Exploration of a three-dimensional matrix as micro-reactor in the form of reactive polyaminosaccharide hydrogel beads using multipoint covalent interaction approach.

OBJECTIVES: Diversity in backbone polymer composition makes hydrogel-based resources open to broad spectrum of applications. Biomacromolecules which have reactive functional groups in their structural frame and can also exhibit hydrogel properties could be utilized in biomedical, pharmaceutical and drug delivery applications after some chemical modifications. RESULTS: Present study aims towards development of chitosan-based hydrogel system crosslinked together with glucosyltransferase. Hydrogel structure worked as an immobilization matrix and as a microreactor system to catalyze the cleavage of a disaccharide. Uniform chitosan hydrogel beads were prepared and dextransucrase was attached using multipoint covalent interaction approach. Strong interaction was developed by linking polymeric hydrogel with the biocatalyst utilizing glutaraldehyde as spacer arms. This bifunctional crosslinking agent performed two important tasks that includes functionalization of hydrogel beads and crosslinking of this activated matrix system with enzyme fragments. Hydrogel beads required 18.0 h crosslinking time with enzyme (6.5 mg ml-1, 189.9 DSU) under specific environment (4 °C, 100 rpm) to saturate all available ends. Enzyme fragments were observed bound with hydrogel beads when screened for surface topology indicating successful crosslinking. Steady state kinetics of crosslinked dextransucrase was studied in detail and it was revealed that it can catalyse sucrose in 30.0 min at 35 °C (pH 5.5) with an energy of activation around 15.23 kJ mol-1 with increased Vmax (785 DSU ml-1) and Km (256 mM) values as compared to soluble enzyme version. Thermal stability of the crosslinked dextransucrase also particularly improved 2.5 fold at 45 °C in comparison with soluble enzyme. Improved catalytic performance suggests that multipoint covalent immobilization protocol adapted using hydrogel system could be tailored as microreactor for catalysis of profitable macromolecules.

Thermodynamics, kinetics and optimization of catalytic behavior of polyacrylamide-entrapped carboxymethyl cellulase (CMCase) for prospective industrial use.

In the current study, kinetic and thermodynamic parameters of free and polyacrylamide-immobilized CMCase were analyzed. The maximum immobilization yield of 34 ± 1.7% was achieved at 11% acrylamide. The enthalpy of activation (ΔH) of free and immobilized enzyme was found to be 13.61 and 0.29 kJ mol-1, respectively. Irreversible inactivation energy of free and immobilized CMCase was 96.43 and 99.01 kJ mol-1, respectively. Similarly, the enthalpy of deactivation (ΔHd) values for free and immobilized enzyme were found to be in the range of 93.51-93.76 kJ mol-1 and 96.08-96.33 kJ mol-1, respectively. Michaelis-Menten constant (Km) increased from 1.267 ± 0.06 to 1.5891 ± 0.07 mg ml-1 and the maximum reaction rate (Vmax) value decreased (8319.47 ± 416 to 5643.34 ± 282 U ml-1 min-1) after immobilization. Due to wide pH and temperature stability profile with sufficient reusing efficiency up to three successive cycles, the immobilized CMCase might be useful for various industrial processes.

Inhibitory mechanism of BAC-IB17 against ß-lactamase mediated resistance in methicillin-resistant Staphylococcus aureus and application as an oncolytic agent.

Cancer remains a foremost cause of deaths worldwide, despite several advances in the medical science. The conventional chemotherapeutic methods are not only harmful for normal body cells but also become inactive due to the development of resistance by cancer cells. Therefore, the demand of safe anticancer agents is increasing and enforced the bottomless research on the bacteriocins. Several studies have reported the selective anticancer property of bacteriocins. Current research is the contribution to explore the exact mechanism of action and in vitro application of bacteriocin (BAC-IB17) as an oncolytic agent. In this study, ß-lactamase mediated resistance of methicillin resistant Staphylococcus aureus (MRSA) was studied and inhibitory mechanism of MRSA by BAC-IB17 was investigated. Cytotoxic studies were conducted to analyze the anticancerous potential of BAC-IB17. Results revealed that BAC-IB17 inhibited the ß-lactamase and produced profound effect on the membrane integrity of MRSA confirmed by scanning electron microscope (SEM). FTIR spectroscopic analysis revealed the changes in the functional groups of bacterial cells before and after treatment with BAC-IB17. BAC-IB17 also found anticancer in nature as it kills HeLa cell lines with the IC50 value of 12.5 µg mL-1 with no cytotoxic effect on normal cells at this concentration. This specific anticancer property of BAC-IB17 will make it a promising candidate for the treatment of cancer after further clinical trials. Moreover, BAC-IB17 may control MDR bacteria responsible for the secondary complications in cancer patients.

