Streptococcus Equisimilis Research Articles

Anionic cardiolipin stabilizes the transmembrane region of hyaluronan synthase and promotes catalysis-relevant dynamics

Hyaluronic acid (HA) is a glycosaminoglycan essential for cellular processes and finding increasingly applications in medicine, pharmaceuticals, and cosmetics. While membrane-integrated Class I hyaluronan synthase (HAS) catalyzes HA synthesis in most organisms, the molecular mechanisms by which HAS-lipid interactions impact HAS catalysis remain unclear. This study employed coarse-grained molecular dynamics simulation combined with dimensionality reduction to uncover the interplay between lipids and Streptococcus equisimilis HAS (SeHAS). A minimum of 67 % cardiolipin is necessary for HA synthesis, as determined through simulations using gradient-composed membranes. The anionic cardiolipin stabilizes the cationic transmembrane regions of SeHAS and thereby maintains its conformation. Moreover, the highly dynamic cardiolipin is required to modulate the catalysis-relevant motions in HAS and thus facilitate HA synthesis. These findings provide molecular insights essential not only for understanding the physiological functions of HAS, but also for the development of cell factories and enzyme catalysts for HA production.

Enzyme variants in biosynthesis and biological assessment of different molecular weight hyaluronan

In the present study, low- and high-molecular-weight hyaluronic acids (LMW-HA and HMW-HA) were synthesized in vitro by truncated Streptococcus equisimilis hyaluronan synthases (SeHAS). The enzyme kinetic parameters were determined for each enzyme variant. The MW, structure, dispersity, and biological activity of polymers were determined by electrophoresis, FTIR spectroscopy, carbazole, cell proliferation, and cell migration assay, respectively. The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 µgHA µgenzyme−1 min−1 for SeHAS, HAS123, HAS23, and HASIntra, respectively. The results revealed SeHAS produced a polydisperse HMW-HA (268 kDa), while HAS123 and HAS23 produced a polydisperse LMW-HA (< 30 kDa). Interestingly, HASIntra produced a low-disperse LMW-HA. Kinetics studies revealed the truncated variants displayed increased Km values for two substrates when compared to the wild-type enzyme. Biological assessments indicated all LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest cell migration effect at 10 µg/mL, while at 200 µg/mL, both LMW- and HMW-HAs postponed the healing recovery rate. The study elucidated that the transmembrane domains (TMDs) of SeHAS affect the enzyme kinetics, HA-titer, HA-size, and HA-dispersity. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.Graphical abstract

Vaginal Bacteria in Mares and the Occurrence of Antimicrobial Resistance.

Antibiotics are added to semen extenders in insemination doses but their effect on the vaginal microbiota of the inseminated female is unknown. The objectives of this study were to define the equine vaginal microbiota and its antimicrobial resistance, and to determine whether it changes after exposure to antibiotics in semen extenders. Vaginal swabs were taken prior to sham-insemination (day 0), and again on days 3, 7, and 14 after insemination. Isolated bacteria were identified by MALDI-TOF and tested for antimicrobial susceptibility by microdilution. The bacteria isolated from the vagina differed according to reproductive status (brood mare or maiden mare), location (north or middle of Sweden), and the stage of the estrous cycle. Five bacterial species were frequently isolated from mares in both locations: Escherichia coli, Staphylococcus capitis, Streptococcus equisimilis, Streptococcus thoraltensis, and Streptococcus zooepidemicus. Overall, vaginal bacteria isolated from inseminated mares showed higher antibiotic resistance than from non-inseminated mares, suggesting a possible link between exposure to antibiotics in the semen extender and the appearance of antimicrobial resistance. The whole-genome sequencing of E. coli isolates from inseminated mares revealed some genes which are known to confer antimicrobial resistance; however, some instances of resistance in these isolates were not characteristic of induced AMR.

Development of Streptococcus equisimilis group G mutant strains produce low polydisperse and low molecular weight Hyaluronic Acid.

