44 research outputs found
Characterization of immobilized alkaline cyclodextringlycosyltransferase from a newly isolated Bacillus agaradhaerens KSU-A11
Alkaliphilic bacteria were isolated from soil and water samples obtained from Egyptian soda lakes (Wadi Natrun area, Egypt). Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria resulted in isolation of 10 positive strains. Strain KSU-A11 was selected as the best CGTase producer (2.1 U/ml). 16S rDNA sequence analysis identified the KSU-A11strain as Bacillus agaradhaerens. CGTase was partially purified using starch adsorption technique. The partially purified CGTase was immobilized on chitin by covalent binding tecnique using cross linking reaction with high immobilization yield (85%). The properties of the free and immobilized CGTase were determined. The optimum pH of the immobilized enzyme was slightly higher than that of the free enzyme at pH 10 and 10.5, respectively. In addition, both free and immobilized enzyme retained 94 to 100% of its initial activity over a wide pH range (pH 6.0 to 11.0). The enzymatic activity of both free and immobilized CGTase was highest at temperature 50°C; however, the relative activities of the immobilized CGTase were slightly higher than those of the free enzyme. Furthermore, investigation of thermostability of the enzyme indicated that the immobilization process of CGTase on chitin significantly protected the enzyme against thermo-inactivation. Kinetic parameters, Km and Vmax, values for free and immobilized enzymes were estimated and while there was no change in the Vmax value (83.3 μmol/min. mg) for both free and immobilized CGTase, the Km of the enzyme increased from 14.28 to 20 mg/ml upon immobilization. The immobilization of the enzyme showed high operational stability by retaining almost 50% of the initial activity after nine uses.Key words: Cyclodextrin glycosyltransferase, Bacillus agaradhaerens, immobilization, chitin, alkaliphiles
Isolation and characterization of novel potent Cr(VI) reducing alkaliphilic Amphibacillus sp. KSUCr3 from hypersaline soda lakes
A strain KSUCr3 with extremely high Cr(VI)-reducing ability under
alkaline conditions was isolated from hypersaline soda lakes and
identified as Amphibacillus sp. on the basis of 16S rRNA gene sequence
analysis. The results showed that Amphibacillus sp. strain KSUCr3 was
tolerance to very high Cr(VI) concentration (75 mM) in addition to high
tolerance to other heavy metals including Ni2+ (100 mM), Mo2+ (75 mM),
Co2+ (5 mM), Mn2+ (100 mM), Zn2+ (2 mM), Cu2+ (2 mM) and Pb (75 mM).
Strain KSUCr3 was shown to be of a high efficiency in detoxifying
chromate, as it could rapidly reduce 5 mM of Cr(VI) to a non detectable
level over 24 hrs. In addition, strain KSUCr3 could reduce Cr(VI)
efficiently over a wide range of initial Cr(VI) concentrations (1-10
mM) in alkaline medium under aerobic conditions without significant
effect on the bacterial growth. Addition of glucose, NaCl and Na2CO3 to
the culture medium caused a dramatic increase in Cr(VI)-reduction by
Amphibacillus sp. strain KSUCr3. The maximum chromate removal was
exhibited in alkaline medium containing 1.5% Na2CO3, 0.8% glucose, and
1.2% NaCl, at incubation temperature of 40\ub0C and shaking of 100
rpm. Under optimum Cr(VI) reduction conditions, Cr(VI) reduction rate
reached 237 \u3bcMh1 which is one of the highest Cr(VI) reduction
rate, under alkaline conditions and high salt concentration, compared
to other microorganisms that has been reported so far. Furthermore, the
presence of other metals, such as Ni2+, Co2+, Cu2+ and Mn2+ slightly
stimulated Cr(VI)-reduction ability by the strain KSUCr3.The isolate,
Amphibacillus sp. strain KSUCr3, exhibited an ability to repeatedly
reduce hexavalent chromium without any amendment of nutrients,
suggesting its potential application in continuous bioremediation of
Cr(VI). The results also revealed the possible isolation of potent
heavy metals resistant bacteria from extreme environment such as
hypersaline soda lakes
Tapping uncultured microorganisms through metagenomics for drug discovery
Natural products have been an important historical source of therapeutic agents. Microorganisms are major source of bioactive natural products, and several microbial products including antibiotics, anti-inflammatory, anti-tumour, immunosuppressants and others are currently used as therapeutic agents for human and domestic animals. Most of these products were obtained from cultured environmental microorganisms. However, it is widely accepted that a very large majority of the microorganisms present in natural environments are not readily cultured under laboratory conditions, and therefore are not accessible for drug discovery. Metagenomics is a recent culture-independent approach that has been developed to access the collective genomes of natural bacterial populations. It enables discovery of the diverse biosynthetic pathways encoded by diverse microbial assemblages that are known to be present in the environment but not-yet cultured. Recently, several new bioactive molecules and proteins have been discovered using a metagenomic approach. This review highlights the recent methodologies, limitations, and applications of metagenomics for the discovery of new drugs. Moreover, it shows how a multidisciplinary approach combining metagenomics with other technologies can expedite and revolutionize drug discovery from diverse environmental microorganisms.Key words: Microbial diversity, metagenomics, natural products, drug discovery, microbial ecology
