120 rpm, the cultured broth could reach 98.30 unit/mL agarase activity and pH value of 6.21 after 48 hr. P. vesicularis MA103 was cultivated in various incubation conditions as (1) 500 mL MM-P broth in a 1 L fermenter, (2) 3 L MM-P broth in a 5 L fermenter, or (3) 3 L MM-P broth with controlled pH value at 6.20 in a 5 L fermenter. While being cultivated in (1) 500 mL MM-P broth, strain MA103 performed 51.91 unit/mL agarase activity. As incubated in (3) 3 L MM-P broth, strain MA103 achieved a higher agarase activity, 101.20 unit/mL. Total carbohydrate contents of algal polysaccharides that extracts from Gracilaria, Porphyra dentata, and Monostroma nitidium with different methods are ranged from 23.34 to 49.25%. The highest content of sulfate (1.41%) was observed in the polysaccharides extracted from Porphyra dentata as treated with hot water. The highest content of 3, 6-anhydro- galactose (31.26%) was revealed on the polysaccharides extracted from Gracilaria as treated with enzymes or mixture. The highest yield 50.16% of polysaccharide extract was obtained when Porphyra dentata was treated with enzymes or mixture. Total sugar contents of oligosaccharide lysates derived from algal polysaccharides extracts following digested by P. vesicularis MA103 agarases at 26oC for 48 hr are ranged from 5.74 to 31.26%. Molecular weight of the oligosaccharide mixture, derived from eight algal polysaccharides extracts that digested by P. vesicularis MA103 agarases at 26oC for 48 hr, were determined by the gel permeation chromatography (with Sephadex G-10) are in the range of 292.0 - 1689.6 Da. In the experiment of emulsifying properties of algal polysaccharide extracts, as the concentration of algal polysaccharide was increased from 0.0 to 1.0% (w/v), the emulsifying activity of the reacting mixture was also increased. When 1.0% polysaccharide extract, derived from Porphyra dentata with hot water treatment, were added to the emulsifying mixture, the best performance of emulsifying activity and emulsion stability were measured as 100% and 90%, respectively. Porphyra oligosaccharide mixtures, which obtained from the porphyral polysaccharide extract with enzyme or hot water treatment followed by digested by P. vesicularis MA103 agarases, were added (2.0%) individually to the emulsifying reacting solution, and the resulting emulsifying activity was 52.63 and 56.25%, repectively. The emulsifying activity of the rest oligosaccharide mixtures were measured between 45.00 and 46.66%. As to the emulsion stability, various oligosaccharide lysates did not show significant differences, which are among 40.00 and 46.15%. After dehydration under 60oC for 8 hr, eight algal polysaccharides extracts showed the capability of film formation. The higher penetration force (796.96 g) was observed happened on the film made by the polysaccharide extract derived from Gracilaria that treated with enzymes. Among various oligosaccharide hydrolysates, film made by oligosaccharides derived from Gracilaria polysaccharide extract treated with enzyme and then digested by P. vesicularis MA103 agarases, showed a higher penetration force (288.66 g).
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Ho, Yi-Jui, and 何宜叡. "Purification and characterization of native and recombinant agarases from Alterococcus agarolytics S3PY." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/53203111373791405999.
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Shao-Chi, Wu, and 吳紹祺. "The Studies on the Production Conditions of Agarases Produced from Marine Bacteria and Profiles of Oligosaccharides Derived from the Digestion of the Algal Polysaccharides by Agarase." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/35545473222814549825.
Full textAbstract:
碩士國立海洋大學
食品科學系
87
Abstract There are thirteen marine bacteria, which isolated from biofouling material of coupons that immersed into sea water, have the capability to hydrolyze agar. These agarase producing strains are belong to Aeromonas salmonicida Aga06、Aga08 and MAEF108, Flavobacterium meingosepticum YBC11A2，Moraxella spp. Aga14 and MAEF102, Pasteurella pneumotropica SCC21, Pseudomonas vesicularis B05, B10, MA103, MAEF103 and YBC12. And strain B13 has not been exclusively identified as the results of inconsistency between traditional and API 20 NE methods, and it is temporally designated as Flavobacterium spp. B13. The effect of agarase production from Moraxella spp. Aga14, Flavobacterium spp. B13, Pseudomonas vesicularis MA103, and Aeromonas salmonicida MAEF108 were studied on the cultural conditions such as initial pH value, shaking speed, and incubation temperature as well as the composition of cultural medium such as the addition of agar, various nitrogen source, and various metal ions. The experimental results showed the addition of 0.05% NH4NO3 in the basal medium is as found to reach the highest agarase activity for Moraxella spp. For Flavobacterium spp. B13, basal medium containing 1 mM MnCl2.reached the highest agarase activity (13.94 unit). And, the addition of 0.1% yeast extract in the basal medium is required for the highest agarase production for P. vesicularis MA103 (15.73 unit agarase activity). Also, a higher shaking speed employed in the incubation for A. salmonicida MAEF108 at 26oC, the agarase activity of its cultured broth could reach 14.55 unit. By using the combination of individual optimal cultural conditions and components of medium, the agarase production of strain MAEF108 could reach 71.35 unit agarase activity after 24 hr shaking incubation (240 rpm). Then, the agarase production was lower to 59.98 unit as the incubation period extended to 48 hr. The optimal temperature and pH value of the agarase (primary purified by UF) derived from Moraxella spp. Aga14, Flavobacterium spp. B13, and Pseudomonas vesicularis MA103 are 48oC/8.0, 45oC/6.0, and 45oC/6.0, respectively. For these agarases, then thermal stability at 30oC and stored at -70oC (for 60 days), showed higher stability than stored at -20 and 4oC on the enzymatic activity. The polysaccharide distributions of the extracts obtained from Gracilaria, enzyme treated Gracilaria, Monostroma, Porphyra, or Porphyra are broad, indicating the nature of heterogeneous polysaccharides. These five algal extracts append only one or two peaks of protein components on the gel permeation chromatograms with a molecular weight ranged from 8.0 103 to 7.0 105 Da. These five algal extracts were also treated by agarase from Pseudomonas vesicularis MA103 for 72, 144, and 216 hr. The protein distributions of these agarase digests were similar to that of untreated algal extracts. As digesting time increased, higher sugar contents present in the late gel permeation chromatograms elution (60-80 fractions) also increased in these five algal extracts. As the 60-80 fractions of gel permeation chromatogram elutions of these five algal extracts were collected and concentrated, mono- and oligosaccharide compositions were analyzed by an analytical NH2 HPLC column. The chromatogram indicated that D-galactose, 3,6-anhydro-galactose, and several oligosaccharides were present detected. More study was necessary before the identification of these oligosaccharides is completed.
