13 research outputs found
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
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
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
A new low molecular mass alkaline cyclodextrin glucanotransferase from Amphibacillus sp. NRC-WN isolated from an Egyptian soda lake
Background: Cyclodextrin glucanotransferase (CGTase) is one of the most
industrially important enzymes used in the commercial production of
cyclodextrins (CDs). Alkaliphilic bacteria have attracted much interest
in the last few decades because of their ability to produce
extracellular enzymes that are active and stable at high pH values.
Here, we report the isolation of a new CGTase from alkaliphilic
bacteria collected from Egyptian soda lakes and describe the
purification and biochemical characterization of this CGTase. Results:
Screening for CGTase-producing alkaliphilic bacteria from sediment and
water samples collected from Egyptian soda lakes located in the Wadi
Natrun valley resulted in the isolation of a potent CGTase-producing
alkaliphilic bacterial strain, designated NRC-WN. Strain NRC-WN was
belonging to genus Amplibacullus by 16S rDNA sequence analysis
(similarity: ca. 98%). Among the tested nitrogen and carbon sources,
peptone (0.15%, w/v) and soluble starch (0.4%, w/v) allowed maximal
CGTase production by Amphibacillus sp. NRC-WN. CGTase was
successfully purified from Amphibacillus sp. NRC-WN up to 159.7-fold
through a combination of starch adsorption and anion exchange
chromatography, resulting in a yield of 84.7%. SDS-PAGE analysis
indicated that the enzyme was purified to homogeneity and revealed an
estimated molecular mass of 36 kDa, which makes it one of the smallest
CGTases reported in the literature. The purified enzyme exhibited
maximum activity at 50oC and was stable up to 70oC, retaining 93% of
its initial activity after treatment for 1 hr. Furthermore, Ca2+ ions
(10 mM) significantly enhanced the thermal stability of the CGTase. The
purified enzyme was active and stable over a wide pH range, showing
maximal activity at pH 9.5. The enzyme was significantly stimulated by
Zn2+, Ca2+ and Co2+ but was completely inhibited in the presence of
Fe3+ and mercaptoethanol. The Km and Vmax values of the purified CGTase
were estimated to be 0.0434 mg/ml and 3,333.3 mg \u3b2-CD/ml/min,
respectively. \u3b2-CD was the predominant product of starch
degradation by the Amphibacillus sp. NRC-WN CGTase, followed by
\u3b1-and \u3b3-CDs. Conclusions: A new low molecular mass alkaline
CGTase was purified from a newly identified alkaliphilic Amphibacillus
sp. NRC-WN isolate from the Egyptian soda lakes. The enzyme showed
promising thermal and pH stability and a high affinity toward starch as
a natural substrate
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
Clonal diversity and antimicrobial resistance of Enterococcus faecalis isolated from endodontic infections
Background: Enterococcus faecalis is considered to be one of most prevalent species in the oral cavity, particularly in endodontic infections. The aim of the present study was to investigate the prevalence of E. faecalis in dental root canals, clonal diversity by restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) analysis, and the antibiotic susceptibility of E. faecalis isolates.
Results: Among the bacterial strains isolated from dental root canal specimens (n = 82), E. faecalis was determined to have the highest prevalence followed by Streptococcus viridians, Leuconostoc mesenteroides, Staphylococcus aureus, Streptococcus mitis, and Pediococcus pentosaceus. Cluster analysis of RAPD-PCR and RFLP patterns of the E. faecalis isolates discriminated five and six different genotypes, respectively. Among the tested strains, 43%, 52% and 5% were susceptible, intermediate resistant, and resistant to erythromycin, respectively. In addition, one strain (E-12) was intermediate resistant to linezolid, and one isolate (E-16) was resistant to tetracycline. Interestingly, many of the intermediate resistant/resistant strains were grouped in clusters 5 and 6, according RAPD and to RFLP, respectively.
Conclusions: E. faecalis demonstrated the highest prevalence in the tested dental root canal specimens collected from Saudi patients and were grouped into five to six different genotypes. Different levels of antimicrobial susceptibility were observed in the tested E. faecalis strains, which clearly indicated that although bacterial strains may be similar, point mutations can result in extreme susceptibility or resistance to various antibiotics. This phenomenon is a cause for concern for clinicians in the treatment of dental infections caused by E. faecalis
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 γ-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°C. Lower reaction temperature
led to higher proportion of γ-CD with maximum percentage at
35°C. 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. ß-CD was the major product up to 1 hr reaction
period with traces of α-CD and no detectable γ-CD. However,
as the reaction proceed, γ-CD started to be synthesised and
α-CD concentration increased up to 4 hrs, where the CDs ratios
were 0.27:0.65:0.07 for α-CD:ß-CD:γ-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
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°C and shaking of 100
rpm. Under optimum Cr(VI) reduction conditions, Cr(VI) reduction rate
reached 237 μMh1 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
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 (0–20%), showing maximal growth and enzyme production at 0–5%, 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°C 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 new halotolerant 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