Mango processing generates significant amounts of residues (35-65%) that may represent environmental problems owed to improper disposal. The use of mango byproducts as substrates to produce hyaluronic acid (HA) is an attractive alternative to reduce the cost of substrate. In this study, we evaluated the potential of hydrolyzates from mango peels and seeds to produce HA by Streptococcus equi. subsp. zooepidemicus. The physicochemical characterization of mango residues showed that the seeds contain a higher amount of holocellulose (cellulose and hemicellulose), which amounts 54.2% (w/w) whereas it only represents 15.5% (w/w) in the peels. Mango peels, however, are composed mainly of hot water-extractives (62% w/w, that include sucrose, fructose, glucose and organic acids). A higher concentration of monosaccharides (39.8 g/L) was obtained from the enzymatic hydrolysis (with Macerex) of peels as compared to seeds (24.8 g/L with Celuzyme). From mango peels, hydrolyzates were obtained 0.6 g/L HA, while 0.9 g/L HA were obtained with hydrolyzates from mango seeds. These results demonstrate that mango byproducts have the potential to be used for production of HA.
- MeSH
- celulosa metabolismus MeSH
- fermentace MeSH
- hydrolýza MeSH
- kyselina hyaluronová * biosyntéza metabolismus MeSH
- Mangifera * mikrobiologie chemie MeSH
- monosacharidy metabolismus MeSH
- semena rostlinná chemie mikrobiologie metabolismus MeSH
- Streptococcus equi * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Hyaluronan (HA) effects on immune response are suggested to be dependent on HA molecular weight (MW), as low MW HA should activate immune cells in contrast to high MW HA. However, some current studies do not support this conception and emphasize the importance of the form of preparation of HA, particularly with respect to its purity and origin. We compared the activation of mouse immune cells by HA samples (100kDa, 500kDa, and 997kDa) prepared from HA originating from rooster comb, and HA samples (71kDa, 500kDa, and 1000kDa) prepared from pharmacological grade HA originating from Streptococcus equi. Interestingly, in contrast to established theory, only middle and high MW HA originating from rooster comb induced the production of tumor necrosis factor-α by macrophages and in whole blood. Further, all tested preparations of HA failed to induce the expression of inducible nitric oxide synthase, the production of nitric oxide, or the expression of cyclooxygenase 2 in macrophages and splenocytes. Importantly, all HA samples originating from rooster comb were found to be contaminated by endotoxin (up to 1.23EU/ml). Hence, low MW HA did not reveal itself to have significantly higher immunostimulatory activity compared to HA of higher MW.
- MeSH
- buněčná imunita účinky léků genetika MeSH
- cyklooxygenasa 2 genetika MeSH
- kur domácí MeSH
- kyselina hyaluronová farmakologie MeSH
- makrofágy účinky léků imunologie MeSH
- molekulová hmotnost MeSH
- myši MeSH
- oxid dusnatý genetika MeSH
- RAW 264.7 buňky MeSH
- regulace genové exprese účinky léků MeSH
- Streptococcus equi chemie MeSH
- synthasa oxidu dusnatého genetika MeSH
- TNF-alfa genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells.
- MeSH
- chromatografie iontoměničová MeSH
- intracelulární prostor chemie MeSH
- kyselina hyaluronová metabolismus MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Streptococcus equi chemie metabolismus MeSH
- uridindifosfátové cukry analýza MeSH
- Publikační typ
- časopisecké články MeSH
This study addresses the influence of upstream region sequence on the strength of has operon promoter in highly encapsulated S. equi subsp. zooepidemicus (SEZ). For this purpose, seven different strains were constructed. Each strain carries a point mutation in one of the following positions upstream of the has promoter: -43, -44, -49, and -50 bp. To facilitate measuring of the recombinant promoter relative strength, ß-glucuronidase gene was used as a reporter gene. Three mutations located in positions -49 and -50: AT, GT, and AG, positively impacted has promoter strength when compared to the wild type sequence GG. Conversely, two other mutations: TG and TT, exhibited a slight inhibitory effect. Further, three different strains carrying chromosomal mutations in the has promoter region were constructed. In two cases, the has operon is under the control of a stronger promoter and in the third strain the has operon is controlled by a weaker promoter. The laboratory fermenter scale cultivations confirmed the increase of hyaluronan yields for SEZPhasAG and SEZPhas2G, resulting 116 and 105 %, respectively. As expected, the yield of the hyaluronic acid of SEZPhas2B strain fell to 41 %.