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Wu, Shao-Chi, and 吳紹祺. "Studies on Biological Activity of Algal Lysates Derived from Agarases Digested and Their Fermentation Products." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/07077538308548895657.
Full textAbstract:
博士國立臺灣海洋大學
食品科學系
93
In ten algal polysaccharides, the algal polysaccharide extracts (APEs) of Porphyra (Por.) dentate possess greatest effects on five kinds antioxidative and four types antimutagenic experiment. And the antioxidative properties and antimutagenicity of ten algal polysaccharides were related to their polyphenol contents. The ten algal polysaccharides had not observed the ability on the ACE inhibitory. On six antioxidative experiment, the 120 algal-oligosaccharide- lysates (AOLs) had scavenging abilities, except for hydroxyl radicals scavenging effect. Without the addition of S9 mixtrues in Ames test, the antimutagenicity of 60 AOLs against 4NQO to Salm. typhimurium TA98 or TA100 were showed from12-94% or 4-98%, respectively. With the addition of S9 mixtrues in Ames test, the antimutagenicity of 60 AOLs against B[a]P to Salm. typhimurium TA98 or TA100 were reveal from 18-89% or 24-99%, respectively. The effect of antioxidative properties and antimutagenicity of 120 AOLs were relations with their polyphenol contents. The AOL derived from APEs of Mon. nitidum digested by B500 treatment had 17.1 �b 1.2% inhibitory of ACE was better than other AOL. The algal-oligosaccharides solution (108-Por500) derived from3 L of 0.5% APEs of Por. dentate digested by 15,000 AU of MAEF108-agarases were categorized as following 5 fractions: (i) > 5 KDa agar-lytic, (ii) 5-3 KDa agar-lytic, (iii) 3-1 KDa agar-lytic, (iv) < 1 KDa agar-lytic and (v) polyphenolic fraction. The (i)-(iv) agar-lytic fractions without exist polyphenol contents, and the (v) polyphenolic fraction exist polyphenol. The neoagarooligosaccharides, such as neoagarobiose, neoagarotetraose, and neoagarohexaose colud be able to separate from 1 KDa agar-lytic fraction. The (i)-(iv) agar-lytic fractions displayed 2.48-17.85% on chelating effect in ferrous ion, and whitout antioxidative abilities on the other five antioxidative tests. On the other hand, the polyphenolic fraction exist scavenging abilities on six antioxidative properties. In four types antimutagenic experiment, the polyphenolic fraction present antimutagenicity were greater than 90%. The 90 lactic acid fermentation solutions derive from (1) algal polysaccharide lactic acid fermentation substrate (APsLAFSs), (2) algal oligosaccharide lactic acid fermentation substrate (AOsLAFSs), or (3) algal oligosaccharide plus galactose lactic acid fermentation substrate (AOsGLAFSs) which fermented by Streptococcus (Strep.) faecalis BCRC13076 and Lactobacillus (Lact.) plantarum BCRC1406 could decreased pH value below 4.6 within 24 hr at 37oC. During storage at 4oC, 90 lactic acid fermentation solutions, exhibited decrease pH, increase TA, decrease viability, and an increasing reduction in sugar contents from 1 to 2 weeks. In the 90 lactic acid fermentation products, except for DPPH radical scavenging effect, the other five kinds antioxidative tests had effect increasing with reducing sugar content. Without the addition of S9 mixtrues in Ames test, the antimutagenicity of 90 lactic acid fermentation products against 4NQO to Salm. typhimurium TA98 or TA100 were ranged from 26-99% or 33-97%, respectively. With the addition of S9 mixtrues in Ames test, the antimutagenicity of 90 lactic acid fermentation products against B[a]P to Salm. typhimurium TA98 or TA100 were exhibited from 4-98% or 33-99%, respectively. The ACE inhibitory of AOsGLAFSs-Mon was 36.6 �b 6.6% and better than other lactic acid fermentation products. The 30 acetic acid fermentation solutions derive from (1) algal polysaccharide acetic acid fermentation substrate (APsAAFSs), (2) algal oligosaccharide acetic acid fermentation substrate (AOsAAFSs), or (3) algal oligosaccharide plus galactose acetic acid fermentation substrate (AOsGAAFSs) which fermented by Acetobacter (Acet.) pasteurianus BCRC11070 could increased TA above 1% within 48 hr at 26oC. During storage at 4oC, 90 lactic acid fermentation solutions, exhibited decrease pH, increase TA, decrease viability, and an increasing reduction in sugar contents from 1 to 2 weeks. In the 30 acetic acid fermentation products, except for without the hydroxyl raducals scavenging effect, other five kinds antioxidative tests had scavenging effect increasing with reducing sugar content. Without the addition of S9 mixtrues in Ames test, the antimutagenicity of 30 acetic acid fermentation products against 4NQO to Salm. typhimurium TA98 or TA100 were ranged from 2-56% or 3-76%, respectively. With the addition of S9 mixtrues in Ames test, the antimutagenicity of 30 acetic acid fermentation products against B[a]P to Salm. typhimurium TA98 or TA100 were revealed from 6-88% or 3-75%, respectively. The ACE inhibitory of 30 acetic acid fermentation products were revealed 3.4 �b 0.7% to 53.9 �b 3.7%
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Chiu, Shih-Ming, and 邱士明. "Studies on the change of multiple bands using active stain of agarases from Alterococcus agarolytics S3PY." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/80220560542042853655.
Full textAbstract:
碩士東吳大學
微生物學系
99
The thermophilic marine bacterium Alterococcus agarolyticus S3PY was isolated from a coast hot spring in Lutao by our laboratory many years ago. When the crude agarase of this strain was subjected to electrophoresis and activity staining, multiple active bands were found. The agarase gene had been cloned and expressed in Escherichia coli. Although a putative agarase gene encoded by a single open reading frame was identified, multiple active bands were still found. After heating at 95 oC for 5 min, both of the agarase samples of A. agarolyticus and E. coli showed multiple active bands, but the position of each band was changed. These results revealed that there were multiple ploypeptides having agarolytic activity. This may be due to the presence of some protease in the agarase samples and the possibility of protein conformation change was ruled out. When the fresh agarase sample of A. agarolyticus was incubated at 30-37 oC for one month, the strongest active band at 83 kDa position vanished, and the active band at 50 kDa position became the strongest one. This change had been proved to be related to protease activity.
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Wu, Xin-Hui, and 吳欣蕙. "Sudies on Purification and Characterization of Agarases PV-1 and PV-2 from Pseudomonas vesicularis MA103." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/40880964794840574064.
Full textAbstract:
碩士國立臺灣海洋大學
食品科學系
95
Abstract Pseudomonas vesicularis MA103 is a marine bacterium, which has the ability to degrade agar. Strain MA103 was grown at 26oC for 48 hr in MMB-MA103 broth with 150 rpm, pH 6.2, air flow rate 4.0 L/min in fermenter. After removing the cells, collected crude enzymes proceed in the separation and purification of the agarases. The chromatograph of the wide enzymes with DE-52 gel achieved the separation of three agarases, they are named PV-1, PV-2, and PV-3 by their elution order in NaCl gradient. The following properties of these PV-1, PV-2, and PV-3 agarases were: specific activity, 5008.73, 5801.76, and 6604 U/mg; purification fold, 18.89, 20.96, and 23.85 fold; respectively. Agarases PV-1 and PV-2 were purified further by Bio-Gel P-100 chromatography. The estimated molecular mass of agarases PV-1 and PV-2 were 62.9 kDa and 86.4 kDa, respectively. The optimum pH for agarase PV-1 was found to be 6.0. The optimum temperature for PV-1 was 40oC, but 80% of the enzyme activity was inactivated in 15 minutes under 50oC. The optimum pH for agarase PV-2 was found to be 7.0. The optimum temperature for PV-2 was 40oC or 45oC, but 58.86% of the enzyme activity was inactivated in 15 minutes under 50oC. The best substrate specific activities of agarases PV-1 and PV-2 toward HIMEDIATM agar were 1.42 and 1.24 folds digestion activity compare to agarose, respectively. Agarase PV-2 did not have the capability to degrade Sargassum sp. polysaccharide. Hydrolytic products of agarose digested by either agarases PV-1 or PV-2 were analyzed by HPLC, and the results showed that they could be oligosaccharides, which have molecular size between neoagarohexaose and neoagarobiose. Products of neoagarohexaose reacted with agarases PV-1 and PV-2 were analyzed by HPLC. The products examined could be oligosaccharides, whose molecular size was larger than neoagarohexaose. Based on these results, agarases PV-1 and PV-2 are enzymes that could have both hydrolytic and polymeric activitires.
22
Li, Cheng-Fang, and 李承芳. "Molecular cloning and analysis of Alterococcus agarase gene." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/94976591728616652853.
Full textAbstract:
碩士東吳大學
微生物學系
93
Agar is an important gelifying agent for biochemical use and the food industry, mainly extracted from the red seaweeds belonging to the family of Rhodophyceae. Agarose is the gelling component comprising a linear chain of alternating 3-O-linked-α-D-galactopyranose and 4-O-linked 3,6-anhydro-β-L-galactopyranose. A moderately thermophilic marine bacteria S3PY (lab code) which was isolated from hot springs in the intertidal zone of Lutao, Taiwan, produce at least 8 different extracellular agarases in molecular weight. That was Gram-negative halophile growing optimally at 0-5％NaCl. The optimal growth temperature range was approximate 50-55℃. The strains tolerated a relatively narrow pH range from 6 to 6.5 and had 64.5 mo1％G + C contents. Several physiology test and 16s rDNA sequence prove that S3PY was identical to the Alterococcus agarlyticus published by Shien in 1998. Partially Sau3A-digested genomic DNA of Alterococcus agarlyticus was cloned into BamHI-digested pUC19 vector and transformed to E.coli DH5α MCR‘. After 40,000-50,000 transformants were isolated, one with agarolytic activity clone was picked out. The pUC19 plasmid with a 5.2 kb insert harbored by the clone was designated as pUC3A. Multiple agarases expressed by the clone exactly match the agarases in S3PY, except 2-3Kda larger in molecular mass. SDS-PAGE containing 6M urea and 5 times of SDS showed those multiple agarases are individual monomer. Single open reading frame of 3,411bp was found and designated as agoI beginning with ATG at nucleotide 3409 (Met1) and ending with TAG at XbaI site of pUC19. Based on BLAST, the putative agarase gene belongs to GH 86; The search of Conserved domain shows that AgoI is consist of several module protein may served as substrate binding or surface adhesion. The functional fragments subcloning by PCR amplification implied those multiple agarases were originated from the single ORF agoI. The ORF subcloned in pET25b expression vector validate the comsuption. To further understand the regulation of Alterococcus Agarolyticus agarases, several hypotheses have been proposed.
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Chen, Chih-Jung, and 陳芝蓉. "Characterization of agarase from a marine bacterium FY." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/zr9s9b.
Full textAbstract:
碩士國立高雄海洋科技大學
水產食品科學研究所
102
Hydrolytic products of red algal polysaccharides, agar, are confirmed to have many biological activities. An agarase-producing bacterium FY is studied and characteristics of its agarases are investigated in this thesis. The enzyme activitystaining after electrophoresis showed that agarases with molecular weight about 43-52 kDa display higher enzyme activity. Therefore, agarases derived from FY fermrntatuon broth after desaiting are purified by gel filtration and anion exchange chromatography, purified molecule of agar about 50 kDa enzyme, and its characteristics were discussed. The results indicate that the optium cultivation temperature and pH of the purification agarase are 30 oC and 3, respectively. The metal ions Co+ 2 and Mn+ 2 increase FY agarase activity, but Hg+2 inhibit its activity. The activity of the purification agarases not reduced by addition of citric acid, EDTA, magnesium sulfste, and potassium dihydrogen phosphate. In addition, FY agarases display detergent-tolerant activity because many detergents have no significant effect on their agarase activity. Moreove, the FY agarases are salt-tolerant when the NaCl concentration increased to 6 M, the further 99% activity. In addition, that thermal stability and pH stability of the purified agarase are 50 oC and 9, respectively. In this study, the purified enzyme FY km and Vmax were 0.56 μg/ ml and 14.36 mg/ ml/ hr, and the purified enzyme was β-agarase, as it was able to hydrolyze agarose, releasing neoagarotetraose, neoagarohexaose and neoagarooctaose as the products. These oligosaccharides may be further utilized to add functional food.
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Kang, Sheen-Kye, and 康新楷. "Studies on Purification and Characterization of Aeromonas salmonicida MAEF108 Agarase AS-II and Bioactivities of Agarase AS-II Digested Algal Oligosaccharides." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/26722114634038934390.
Full textAbstract:
碩士國立臺灣海洋大學
食品科學系
95
Agarase AS-II was purified from the four fractions having agarase activities of marine bacteria Aeromonas salmonicida MAEF108 agarases, and was used to digest Gracilaria sp. or Monostroma nitidum extracted polysaccharides to evaluate its bioactive properties. Culture suspension of A. salmonicida MAEF108 was centrifuged (30,700 x g), ultrafiltration (30 kDa MWCO), then applied to ion-exchange chromatography to obtain Agarase AS-II which was 44.03-fold purified and was estimated as a 95.4 kDa single protein. The optimal temperature and pH to degrade 0.2% agarose were 45oC and 7.0, respectively. It maintained 60% residual activity when Agarase AS-II was stored below 40oC for 2 hours; the stability of Agarase AS-II was better when stored in pH 6.0-8.0 phosphate buffer. The addition of 5 mM CaCl2 in assay buffer could contribute 30% higher relative activity than no metal ions were added. Activation energy and Km of Agarase AS-II were 46.24 J mol-1 K-1 and 4.39% of agarose, respectively. The purified agarase has substrate specificity on Rhodophyta and its refined products, and could degrade agarose into final products of neoagarohexaose (N6) and neoagarotetraose (N4), indicating that Agarase AS-II could be regarded as a beta-agarase which would directly degrade agarose to produce N6 and N4. In addition, Agarase AS-II has the abilities to biosynthesize N4 and unidentified galactose oligomers from neoagarobiose (N2) and galactose, respectively. 2 mg/mL of (1) either Gracilaria sp. extracted polysaccharides (Gra) or M. nitidum extracted polysaccharides (Mon), Gra or Mon digested by A. salmonicida MAEF108 crude agarases (Gra-C108 and Mon-C108), or (3) Gra or Mon digested by Agarase AS-II (Gra-AS-II or Mon-AS-II) was tested as a carbon source for bifidobacteria growth to evaluate prebiotic potentials. Viable bacteria counts of Bifidobacterium pseudolongum subsp. pseudolongum BCRC 14673 could increase more than 3 log cfu/mL in 24 hour-incubation when Gra-C108 was utilized. M. nitidum extracted polysaccharides and its agarase lysates were also able to be utilized by bifidobacteria as carbon source. B. adolescentis BCRC 14608 and B. pseudolongum subsp. pseudolongum BCRC 14673 could utilize 6 algal samples to lower pH. B. longum BCRC 11847 performed better pH lowering ability when utilizing Gracilaria sp. based samples. Different concentrations of 6 algal samples (Gra, Mon, Gra-C108, Mon-C108, Gra-AS-II, or Mon-AS-II) were cultured with different cell densities of BHK-21 kidney cells in which the algal samples showed no cytotoxicity and the cell viability was higher than 78%. The 6 algal samples showed ability to decrease the effects caused by Japanese Encephalitis Virus (JEV, Beijing-1 strain) infection. It was found that M. nitidum based samples performing better antiviral activity than Gracilaria sp. based samples was due to having higher sulfated group contents. Gra or Mon digested by either A. salmonicida MAEF108 crude agarases or Agarase AS-II, could obtain products containing higher amount of polyphenolic compounds and sulfated group. Gra-C108 and Gra-AS-II performed 93.10 and 78.49% chelating effects on Fe2+, respectively, which were higher than those of Gra (62.52%) or Mon (21.25%).
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Liang, Feng-Yu, and 梁鳳鈺. "Purification and Properties of agarase from Pseudomonas vesicularis MA103." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/88877854804621069339.
Full textAbstract:
碩士國立海洋大學
食品科學系
87
Pseudomonas vesicularis MA103 is a marine bacterium, which has the ability to degrade agar. Strain MA103 was grown at 26℃ for 20 hr in marine broth (containing 0.15% agar) with shaking speed of 120 rpm. Chromatographed on DE-52, Sephacryl S-100 HR and Sepharose CL-6B purified two agarases in the cultured broth. Agarase I was purified to electrophoretic homogeneity by DE-52 and Sephacryl S-100 HR, and agarase II was partially purified with the three column chromatographies. Molecular weight of the agarase I determined by 5%, 7%, 9%, and 11% disc-PAGE and 5-20% gradient SDS-PAGE were 42.7 kDa and 50.3 kDa, respectively. The optimum pH for agarase I was found to be 6.0~6.2. The optimum temperature for agarase I at pH 6.2 is 37℃, but approximately 55% of the enzyme activity was inactivated in 10 minutes under this condition. Ca2+, Na+, K+, Mg2+, Fe2+, Sr2+, Zn2+, Cs2+, Co2+, Cu2+, and Hg2+ showing different degrees inhibited the enzyme. Specific activities of purified agarase I toward Difco-agar, high-melting-point agarose and low-melting-point agarose were 182, 170 and 67 mmole galactose/min/mg, respectively.
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Chiang, Hsin, and 蔣信. "Application of Alterococcus agarolyticus S3PY agarase produced by Escherichia coli." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/24449135213599287726.
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Anggraeni, Santi Rukminita. "The Agarolytic System of Microbulbifer elongatus PORT2, Isolated from Batu Karas, Pangandaran West Java Indonesia." 2020. https://tud.qucosa.de/id/qucosa%3A73107.
Full textAbstract:
Agar is a marine heteropolysaccharide with repeating units consisting of 3,6-α-anhydro-L-galactopyranose and D-galactopyranose linked by α-(1,3) and β-(1,4) linkages. It has been promoted as a prospective replacement for petroleum-based feedstocks and other applications. Enzymatic biotransformation of agar generates high specific products: It is also more environmentally friendly than chemical hydrolysis. In particular, agarolytic bacteria and their agarases are preferred for the processing of agar into sugar derivatives.
Agar-producing macroalgae are one of Indonesia's national commodities. However, agar-based products and technology are rarely developed in Indonesia. This research is aimed to explore the potential of an Indonesian marine bacterium and its agarases as bioagents for agar bioprocessing. The research objectives are to identify the novelty of the isolate among known agarolytic bacteria using microbiology and molecular biology approaches, to elucidate the agarolytic system of the bacterium using in silico genome analysis, to express and characterize the recombinant agarases, and to elucidate their potential for producing agar-derived saccharides from Indonesian natural agar.
Microbulbifer elongatus PORT2 is a gram-negative marine bacterium that had been isolated from Batu Karas seawater, Pangandaran, West Java Indonesia. PORT2 shows potential as biocatalysts for agar saccharides conversion by showing remarkable agar liquefaction. The annotation of the draft genome identifies six putative β-agarases consist of three GH50, two GH86, and one GH16 in M. elongatus PORT2. Those agarases are clustered at two different contigs. Besides agarases, other genes for D-galactose and 3,6 anhydro-L galactose metabolism, sugar transports and regulatory system are found in the vicinity of the agarases clusters. Despite the ability to utilize agar as a sole carbon sole, PORT2 lacks any putative α-agarase GH117 or GH96. Both are responsible for the cleavage of α-glycosidic bonds in agar. Indeed, several hypothetical proteins are in the neighborhood of the agarase gene clusters in M. elongatus PORT2. They probably could have a function as the alternative machinery or pathway for agar monomerization that needs clarification in future research work.
Four recombinant β-agarases from PORT2; AgaA50, AgaB50, AgaC50, and AgaF16A have been successfully overexpressed in E.coli and characterized. The AgaA50 and AgaC50 exhibit metal-dependent activity. They perform exo-agarolytic modes and generates neoagarobiose (NA2). The AgaB50 can act as endo-and exo-β-agarase without any additional activator and produces neoagarohexaose (NA6), neoagarotetraose (NA4), and NA2. AgaF16 produces NA6 and NA4. The enzyme shows pure endo-catalytic action which thiol agents positively affect its activity. The synergetic reaction of AgaF16A and AgaA50 converts Indonesian Gelidium agar into NA2 and Gracilaria agar into modified NA2. The modified NA2 from Gracilaria agar could promise new potential bioactivity that is different from agarose-derived NA2 due to the presence of additional side chains on the saccharide backbone. The NA6, NA4, and NA2 products from agarose have shown potential pharmaceutical applications such as immunomodulator, anti-tumor, antioxidant, anti-diabetic, and moisturizer.
Despite being isolated from a mesophilic marine bacterium, the recombinant agarases from M. elongatus PORT2 are active at 50 °C and pH between 6.5 to 8. They maintain more than 75% of their activities even after 1 h preincubation at 50 °C, except for AgaC50. Their thermostability gives advantages for the effective biocatalytic conversion of agar because the substrate is more accessible at mild pH and the temperature above the sol-gel condition (> 40 °C).:Contents
1. Introduction 1
1.1. Motivation and Scientific Goals 1
1.2. Literature Review 3
2. Materials and Methods 12
2.1. Materials 12
2.2. Methods 13
3. Agarolytic Bacterium Microbulbifer elongatus PORT2 22
3.1. Results 22
3.2. Discussion 28
4. Genome Profiling for In Silico Elucidation of the Agarolytic System 32
4.1. Results 32
4.2. Discussion 41
5. Recombinant Agarases from Microbulbifer elongatus PORT2 44
5.1. Results 44
5.2. Discussion 71
6. Conclusions and Outlooks 78
References 81
Appendices 97
Acknowledgements 110
28
李健瑋. "Identification of an Agarolytic Thermophilic Bacterium and Purification of Its Extracellular Agarase." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/16524416401793932044.
Full textAbstract:
碩士東吳大學
微生物學系
89
Agar, a complex polysaccharide present in the cell wall of some red algae, is composed of polygalatan and agarose. It is usually used as a solidifying agent of microbiological culture media or a gelling agent in some industrial or pharmaceutical applications. Many bacterial isolates that could degrade agar had been found and many agar-degrading enzymes of them had been purified, but most of these bacteria were mesophiles, and most agarase obtained from these isolates lost activity when they were incubated at temperature over 45℃. In a previous work in the summer of 1995, an agar-degrading bacterial strain S3-PY could grow at temperatures approaching 50 to 55℃was isolated from a hot spring in the intertidal zone of Lutao, Taiwan. In the present study, cells of the isolate were found to be Gram-negative cocci or rods with a dimension of 0.1~0.2μm. This bacterium was halophilic thermophile that grew optimally at 3% NaCl and 45℃. Many other physiological and biochemical properties were the same as those of Alterococcus agarolyticus reported by Shieh and Jean in 1998. A better production of agarase of Strain S3-PY was found when cells grew in PYMA medium which consisted of 8 g/L peptone, 2 g/L yeast extract, 0.5 g/L MgSO4·7H2O, 10 g/L NaCl, 0.1 g/L CaCl2, 0.03 g/L ferric ammonium citrate, 4.5 g/L MOPSO, and 0.25 g/L agar (pH 7.0). The optimal temperature of agarase production is 45℃. The optimal temperature of agar- III degrading activity of crue enzyme was in the range of 55~60℃and the optimal pH was in the range of 6~6.5. About 70% of activity remained after an incubation at 60℃ for 3 hours. After conducting ion exchanger chromatography and gel filtration chromatography, a partially purified agarase preparation was obtained. Its optimal temperature and pH were found to be 50~55℃ and 6~6.5, respectively. After incubated at 60℃ for 2 hours, about 80﹪of activity still remained.
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CHENG, YUNG HSI, and 鄭永熙. "Purification and Characterization of Thermostable High Molecular Weight Agarase from Alterococcus Agarolyticus S3PY." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/60457770619322476339.
Full textAbstract:
碩士東吳大學
微生物學系
104
The agarolytic bacterium Alterococcus agarolyticus S3PY was isolated from hot spring in Ludao by our laboratory. Previous research revealed that product are multiple agarases, they were of the opinion that it caused by protease. This study confirmed the presence of protease, and it can be inhibited by EDTA. In this research, high molecular weight agarase can be obtained in fermenter culture without aeration and after purification in the presence of EDTA. A new thermostable 53kDa-agarase was found after purification with ion exchange column. The 53kDa-agarase has high thermal stability that retained approximately 99 % of the initial activity after incubation for 1 hour at 60 ℃, and retained 83 % activity after 12 hours.
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馮家輝. "Studies on Gene Cloning and Characterization of Aeromonas salmonicida MAEF108 Agarase AS-I." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/77014079513879554976.
Full textAbstract:
碩士國立臺灣海洋大學
食品科學系
96
The aim of this study is to separate and purify Agarase AS-I, which produced by Aeromonas salmonicida MAEF108, and its biochemical properties and digested oligomeric products from algal polysaccharides were also being investigated. Three agarase genes (Aga-2769, Aga-2379, and Aga-2400) were picked up from the genomic of strain MAEF108 and trials have been done for cloning them into E. coli DH5�� host cell for expression of agarase activity. A. salmonicida MAEF108 crude agarase solution was centrifuged and ultrafiltration, then applied to anion-exchange and gel premeation chromatography, to obtain Agarase AS-I which was 53 fold and estimated molecular mass as a 86 kDa. The optimal temperature and pH to hydrolytic 0.2% agarose were 45oC and 6.0, respectively. Mn2+ showed the highest relative activity a comparison other metal ions, Fe3+ and Sn2+ could make Agarase AS-I inactivity. Activation energy and Km of Agarase AS-I were 25.46 J K-1 mol-1 (6.09 cal K-1 mol-1) and 0.49 mg/mL of hydrolytic agarose, respectively. Agarase AS-I could degrade Rhodphyta extracted polysacchariedses (Gelidium sp. and Gracilaria sp.) and commercial agar. While agarose was degraded by Agarase AS-I, hydrolysed products include mainly neoagarohexaose (DP6) and neoagarotetraose (DP4). In addition, Agarase AS-I showed no effect to biosynthesize any product from galactose. In restriction-free (RF) cloning experiment, agarase gene of Aga-2769, Aga-2379, and Aga-2400 used polymerase chain reaction (PCR) with pET21b, and then utilized DpnI digestion and transformation in E. coli DH5��, none of transformant found in medium. More trials have been done on adjust annealing temperature and concentration of magnesium ion (Mg2+), but there is no transformant observed in medium. In tradition of restriction splicing experiment, at first, agarase gene of Aga-2769 and Aga-2379 as well as pET21b utilized NdeI and SacI digestion, and then used T4 ligase ligation and transformation in E. coli DH5��, the result as same as RF cloning, none of transformant found on ampicillin selective agar plate.
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Yao, Chuan-Chi, and 姚筌棋. "Optimization of Culture Conditions and Scale-up Fermentation Processes of an Agarase-producing Bacterium FY." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/587gd2.
Full textAbstract:
碩士國立高雄海洋科技大學
水產食品科學研究所
102
The effect of five parameters including yeast extract concentration, culture medium volume, shake speed, peptone concentration and agar concentration on the activity of agar-degrading enzyme (ADE) from bacterium FY by using experimental designs was investigated in this study. In 2-level factorial screening test, significant factors: yeast extract concentration, culture medium volume and shake speed were identified. To optimize the activity of ADE, the test ranges of yeast extract concentrations (%), culture medium volume (ml) and shake speed (rpm) were set at 0.1~0.3%, 25~125 ml and 75~175 rpm in response surface methodology (RSM) experiments, respectively. The result indicated that the ADE activity was increased from 265.9 (initial) to 600.1 U after optimization. In addition, 5-liter fermenter was employed for scale-up culture. Although the activity of ADE was only 382.2 U by the fermenter culture, enzyme harvest (volume) was increased 80-fold and cultivation time was reduced from 48 to 12 hours. By using TLC and HPLC analysis, it revealed that FY agarase belongs to β-agarase because the hydrolytic products of agarose digested by FY agarase are neoagaro- oligosaccharides. The oligosaccharides have been reportede to have biological activities. Because we found that the hydrolytic products of food-grade agar hydrolyzed by FY agarase are the same as those of analytical grade agarose, the use of food-grade agar for the neoagaro-oligosaccharide production can reduce cost. Moreover, it indicated that hydrolysis time plays an important role in the types of oligosaccharide dervatives from hydrolytic process.
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Sie, Yu-Fong, and 謝育峰. "Purification and some properties of agarase from a novel agarolytic bacterium, Alcaligenes sp. Strain Yen." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/9jnmnt.
Full textAbstract:
碩士國立臺東大學
生命科學系碩士班
97
The agar-degrading bacterium was isolated from Taitung, Taiwan. After biochemical tests and 16S rDNA sequencing, we found the agar-degrading bacterium was a bacillus, gram-negative, mobile, aerobic, catalase and oxidase positive, didn`t produce H2S and Indole. This bacterium was identified as Alcaligenes sp. strain Yen (AY744384). Strain Yen was grown at RT for 48 hr in C-10 broth with shaking(150 rpm). After removing the cell debries, crude enzyme was collected, and then proceeded to separation and purification. First step was to concentrate and dialyse the crude enzyme, and to purify it, and yielded a solution with 157.33 U/mg(purified 2.19 fold), and production rate was 54.61%. Then Sephadex G-100 chromatographs was used to final purify Yen-agarase. The results showed its specific activity was 406.44 U/mg(purified 5.66 fold), and 7.96% production rate. The optimum pH for Yen-agarase
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Hsu, Chih-Wei, and 許志維. "Molecular Cloning and Expression of Aeromonas salmonicida MAEF108 Agarase AS-IIIb in Escherichia coli and Yeast." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/97212759720734155181.
Full textAbstract:
碩士國立臺灣海洋大學
食品科學系
96
The study was aimed to purify Agarase AS-IIIb (AS-IIIb) produced from Aeromonas salmonicida MAEF108 and identified the N-terminal sequence of AS-IIIb. According to the full genomic DNA sequencing results of Aeromonas salmonicida MAEF108 and N-terminal amino acid sequencing result of AS-IIIb, the full DNA sequence of AS-IIIb gene was confirmed. Specific primers were designed to clone the AS-IIIb gene into E. coli and yeast by restriction-free (RF) cloning. The recombinant Agarase AS-IIIb (rAS-IIIb) was extracted from expression host to analyze the biochemical properties and the agarose hydrolytic products composition. The Agarase AS-IIIb gene consists of 834 bp, and encodes a protein of 269 amino acids in mature type with molecular weight about 27.6 kDa. By using pET expression system and RF cloning method, two rAS-IIIb clones, agaI and agaII were constructed; both have extra- and intracellular agarase activity. Further study was focused on the agaII clone. The agarase can be produced without adding IPTG as inducer, but its addition will improve the expression on agarase activity. The extracellular agarase activity is too low to be detected in culture broth before being concentrated into 80-100 folds. The optimal agarase expression induced condition is at 30oC for 3 hr, the enzyme activity is 1.57 U/mg. AS-IIIb and rAS-IIIb are optimally active at 40oC and 50oC, respectively; both are optimally active at pH 6. The activities of two agarases were both increased by 5 mM Mn2+, while decreased by 5m M Fe3+. Ca2+ ion of 5mM concentration increases the activity of rAS-IIIb, but decreased 27.6% activity of AS-IIIb. Hydrolytic products of agarose by either AS-IIIb or rAS-IIIb were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Both of the two agarases have two main hydrolytic products, which have molecular size between neoagarohexaose (NA6) and neoagarobiose (NA2); and have the similar retention time of neoagarotetraose (NA4) and NA6 separately in HPLC analysis. The Agarase AS-IIIb gene was cloned into vector pPICZ�� B (for yeast expression system) by RF cloning, and obtained a successful clone Y-aga within E. coli with correct DNA sequence. The Y-aga clone vector was transformed into X-33 Pichia strain by electroporation, the electrotransformation frequency calculated in the experiment is 10.9 transformants obtained per �慊 DNA..
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Wei, Chien-Han, and 危建翰. "Sequences alignment analysis of the putative agarase AgaA from Pseudomonas vesicularis MA103 and the optimization of its overexpression in Escherichia coli on the yields and activity." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/59r6ps.
Full textAbstract:
碩士國立臺灣海洋大學
食品科學系
102
garases are the enzymes which catalyze the hydrolysis of agar. Based on the cleavage pattern, they are classified into α-agarase (EC 3.2.1.158) and β-agarase (EC 3.2.1.81). Agarases have been mainly isolated from many kinds of bacteria in seawater and marine sediments because agar is the main constituents of red algea. Agarases have wide applications in many fields due to the physiological activity of the oligosaccharides produced by them. They also act as an important enzyme tool of biological studies. Pseudomonas vesicularis MA103, having the ability to degrade algal polysaccharides, was isolated from sea water (吳，1999). Its genomic sequence profile was identified and revealed many kinds of possible polysaccharides degrading enzyme sequence when comparing with database and the agaA (originally carIII) was annotated to encode a putative carrageenase. agaA*—an β-agarase sequence from Vibrio sp. PO-303, showed 95.7% identity in DNA sequence and 98.0% identity in protein sequence with AgaA. This result indicated that AgaA might be an agarase, possessing the ability to degrade agarose. So we cloned different constructs of agaA gene onto pET-21a and pET-26b vector and, transformed the plasmids into E. coli C43 (DE3) for overexpression. Then we used cell disruption and osmotic shock methods to purify AgaA protein, aiming to improve its yield and activity. The results of TLC and DNS tests showed AgaA had hydrolytic activity for agar and agarose, but not for reaction with κ-carrageenan and ι-carrageenan. The determination of the AgaA activity via DNS assay found that purified AgaA by using the way of osmotic shock, compared to the use of cell disruption methods, gave a higher activity but lower protein purification fold.
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Chen, Po-Hsuan, and 陳柏璇. "Studies on Cloning and Expression of MAEF108 Agarase AS-IIIb Gene from Aeromonas salmonicida to Lactic Acid Bacteria and Lactic Acid Bacteria Fermented with Yam and Algal Oligosaccharides Dairy Product." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/65144133486276816540.