Effects of substrates and reaction conditions on production of cyclodextrins using cyclodextrin glucanotransferase from newly isolated Bacillus agaradhaerens KSU-A11
The effects of reaction conditions on cyclodextrins (CDs) production by
CGTase from newly isolated Bacillus agaradhaerens KSU-A11 is
reported. Among six types of starch tested, potato starch gave highest
starch conversion into CDs. In addition, CDs yield was about three fold
higher when using gelatinized potato starch in comparison to raw
starch. The total CDs production was increased with increasing pH,
showing maximum starch conversion at pH 10. Furthermore, the proportion
of \u3b3-CD was relatively higher under slightly acidic-neutral
conditions than at alkaline pH with a maximum proportion of 35.6% at pH
7 compared to 7.6% at pH 10. Maximum starch conversion into CDs was
seen at reaction temperature of 55\ub0C. Lower reaction temperature
led to higher proportion of \u3b3-CD with maximum percentage at
35\ub0C. Cyclization reaction was significantly promoted in the
presence CaCl2 (10 mM), while in the presence of ethyl alcohol there
was significant decrease in CD production particularly at high
concentration.
f-CD was the major product up to 1 hr reaction
period with traces of \u3b1-CD and no detectable \u3b3-CD. However,
as the reaction proceed, \u3b3-CD started to be synthesised and
\u3b1-CD concentration increased up to 4 hrs, where the CDs ratios
were 0.27:0.65:0.07 for \u3b1-CD:
f-CD:\u3b3-CD, respectively. In
addition, optimum CGTase/starch ratio was obtained at 80 U/g starch,
showing highest starch conversion into CDs. All the parameters involved
have been shown to affect the products yield and/or specificity of B.
agaradhaerens KSU-A11 CGTase
Immobilization of cyclodextrin glucanotransferase on aminopropyl-functionalized silica-coated superparamagnetic nanoparticles
Background: Cyclodextrin glycosyltransferase (CGTase) from
Amphibacillus sp. NPST-10 was successfully covalently immobilized on
aminopropyl-functionalized silica coated superparamagnetic
nanoparticles; and the properties of immobilized enzyme were
investigated. The synthesis process included preparing of core magnetic
magnetite (Fe3O4) nanoparticles using solvothermal synthesis; followed
by coating of Fe3O4 nanoparticles with dense amino-functionalized
silica (NH2-SiO2) layer using in situ functionalization method. The
structure of synthesized Fe3O4@NH2-SiO2 nanoparticles was characterized
using TEM, XRD, and FT-IR analysis. Fe3O4@NH2-SiO2 nanoparticles were
further activated by gluteraaldehyde as bifunctional cross linker, and
the activated nanoparticles were used for CGTase immobilization by
covalent attachment. Results: Magnetite nanoparticles was successfully
synthesized and coated with and amino functionalized silica layer
(Fe3O4/NH2-SiO2), with particle size of 50-70 nm. The silica coated
magnetite nanoparticles showed with saturation magnetization of 65
emug-1, and can be quickly recovered from the bulk solution using an
external magnet within 10 sec. The activated support was effective for
CGTase immobilization, which was confirmed by comparison of FT-IR
spectra of free and immobilized enzyme. The applied approach for
support preparation, activation, and optimization of immobilization
conditions, led to high yields of CGTase immobilization (92.3%),
activity recovery (73%), and loading efficiency (95.2%); which is one
of the highest so far reported for CGTase. Immobilized enzyme showed
shift in the optimal temperature from 50 to 55\ubaC, and significant
enhancement in the thermal stability compared with free enzyme. The
optimum pH for enzyme activity was pH 8 and pH 7.5 for free and
immobilized CGTase, respectively, with slight improvement of pH
stability of immobilized enzyme. Furthermore, kinetic studies revealed
that immobilized CGTase had higher affinity toward substrate; with km
values of 1.18 \ub1 0.05 mg/ml and 1.75 \ub1 0.07 mg/ml for
immobilized and free CGTase, respectively. Immobilized CGTase retained
87% and 67 of its initial activity after 5 and 10 repeated batches
reaction, indicating that immobilized CGTase on Fe3O4/NH2-SiO2 had good
durability and magnetic recovery. Conclusion: The improvement in
kinetic and stability parameters of immobilized CGTase makes the
proposed method a suitable candidate for industrial applications of
CGTase. To best of our knowledge, this is the first report about CGTase
immobilization on silica coated magnetite nanoparticles