- MeSH
- bakteriální geny MeSH
- biomasa MeSH
- biotechnologie MeSH
- kyselina hyaluronová chemie metabolismus MeSH
- mutace * MeSH
- mutageneze cílená metody MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) MeSH
- Streptococcus equi genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The purpose of this study is to determine the effect of the corresponding nucleotides from Streptococcus pyogenes on the has promoter strength in highly encapsulated strain S. equi subsp. zooepidemicus (SEZ) and detect an empowering mutations in SEZ. Eight different strains of SEZ carrying nucleotide mutations in the -73 to -38 region upstream of the has promoter were constructed. The significant activity decrease to 36-1% was observed after the introduction of mutations in the promoter region from -44 to -38 site. The exception was observed in mutation in -49 site when no significant decrease was observed. When nucleotides TTT were used in positions -73 the promoter became weaker, whereas no significant effect was observed after using nucleotides CCC (96%). Unfortunately, introduction of these mutations into chromosome SEZ has no empowering effect. Six strains, which carried nucleotide sequences of different lengths upstream from the transcription start of hasA promoter, were constructed to determine the minimum upstream region required for the maximum transcription efficiency of the has operon. No change of the activity of the has promoter constructs containing as few as 101 nucleotides upstream from the transcription start point was observed.
- MeSH
- bakteriální geny genetika MeSH
- bakteriální proteiny MeSH
- glukuronidasa MeSH
- klonování DNA MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- mutageneze MeSH
- operon MeSH
- plazmidy MeSH
- promotorové oblasti (genetika) genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- Streptococcus equi genetika MeSH
- Streptococcus pyogenes genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Kyselina hyaluronová je nesulfátovaný glykosaminoglykan, který se skládá z opakujících se jednotek disacharidů kyseliny D -glukuronové a N -acetyl- D -glukosaminu, spojených (1 ➝ 3)- a (1 ➝ 4)- β - D -glykosidovou vazbou. Tento polysacharid je hlavní složkou mezibuněčné hmoty. Kyselina hyaluronová se jako signální molekula také účastní imunologických procesů. Díky svým unikátním vlastnostem jako je biokompatibilita, hydrofilita nebo viskoelasticita je kyselina hyaluronová velice cenným biopolymerem v medicíně, například v oční a plastické chirurgii, dále je také využívána v kosmetickém průmyslu a potravinářství. V současné době se kyselina hyaluronová komerčně získává především mikrobiální fermentací pomocí bakteriálního kmene Streptococcus equi subsp. zooepidemicus . Ke kultivaci se obecně používá syntetické médium obsahující glukosu a kvasničný extrakt. Byly testovány také některé zdroje zemědělského původu, které by mohly nahradit toto médium a být dále využity jako zdroj bioenergie nebo pro přípravu cenných bioproduktů.
Hyaluronic acid is non-sulfated glycosaminoglycan which is composed of alternating disaccharide units of D -glucuronic acid and N -ace- tyl- D -glucosamine joined by (1 ➝ 3)- and (1 ➝ 4)- β - D -glycosidic bonds. This polysaccharide is abundant in the extracellular matrix. Hyaluronic acid as a signal molecule also participates immunological process. Thanks to its unique properties such as biocompatibility, hydrofility or viscoelasticity hyaluronic acid is very valuable medical biopolymer (e.g. in eye surgery and plastic surgery) and it is also used in the cosmetic and food industry. Currently hyaluronic acid is commercially obtained by microbial fermentation of the path ogenic Streptococcus equi subsp. zooepidemicus . For cultivation is generally used synthetic cultivation medium containing glucose and yeast extrakt. Some agricultural resources derivates which can be converted into bioenergy or valuable bioproducts were also tested as a compensation for synthetic medium.
- MeSH
- farmaceutický průmysl MeSH
- fermentace imunologie MeSH
- imunitní systém MeSH
- kosmetické přípravky MeSH
- kyselina hyaluronová farmakologie MeSH
- lidé MeSH
- oftalmologie MeSH
- ortopedické výkony MeSH
- potravinářské přísady MeSH
- průmyslová mikrobiologie metody trendy MeSH
- Streptococcus equi růst a vývoj MeSH
- Check Tag
- lidé MeSH
Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.