Full textAbstract:
碩士國立臺灣海洋大學
食品科學系
98
The object of this study was using TA-cloning to clone gram negative Agarase gene AS-IIIb into lactic acid bacteria (LAB) with food grade vector pNZ8149 of Lactococcus lactis express system. Another way utilized LAB to ferment Keelung yam and Monostroma nitidum oligosaccharides (MnOS) cultured milk and examined the effect of their storage period at 4oC. 48 lactic acid bacteria mutant were found from Lc. lactis subsp. lactis BCRC 10791, BCRC 12322, and LM2301. 10 LAB survived after the protoplast electroporated to lactis acid host cell. Specific primers include restricted enzyme were designed to clone the Agerase AS-IIIb gene into E.coli DH5 by TA-cloning,used heat shock and electroporated to obtained AS-IIIb clone. By using lactic acid bacteria expression system and restriction enzyme SpeI, SacI with T4 polymerase mothed, three rAS-IIIb clones L-aga I, L-aga II, and L-aga III were contructed; both have extra- and intra-cellular agarase activity, but the extracellular agarase activity is too low to be detected. Hot water extact yam solution degraded by used four group, A: MAEF108 + MA103-crude agarases, B: MA103-crude amylase, C: MA103-crude alginate lyase, and D: commeicial agarase. group A, B, C to obtained oligosaccharides that molecular weight larger than lactose after reacting 1 and 6 hr, commeicial agarase group to obtained saccharides similar to galactose and maltose. In the fermentation experiment, yam cultured milk was fermented by Lb. plantarum BCRC 12250 + BCRC 10069, Ped. pentosaceus MFS + MFL, and Lb. bulgaricus BCRC 10696 + Strep. thermophilus BCRC 12268, respectively. The pH could reach 4.6 in 24 hr, LAB counts were 8.53-9.47 CFU/mL and titratable acidity rised 0.87%-1.23%. The pH of yam and MnOS cultured milk by Lb. plantarum BCRC 10069 + 12250 group and Ped. pentosaceus MFL + MFS group could reach 4.6 in 12 hr, LAB counts were 10.19-11.52 log CFU/mL, titratable acidity rised 0.64-0.76%. Both mold/yeast count and coliforms count have not been detected during the fermentation period. In the storage period at 4oC experiment of yam cultured milk and yam with MnOS cultured milk, the lactic acid bacteria and pH value decreased and titratable acidity increased after 14 days. Both mold/yeast count and coliforms count have not been detected during the storage period at 4oC.
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Liao, You-Hao, and 廖友豪. "Studies on Cloning of Agarase Genes of AS-IIIb from Aeromonas salmonicida MAEF108 and PV-Ib fromPseudomonas vesicularis MA103 in Lactic Acid Bacteria and Production of Lactic Fermented Product with Algal Oligosaccharides." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/07321473886627739053.
Full textAbstract:
碩士國立臺灣海洋大學
食品科學系
97
The object of this study was clone the Agarase gene AS-IIIb and PV-Ib into the lactic acid bacteria (LAB) with shuttle vector. And investigate LAB ferment Monostroma nitidum oligosaccharides (MnOS) contained milk on the effect of MnOS of their antioxidant properties, anticoagulant properties, and whitening properties. The results showed that the effectively of protoplast formation of Ped. pentosaceus MFS, MFL, Lb. bulgaricus BCRC 10696, and Lb. plantarum BCRC 10069 treated with mutanolysin were 46.3%, 3.4%, 5.6%, and 9.9% respectively. There was more than 7.3 log CFU/mL LAB kept alive after the protoplast electroporated by 2.0 kV voltage. The constructions of Agarase AS-IIb and PV-IB genes and vectors pVA838 and pDG148-Stu by restriction-free (RF) cloning were not completed. The constructed gene of pDG148-Stu vector were treated with restricted enzyme Stu, T4 polymerase, and dTTP but the clone was not observed yet. MnOS was separated by ultrafiltration to obtain (A) 3-5 kDa fraction contained degree of polymerization (DP) 6, 12, and 30 with the highest reducing sugar content and sulftate content (0.28 mg/mL and 0.68 mg/mg) and (B) 1-3 kDa fraction contained DP 6. In the fermented experiment of 0.10%, 0.05%, and 0.02% MnOS contained milks, Lb. plantarum BCRC 10069 + 12250 group took 16 hr to reached the pH < 4.6, titratable acidity were 0.67-0.68%, and LAB counts were above 9 log CFU/mL. Ped. pentosaceus MFL + MFS group took 24 hr to reached the pH < pH 4.6, titratable acidity were 0.60-0.70%, and LAB counts were above 8.5 log CFU/mL. The MnOS-GAI-0.1% cultured milk had the best scavenging DPPH free radicals ability 21.73% and MnOS-GBII-0.1% had the best chelating Fe2+ ability 72.56%. The pH value of two MnOS cultured milks were fell to 4.47-4.07, and titratable acidity were increased to 0.76%-0.89% during 4oC for 14 days. The abilities of antioxidation were decreased as storage time increased. The APTT anticoagulants activity test of rabbit plasma showed the MnOS-GAI-100 �慊/mL group compared with control which could delay the coagulant time for 20 sec, and decrease the coagulation effect for 32.4%. The rabbit plasma PT anticoagulants activity test result showed it could decrease the coagulation effect for 2.7%, and on human plasma it decreased 8.7% and 7.5% coagulation effect for APTT and PT. The MnOS cultured milks with better antioxidative ability could increase the cell viability of NIH-3T3 and CCD-966SK of 124-128% and 122-138%. The inhibition of tyrosinase was increased with increasing the concentration of MnOS cultured milk, and the inhibition were 35-37%. The MnOS cultured milks could inhibited the A375 cell, and the cell viability were 46-87%. The experiment results did not obtain the recombined clone, but the result of protoplast formation and electroporation conditions could be consulted. The MnOS cultured milk had the potential to be developed as health food product and used as whitening ingredient in cosmetic product.