Cyclodextrin glucanotransferase immobilization onto functionalized magnetic double mesoporous core\u2013shell silica nanospheres
Background: Cyclodextrin glucanotransferase (CGTase) from Amphibacillus
sp. NPST-10 was covalently immobilized onto amino-functionalized
magnetic double mesoporous core\u2013shell silica nanospheres
(mag@d-SiO2@m-SiO2-NH2), and the properties of the immobilized enzyme
were investigated. The synthesis process of the nanospheres included
preparing core magnetic magnetite (Fe3O4) nanoparticles, coating the
Fe3O4 with a dense silica layer, followed by further coating with
functionalized or non-functionalized mesoporous silica shell. The
structure of the synthesized nanospheres was characterized using TEM,
XRD, and FT-IR analyses. CGTase was immobilized onto the functionalized
and non-functionalized nanospheres by covalent attachment and physical
adsorption. Results: The results indicated that the enzyme
immobilization by covalent attachment onto the activated
mag@d-SiO2@m-SiO2-NH2, prepared using anionic surfactant, showed
highest immobilization yield (98.1%), loading efficiency (96.2%), and
loading capacity 58 \u3bcg protein [CGTase]/mg [nanoparticles]) which
were among the highest yields reported so far for CGTase. Compared with
the free enzyme, the immobilized CGTase demonstrated a shift in the
optimal temperature from 50\ub0C to 50\u201355\ub0C, and showed a
significant enhancement in the enzyme thermal stability. The optimum pH
values for the activity of the free and immobilized CGTase were pH 8
and pH 8.5, respectively, and there was a significant improvement in pH
stability of the immobilized enzyme. Moreover, the immobilized CGTase
exhibited good operational stability, retaining 56% of the initial
activity after reutilizations of ten successive cycles. Conclusion: The
enhancement of CGTase properties upon immobilization suggested that the
applied nano-structured carriers and immobilization protocol are
promising approach for industrial bioprocess for production of
cyclodextrins using immobilized CGTase
Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes
Background: Alkaline proteases are among the most important classes of
industrial hydrolytic enzymes. The industrial demand for alkaline
proteases with favorable properties continues to enhance the search for
new enzymes. The present study focused on isolation of new alkaline
producing alkaliphilic bacteria from hyper saline soda lakes and
optimization of the enzyme production. Results: A new potent alkaline
protease producing halotolerant alkaliphilic isolate NPST-AK15 was
isolated from hyper saline soda lakes, which affiliated to Bacillus
sp. based on 16S rRNA gene analysis. Organic nitrogen supported enzyme
production showing maximum yield using yeast extract, and as a carbon
source, fructose gave maximum protease production. NPST-AK15 can grow
over a broad range of NaCl concentrations (0\u201320%), showing
maximal growth and enzyme production at 0\u20135%, indicated the
halotolerant nature of this bacterium. Ba and Ca enhanced enzyme
production by 1.6 and 1.3 fold respectively. The optimum temperature
and pH for both enzyme production and cell growth were at 40\ub0C and
pH 11, respectively. Alkaline protease secretion was coherent with the
growth pattern, started at beginning of the exponential phase and
reached maximal in mid stationary phase (36 h). Conclusions: A
newhalotolerant alkaliphilic alkaline protease producing Bacillus
sp.NPST-AK15 was isolated from soda lakes. Optimization of various
fermentation parameters resulted in an increase of enzyme yield by 22.8
fold, indicating the significance of optimization of the fermentation
parameters to obtain commercial yield of the enzyme. NPST-AK15 and its
extracellular alkaline protease with salt tolerance signify their
potential applicability in the laundry industry and other applications
Mortality and pulmonary complications in patients undergoing surgery with perioperative sars-cov-2 infection: An international cohort study
Background The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (740%) had emergency surgery and 280 (248%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (261%) patients. 30-day mortality was 238% (268 of 1128). Pulmonary complications occurred in 577 (512%) of 1128 patients; 30-day mortality in these patients was 380% (219 of 577), accounting for 817% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 175 [95% CI 128-240], p<00001), age 70 years or older versus younger than 70 years (230 [165-322], p<00001), American Society of Anesthesiologists grades 3-5 versus grades 1-2 (235 [157-353], p<00001), malignant versus benign or obstetric diagnosis (155 [101-239], p=0046), emergency versus elective surgery (167 [106-263], p=0026), and major versus minor surgery (152 [101-231], p=0047). Interpretation Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research
Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.
PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks