Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India) with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg 2+ , acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40˚C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production.
Keeping
in view various pharmacological attributes of indole and coumarin
derivatives, a new series of indolindione–coumarin molecular
hybrids was rationally designed and synthesized. All synthesized hybrid
molecules were evaluated for their antimicrobial potential against
Gram-negative bacterial strains (
Escherichia coli
and
Salmonella enterica
), Gram-positive
bacterial strains (
Staphylococcus aureus
and
Mycobacterium smegmatis
), and
four fungal strains (
Candida albicans
,
Alternaria mali
,
Penicillium
sp., and
Fusarium oxysporum
) by using the agar gel diffusion method. Among all synthetics, compounds
K-1
and
K-2
were found to be the best antimicrobial
agents with the minimum inhibitory concentration values of 30 and
312 μg/mL, against
Penicillium
sp. and
S. aureus
, respectively.
The biological data revealed some interesting facts about the structure–activity
relationship which state that the electronic environment on the indolinedione
moiety and carbon chain length between indolinedione and triazole
moieties considerably affect the antimicrobial potential of the synthesized
hybrids. Various types of binding interactions of
K-2
within the active site of
S. aureus
dihydrofolate reductase were also streamlined by molecular modeling
studies, which revealed the possible mechanism for potent antibacterial
activity of the compound.
Keeping in view various
pharmacological attributes of curcumin, coumarin, and isatin derivatives,
triazole-tethered monocarbonyl curcumin–coumarin and curcumin–isatin
molecular hybrids have been synthesized and evaluated for their antibacterial
potential against Gram-positive (
Enterococcus faecalis
and
Staphylococcus aureus
) and Gram-negative
(
Pseudomonas aeruginosa
and
Escherichia coli
) human pathogenic bacterial strains.
Among all hybrid molecules,
A-4
and
B-38
showed the most potent antibacterial activity with inhibition zones
of 29 and 31 mm along with MIC values of 12.50 and 6.25 μg/mL,
respectively. Structure–activity relationship that emerged
from biological data revealed that the two-carbon alkyl chain between
triazole and coumarin/isatin moiety is well tolerable for the activity.
Bromo substitution at the fifth position of isatin, para-cholo substitution
in the case of curcumin–isatin, and para-methoxy in the case
of curcumin–coumarin hybrids on ring A of curcumin are most
suitable groups for the antibacterial activity. Various types of binding
interactions of
A-4
and
B-38
within the
active site of dihydrofolate reductase (DHFR) of
S.
aureus
are also streamlined by molecular modeling
studies, suggesting their capability in completely blocking DHFR.
Ten different strains of Thermomyces lanuginosus, isolated from composting soils were found to produce phytase when grown on PSM medium. The wild type strain CM was found to produce maximum amount ofphytase (4.33 units/g DW substrate). Culturing T. lanuginosus strain CM on medium containing wheat bran and optimizing other culture conditions (carbon source, media type, nitrogen source, level of nitrogen, temperature, pH, inoculum age, inoculum level and moisture), increased the phytase yield to 13.26 units/g substrate. This culture was further subjected to UV mutagenesis for developing phytase hyperproducing mutants. The mutant (TL-7) showed 2.29-fold increase in phytase activity as compared to the parental strain. Employing Box-Behnken factor factorial design of response surface methodology resulted in optimized phytase production (32.19 units/g of substrate) by mutant TL-7. A simple two-step purification (40.75-folds) ofphytase from mutant TL-7 was achieved by anion exchange and gel filtration chromatography. The purified phytase (approximately 54 kDa) was characterized to be optimally active at pH 5.0 and temperature 70 degrees C, though the enzyme showed approximately 70% activity over a wide pH and temperature range (2.0-10.0 and 30-90 degrees C, respectively). The phytase showed broad substrate specificity with activity against sodium phytate, ADP and riboflavin phosphate. The phytase from T. lanuginosus was thermoacidstable as it showed up to 70% residual activity after exposure to 70 degrees C at pH 3.0 for 120 min. The enzyme showed Km 4.55 microM and Vmax 0.833 microM/min/mg against sodium phytate as substrate.
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