- MeSH
- glukuronidasa genetika MeSH
- koně MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční homologie nukleových kyselin MeSH
- shluková analýza MeSH
- Streptococcus equi genetika MeSH
- terciární struktura proteinů genetika MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Recently, a new gene encoding beta-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37 degrees C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52 degrees C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of beta-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of beta-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified beta-glucuronidase from SEZ glcHis was four times as low as beta-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal beta-glucuronidase from E. coli with E. coli beta-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.
Streptokoky sérologické skupiny C představují 9-17 % všech kmenů beta-hemolytických streptokoků získaných z klinických vzorků od člověka. Jsou izolovány od pacientů s invazivními infekcemi méně často, než je tomu u streptokoků sérologických skupin A, B a G. Nejčastěji nalézaným streptokokem skupiny C je Streptococcus dysgalactiae subsp. equisimilis. Vyvolává jak infekce mírné a povrchové, tak i onemocnění závažná - bakteriémie, septikémie, streptokokový syndrom toxického šoku, hnisavé infekce kůže a měkkých tkání, nekrotizující fasciitidu, infekce pleuropulmonární a metastaticky rozšířené. Spektrum onemocnění zahrnuje i post-streptokokové následky - peritonzilární absces, akutní glomerulonefritidu (AGN) a reaktivní artritidu. Infekce jsou jak endogenního, tak exogennflio (zvířecího) původu. Jejich manifestace je prakticky neodlišitelná od infekcí vyvolaných streptokoky skupiny A. Až v poslední době byla potvrzena účast Streptococcus constellatus subsp. pharyngis v etiologii akutní faryngitidy. Streptococcus anginosus je významným agens zejména purulentních infekcí - abscesů vznikajících na různých místech těla (včetně dutiny ústní, jater a sleziny) a endokarditid. Nákazy vyvolané Streptococcus equi subsp. zooepidemicus a Streptococcus equi subsp. equi mají charakter zoonóz a jsou vzácné. (Nemocnění působená S. equi subsp. zooepidemicus zahrnuje záněty horních dýchacích cest, cervikální lymfadenitidy, pneumonie, bakteriémie, endokardi-tidy, meningitidy, septikémie, septické artritidy a celulitidy. Infekce horních dýchacích cest mohou být následovány AGN. (Nemocnění S. equi subsp. equi se prezentuje zejména jako bakteriémie. Lékem volby pro beta-hemolytické streptokoky skupiny C je penicilin G. Všechny kmeny jsou k penicilinu citlivé, penicilin tolerantní izoláty byly popsány. Kombinovaná terapie, penicilin a gentamicin, je doporučena pro léčbu endokarditid, meningitid, septických artritid a bakteriémií u neutropenických pacientů.
Streptococci of the serological group C represent 9-17 % of all beta-hemolytic streptococcus strains obtained from clinical samplings in humans. They háve been isolated in patients down with invasive infections less often than in streptococci of serological groups A, B and G. The most frequently found group C streptococcus is Streptococcus dysgalactiae subsp. equisimilis. It causes mild and superficial infections, as well as serious ones - bacteremia, septicemia, streptococcal toxic shock syndrome, suppurative infections of the skin and soft tissue, necroti-sing fasciitis, pleuropulmonary and metastatically spread infections. The spectrum of diseases also includes post-streptococcal sequelae - peritonsillar abscess, acute glomerulonephritis (AGN) and reactive arthritis. The infections are either endogenous or exogenous (animal) in origin. Their manifestations are practically indiscernible from infections caused by group A streptococci. QÚy recently has there been confirmed the participation of Streptococcus constellatus subsp. pharyngis in the etiology of acute pharyngitis. Streptococcus anginosus is an important agent námely in purulent infections - abscesses appearing in various parts of the body, including the oral cavity, liver and spleen, and endocarditis. Infections caused by Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi háve the character of zoonoses and are rare. Diseases caused by S. equi subsp. zooepidemicus include inflammati-ons of the upper respiratory tract, cervical lymphadenitis, pneumonia, bacteremia, endocarditis, meningitis, septicemia, septic arthritis and cellulitis. Upper respiratory tract infections may be followed by AGN. Diseases caused by S. equi subsp. equi are manifested námely by bacteremias. The remedy of choice for group C beta-hemolytic streptococci is penicillin G. All strains are sensitive to penicillin, there háve been penicillin-resistant isolates. Combined therapy with penicillin and gentamycin is recommended for treating endocarditis, meningitis, septic arthritis and bacteremia in neutropenic patients.