Hyaluronic acid (HA), a natural polymer with unique properties and wide application, is mainly produced by Streptococcal fermentation at industrial scale. In the present work, two rounds of chemical random mutagenesis were applied on Streptococcus equisimilis group G to obtain high-producing strains using 1-methyl-3-nitro-1-nitrosoguanidine. The non-hemolytic and hyaluronidase-negative mutants were screened on blood agar and HA agar, respectively. HA productivity and molecular weight (Mw) was determined by carbazole assay and agarose gel electrophoresis, respectively. Moreover, the stability of strains was assessed within 10 generations in term of HA productivity. The findings showed that the wild type strain produced 1241 ± 2.1µg/ml HA at pH 5.5 and 4 hours of cultivation, while the screened mutants showed a 16.1 - 45.5 % increase in the production. Two mutant strains, named Gm2-120-21-3 (2470± 8.1µg/ml) and Gm2-120-21-4 (2856± 4.2µg/ml), showed the highest titer and a consistent production. The molecular weight of HA for the mutants was less than 160 kDa which considered a low Mw. The mutant strains producing a low polydisperse and low Mw HA at high titer can be considered potential industrial strains for HA production after further safety investigations.

CRISPR-Cas9 Mediated Knockout of SagD Gene for Overexpression of Streptokinase in Streptococcus equisimilis.

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is higher globally than production due to increased incidences of various heart conditions. The main source of streptokinase is various strains of Streptococci. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD gene-mediated post-translational modification of streptolysin, an inhibitor of streptokinase expression through the degradation of FasX small RNA (through CoV/RS), which stabilizes streptokinase mRNA. In order to improve the stability of mRNA and increase the expression of streptokinase, which is inhibited by SagA, we used CRISPR-Cas9 to successfully knockout the SagD gene and observed a 13.58-fold increased expression of streptokinase at the transcript level and 1.48-fold higher expression at the protein level in the mutant strain compared to wild type. We have demonstrated the successful gene knockout of SagD using CRISPR-Cas9 in S. equisimilis, where an engineered strain can be further used for overexpression of streptokinase for therapeutic applications.

Impact of intra-partum azithromycin on carriage of group A streptococcus in the Gambia: a posthoc analysis of a double-blind randomized placebo-controlled trial

BackgroundGroup A Streptococcus (GAS) is a major human pathogen and an important cause of maternal and neonatal sepsis. Asymptomatic bacterial colonization is considered a necessary step towards sepsis. Intra-partum azithromycin may reduce GAS carriage.MethodsA posthoc analysis of a double-blind, placebo-controlled randomized-trial was performed to determine the impact of 2 g oral dose of intra-partum azithromycin on maternal and neonatal GAS carriage and antibiotic resistance. Following screening, 829 mothers were randomized who delivered 843 babies. GAS was determined by obtaining samples from the maternal and newborn nasopharynx, maternal vaginal tract and breastmilk. Whole Genome Sequencing (WGS) of GAS isolates was performed using the Illumina Miseq platform.ResultsGAS carriage was lower in the nasopharynx of both mothers and babies and breast milk among participants in the azithromycin arm. No differences in GAS carriage were found between groups in the vaginal tract. The occurrence of azithromycin-resistant GAS was similar in both arms, except for a higher prevalence in the vaginal tract among women in the azithromycin arm. WGS revealed all macrolide-resistant vaginal tract isolates from the azithromycin arm were Streptococcus dysgalactiae subspecies equisimilis expressing Lancefield group A carbohydrate (SDSE(A)) harbouring macrolide resistant genes msr(D) and mef(A). Ten of the 45 GAS isolates (22.2%) were SDSE(A).ConclusionsOral intra-partum azithromycin reduced GAS carriage among Gambian mothers and neonates however carriage in the maternal vaginal tract was not affected by the intervention due to azithromycin resistant SDSE(A). SDSE(A) resistance must be closely monitored to fully assess the public health impact of intrapartum azithromycin on GAS.Trial registration ClinicalTrials.gov Identifier NCT01800942

Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

Rehabilitation of a complex industrial wastewater containing heavy metals and organic solvents using low cost permeable bio-barriers – From lab-scale to pilot-scale

This work addresses the treatment of a complex industrial effluent containing high concentrations of metals and spiked with two organic solvents (diethylketone – DEK, and methyl ethyl ketone - MEK) using an eco-friendly approach. The treatment system herein proposed consists of a bio-barrier that combines the adsorption capacity of sepiolite with the properties of a Streptococcus equisimilis biofilm with proven ability in the degradation and bioremoval of a wide range of pollutants.Results from the open-systems experiments conducted with raw sepiolite exposed to a binary mixture of DEK and MEK revealed the preference of the sorbent towards DEK. The results from the biodegradation experiments also revealed the preference of S. equisimilis to degrade/bioremove DEK over MEK independently of their initial concentration (100 mg/L to 3200 mg/L). Bioremoval percentages higher than 95% were reached for all the concentrations of DEK tested. The lab-scale experiments conducted in open-system with sepiolite and sepiolite covered with biofilm, and the pilot-scale experiment conducted in closed-loop, revealed similar performances on the rehabilitation of an industrial effluent containing heavy metals and additionally spiked with DEK and MEK. Regarding the selectivity towards the different pollutants, Cu was preferentially removed over Cr and Ni, and DEK over MEK. The presence of the biofilm allowed an improvement on the removal of heavy metals, particularly Cr, besides preventing the leaching of Al, Fe, and Mg from the sepiolite structure, an extremely important advantage in comparison to the system without biofilm. EDS analyses performed in sepiolite samples revealed the presence of several metals (Cr, Cu and Ni), proving thus the occurrence of sorption processes by sepiolite and by sepiolite covered by biofilm. The breakthrough data obtained in the open-systems were properly described by the Dose Response and the Yoon and Nelson mathematical models. More research work needs to be performed with complex industrial effluents aiming the optimization of the treatment systems to be applied in real context scenarios.

Photosynthetic conversion of CO2 to hyaluronic acid by engineered strains of the cyanobacterium Synechococcus sp. PCC 7002

Hyaluronic acid (HA), consisting of alternating N-acetylglucosamine and glucuronic acid units, is a natural polymer with diverse cosmetic and medical applications. Currently, HA is produced by overexpressing HA synthases from gram-negative Pasteurella multocida (encoded by pmHAS) or gram-positive Streptococcus equisimilis (encoded by seHasA) in various heterotrophic microbial production platforms. Here we introduced these two different types of HA synthase into the fast-growing cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) to explore the capacity for producing HA in a photosynthetic system. Our results show that both HA synthases enable Syn7002 to produce HA photoautotrophically, but that overexpression of the soluble HA synthase (PmHAS) is less deleterious to cell growth and results in higher production. Genetic disruption of the competing cellulose biosynthetic pathway increased the HA titer by over 5-fold (from 14mg/L to 80mg/L) and the relative proportion of HA with molecular mass greater than 2MDa. Introduction of glmS and glmU, coding for enzymes involved in the biosynthesis of the precursor UDP-N-acetylglucosamine, in combination with partial glycogen depletion, allowed photosynthetic production of 112mg/L of HA in 5 days, an 8-fold increase in comparison to the initial PmHAS expressing strain. Addition of tuaD and gtaB (coding for genes involved in UDP-glucuronic acid biosynthesis) also improved the HA yield, albeit to a lesser extent. Overall our results have shown that cyanobacteria hold promise for the sustainable production of pharmaceutically important polysaccharides from sunlight and CO2.

Pilot-scale sorption studies of diethylketone in the presence of Cd2+ and Ni2+

ABSTRACTThe effect of pH on the sorption capacity of vermiculite towards cadmium and nickel was tested in batch systems and it was shown that the sorption percentages increase with an increase in the mass of vermiculite and with an increase in the initial pH. Maximum sorption percentages were obtained for a pH of 8 and 4 g of vermiculite (86.5% for Cd2+ and 86.1% for Ni2+, for solutions with 100 mg/L of metal). As a consequence, it was possible to establish a range of optimal pH for biosorption processes, by combining the so determined optimal sorption pH of vermiculite with the optimal growth pH of Streptococcus equisimilis, a bacterium used to treat contaminated water. Pilot-scale experiments with a S. equisimilis biofilm supported on vermiculite were conducted in closed-loop conditions, aiming to treat large volumes of diethylketone aqueous solutions, eventually containing Cd2+ or Ni2+. The excellent capacity of this joint system to simultaneously biodegrade diethylketone and biosorb Cd2+ or Ni2+was proved. The removal percentage and the uptake increase through time, even with the replacement of the initial solution by new ones. The breakthrough curves that best describe the results achieved for Cd2+ and Ni2+ are respectively the Adams–Bohart and the Yoon and Nelson model.

Key Role of the Carboxyl Terminus of Hyaluronan Synthase in Processive Synthesis and Size Control of Hyaluronic Acid Polymers.

The essential pathophysiological roles of hyaluronic acid (HA) strongly depend on HA binding and HA size. Here we deployed the atomic vision of molecular dynamics (MD) simulation to experimentally investigate the influence of C-terminal mutations of Streptococcus equisimilis hyaluronan synthase (SeHAS) on HA product synthesis in Escherichia coli. R413 was vital for HA production, as the removal or mutation of R413 led to inactivation of SeHAS. MD simulations indicated that R406-R413 constituted an HA-binding pattern that stabilized the HA-SeHAS complex. We further increased HA product size via site-directed mutation of the SeHAS C-terminal residues 414-417 based on the hypothesis that higher binding affinity between the SeHAS C-terminus and HA would lead to larger HA size, underlying the important role of the HA-SeHAS interaction in HA size control. W410A and T412A mutations also completely deactivated SeHAS. Moreover, a catalysis-transformation-translocation model was proposed for the HA synthesis and translocation processes.

Bioremoval of Ni and Cd in the presence of diethylketone by fungi and by bacteria – A comparative study

Two fungi (Alternaria sp. and Penicillium sp.) and one gram-positive bacterium (Streptococcus equisimilis) were used to remove Ni and Cd from aqueous solutions in the presence of diethylketone. Individual toxicity assays were performed at an initial stage to evaluate the xenobiotic impact of the initial concentration of those metals on the growth of the microorganisms and allowed to infer that the growth of S. equisimilis is negatively affected by both metals, whereas the growth of both fungi is positively stimulated by the presence of Ni and inhibited by Cd (>40 mg/L). Within the group of microorganisms tested, S. equisimilis presented higher removal efficiency (%) and uptake. In a second stage, biosorption assays were performed using aqueous solutions containing Ni, Cd and diethylketone (mixed solutions) and aimed to infer about the overall effect of the initial metal concentrations on the growth and on the sorption capacity of the microorganisms, as well as to evaluate the interaction between the sorbent matrices. It was demonstrated that despite the mixed solution exert a negative effect on the removal process and on the growth of the three microbial cultures, the system is able to decontaminate aqueous solutions with high concentrations of Ni, Cd and diethylketone.

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(β1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end.

Class I hyaluronan synthases (HAS) assemble [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-β1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcβ1,4)2-5[GlcUA(β1,3)GlcNAc]2-6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2-3 series members. Since lyase releases dehydro-oligos (dHn; M-18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2-6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2-5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(β1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-β1,4)3-4].

Sorption studies of diethylketone in the presence of Al3+, Cd2+, Ni2+ and Mn2+, from lab-scale to pilot scale

ABSTRACTThe toxic effects of diethylketone (DEK) in aqueous solution with different concentrations of Al3+, Cd2+, Ni2+ and Mn2+ were evaluated at lab-scale. It was established that Streptococcus equisimilis is able to efficiently remove DEK with different concentrations with heavy metals. It was proved that this joint-system has excellent capacity to biodegrade high concentrations of DEK in the presence of Al3+, Cd2+, Ni2+ and Mn2+. With the exception of Al3+, the uptake for all metals increased as the initial concentration of each metal in the mixed solution increased. The breakthrough curves are best described by the Adams and Bohart model for Cd2+, by the Yoon and Nelson model for Ni2+ and by the Wolborska model for Mn2+.

Biosorption of nickel and cadmium in the presence of diethylketone by a Streptococcus equisimilis biofilm supported on vermiculite

A Streptococcus equisimilis biofilm supported on clays was used to decontaminate aqueous solutions containing diethylketone (DEK), nickel and/or cadmium, in a batch mode. The interaction between the sorbent matrices and the different sorbates, individually and in binary combinations, was evaluated. It was demonstrated that the growth of S. equisimilis is negatively affected by concentrations higher than 80 mg l−1 of Ni and 5 mg l−1 of Cd. DEK and Cd uptakes are positively correlated with the mass of vermiculite without biofilm, as observed in singular sorbate experiments. The same trend is observed for sorption percentages, reporting to the initial solution concentrations, as values higher than 90% were achieved for both pollutants. Although Ni uptake decreases as the mass of vermiculite increases, the percentage of sorbed Ni increased, reaching a maximum value of 100%. It was demonstrated that Ni and DEK, when mixed, present higher sorption values, suggesting a synergetic interaction between these two pollutants, whereas when DEK and Cd are mixed, the sorption of DEK decreases significantly, revealing a negative effect of Cd over the retention of DEK. It was also observable that for sorbent mass higher than 0.085 g the simultaneous uptake of DEK and Ni by the biofilm supported on vermiculite decreases, but the sorption percentage tends to increase, reaching values of 100% for 40 g of vermiculite. As the mass of sorbent increases the sorption percentage of both sorbates, DEK and Cd, tends to increase, reaching maximum values higher than 98%. These sorption systems are effective and the use of a biofilm may be an important advantage for the treatment of aqueous solutions contaminated with specific pollutants.

Hyaluronan synthase control of synthesis rate and hyaluronan product size are independent functions differentially affected by mutations in a conserved tandem B-X7-B motif.

Hyaluronan synthases (HAS) normally make large (>MDa) hyaluronan (HA) products. Smaller HA fragments (e.g. 100-400 kDa) produced in vivo are associated with inflammation and cell signaling by HA receptors that bind small, but not large, HA. Although HA fragments can arise from breakdown by hyaluronidases, HAS might also be regulated directly to synthesize small HA. Here we examined the Streptococcus equisimilis HAS (SeHAS) C-terminus, which contains a tandem B-X7-B motif (K398-X7-R406-X7-K414), by testing the effects of 27 site-specific scanning mutations and 7 C-terminal truncations on HA synthesis activity and weight-average mass. Although HAS enzymes cannot be HA-binding proteins, these motifs are highly conserved within the Class I HAS family. Fifteen Arg406 mutants made large MDa HA (86-110% wildtype size), with specific activities from 70% to 177% of wildtype. In contrast, 10 of 12 Lys398 mutants made HA that was 8-14% of wildtype size (≤250-480 kDa), with specific activities from 14% to 64% of wildtype. Four nearly inactive (2% wildtype activity) C-terminal truncation mutants made MDa HA (56-71% wildtype). The results confirm earlier findings with Cys-mutants [Weigel PH,Baggenstoss BA. 2012. Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines. Glycobiology 22:1302-1310] that HAS uses two independent activities to control HA size and HA synthesis rate; these are two separate functions. We conclude that HAS regulatory modifications that alter tandem B-X7-B motif conformation could mimic these mutagenesis-induced effects, allowing HAS in vivo to make small HA directly. The results also support a model in which the tandem-motif region is part of the intra-HAS pore and interacts directly with HA.

Erysipelas following breast cancer surgery: 12 case reports

Objective To analyze clinical and bacteriological features and therapeutic methods of erysipelas following breast cancer surgery in 12 hospitalized patients. Methods Clinical data on 12 patients with erysipelas following breast cancer surgery were collected from 3 third-grade class-A hospitals in Ningbo. A retrospective study was carried out. Results Of the 12 patients, 8 were treated with modified radical mastectomy and axillary lymph node dissection, 3 with radical mastectomy and axillary lymph node dissection, and 1 with mammectomy only. All of the 12 patients suffered from postoperative lymphedema on the affected side. Erysipelas mostly occurred within 1 -18 years (mean, 8.0 years) after breast cancer surgery, and on the same side of surgery in all the patients except 2 with multi-site infection. Specifically speaking, erysipelas was located in the upper extremity in 8 patients, in the chest wall in 1 patient, in the lower extremity in 1 patient. Blood culture was performed for 5 patients, of whom, 2 showed positive results, including 1 infected with Streptococcus equisimilis and 1 with Klebsiella pneumonia. Six patients were treated with penicillins, but the primary treatment failed in 2 patients. After replacement of antibiotics, all the 12 patients experienced an improvement of the condition and were discharged from hospital. Conclusions Patients who suffer from lymphedema after breast cancer surgery are prone to erysipelas of the upper limbs and chest wall. Caution should be taken against gram-negative bacterial infections in these pateints. The treatment of lymphedema should be taken into account besides antibiotic therapy. Key words: Erysipelas; Breast neoplasms; Lymphedema; Lymph node excision; Postoperative complications

IMPROVED STREPTOKINASE PRODUCTION

Several strains of beta-hemolytic Streptococci produce streptokinase enzymethat can bind and activate human plasminogen to plasmin. Streptokinase degrades the fibrinlump by its explicit lysine joining site and so it is applied as a remedy in thrombolytic therapy.The purpose of the study was to subject wild strain of Streptococcus equisimilis to straindevelopment technique, using random mutagenesis by UV irradiation for enhanced productionof streptokinase. Objective: To evaluate the hyper production of streptokinase after mutagenesisof wild Streptococcus equisimilis by means of UV irradiation. Study Design: Randomized study.Period: 2012-2014. Setting: Enzyme Biotechnology Laboratory, Department of Biochemistry,University of Agriculture, Faisalabad-Pakistan. Materials and Methods: UV lamp (TUP 40wlamp which has about 90% of its radiation at 2540-2550 A0) was used for the mutation ofStreptococcus equisimilis cells (1x 107 cells mL-1) for enhanced production of streptokinase. 10mL fresh inoculum was transferred to sterile petri plates, which were exposed to UV light for 30,60, 90, 120, 150, 180, 210, 240 and 270 minutes. The exposure was carried out at distance of20cm from the centre of lamp. A dose producing 87% killing was selected as optimum dose,after preparing kill curve. The kill/ survival curve was prepared and time of exposure giving(210 minutes) 3 log kill was selected for mutation of the Streptococcus equisimilis for hyperproduction of streptokinase enzyme. Results: Enzyme assay was performed for both wildand mutant strains. Dose of 210 minutes was selected as best dose which was followed bythe selection using triton X-100. Finally the selected strain S. equisimilis EBL-UV-210 showed480 U mL-1 of streptokinase activity in quantitative blood clot liquefaction test, which is quitehigher than wild strain (370 U mL-1). This maximum yield of streptokinase was obtained after24h, at CSL 4%, pH 7.5, 37oC, KH2PO4 0.04%, K2HPO4 0.05%, MgSO4. 7H2O 0.04%, NaHCO30.15%, CaCO3 0.004%, CH3COONa. 3H2O 0.10%, FeSO4. 7H2O 0.04%, MnCl2. 4H2O 0.02%,glucose 2%, yeast extract 3% and 5% inoculum size in liquid state fermentation. Conclusions:Results showed that mutated strain gave enhanced streptokinase activity in comparison tothe wild strain. Our current study focused on streptokinase production from this UV mutatedstreptococcus equisimilis species and purification of this enzyme by ammonium sulfateprecipitation, Ion exchange and gel filtration chromatography. The activity of streptokinase wasdetermined by using quantitative blood clot liquefaction method.

Biodegradation of Diethylketone by Penicillium sp. and Alternaria sp. – A Comparative Study Biodegradation of Diethylketone by Fungi

Two contaminating fungi were isolated from a bioreactor containing diethylketone and Streptococcus equisimilis, subsequently characterized at molecular level and identified as belonging to the Alternaria and Penicillium genera. The ability of these fungi to biodegrade DEK is evaluated. The kinetic parameters are estimated using four growth kinetic models for biodegradation of organic compounds available in literature. The experimental data for Alternaria sp. and Penicillium sp. was found to be better fitted by the Haldane and the Luong respectively. Biodegradation rate kinetics was evaluated using zero-order, pseudo-first order, pseudo-second order and three-half order models. The pseudo-second-order model was found suitable for all the concentrations of DEK tested for the biodegradation assays using Penicillium sp. whereas for the Alternaria sp. this model just describes properly the assays with initial concentrations of DEK higher than 0.5 g/L. The percentage of biodegraded DEK were approximately 100%, for all the initial concentrations tested. Keywords: Biodegradation, diethylketone, fungi, genetic identification, kinetics.