Purification and catalytic behavior optimization of lactose degrading ß-galactosidase from Aspergillus nidulans.

The ß-galactosidase is an industrially valuable enzyme and used to hydrolyze the lactose into glucose and galactose. Considering the broad utility profile in food industry, ß-galactosidase from Aspergillus nidulans was purified and characterized in term of its catalytic properties and stability. It displayed highest catalytic efficiency at 60 °C after 10.0 min within acidic pH environment (pH 5). The ß-galactosidase exhibited 100% and 60% catalytic activity at 40 °C and 50 °C, respectively even after 120.0 min. The ß-galactosidase activity was remained stable in the presence of Zn2+, Ni2+, and Mg2+ ions. The activity was also retained in all investigated organic solvents except DMSO at various ionic concentrations. The surfactants Triton X-100 and SDS caused positive impact on the catalytic activity of enzyme at 1.0 mM concentration. However, the percent relative activity of ß-galactosidase was significantly reduced when incubated with EDTA. The molecular mass of ß-galactosidase estimated to be 95 kDa. The SEM micrographs of ONPG before and after ß-galactosidase treatment indicated a remarkable difference in the morphology and proved the strong catalytic strength of enzyme. The ß-galactosidase also demonstrated exceptional storage stability at - 80 °C, - 20 °C and 4 °C by retaining 86, 79 and 70% activity even after 100.0 days.

REPORT- Role of metal ions on the catalytic efficiency of dextran hydrolyzing biocatalyst.

Hydrothermal spring isolate Bacillus megaterium KIBGE-IB31was utilized to produce dextranase. Enzyme was partially purified up to 11.8 fold after dialysis. Different metals ions were tested to explore their behavior with dextranase. It was noticed that cobalt (Co+2), copper (Cu+2), magnesium (Mg+2), manganese (Mn+2), nickle (Ni+2) and zinc (Zn+2) act as activator whilst potassium (K+), sodium (Na+), barium (Ba+), calcium (Ca+), mercury (Hg+), vanadium (V+2), aluminum (Al+3) and ferric (Fe+3) ions display inhibitory action.

Screening, purification and characterization of thermostable, protease resistant Bacteriocin active against methicillin resistant Staphylococcus aureus (MRSA).

BACKGROUND: The emergence of serious issues of multidrug resistance in the past few years have enforced the use of bacteriocins for combating infections. Threat posed to public health by various multidrug resistant (MDR) organisms can be resolved by discovering new antimicrobial proteins with broad spectrum of inhibition. RESULTS: In the current study, Bacteriocin (BAC-IB17) produced by Bacillus subtilis KIBGE-IB17 is found to be effective against different strains of methicillin resistant Staphylococcus aureus (MRSA). The approximate molecular mass of BAC-IB17 is 10.7 kDa. This unique bacteriocin is found to be highly thermostable and pH stable in nature. It also showed its stability against various heavy metals, organic solvents, surfactants and proteolytic enzymes. Amino acid profile of BAC-IB17 clearly showed that this protein mainly consists of non-polar and basic amino acids whereas; some acidic amino acids were also detected. Sequence of first 15 amino acid residues obtained from N-terminal sequencing of BAC-IB17 were NKPEALVDYTGVXNS. CONCLUSIONS: The anti-MRSA property of purified bacteriocin may be used to prevent the spread of MRSA infections. Remarkable features of BAC-IB17 suggests its applications in various pharmaceutical and food industries as it can function under a variety of harsh environmental conditions.

Chitosan hydrogel microspheres: an effective covalent matrix for crosslinking of soluble dextranase to increase stability and recycling efficiency.

Dextranase is a unique biocatalyst that has high specificity and stereo-selectivity towards a complex biopolymer known as dextran. Dextranase has wide industrial application, but most of the time harsh environmental conditions adversely affect the functionality and stability of the enzyme. To overcome this issue, a covalent cross-linking immobilization method was adapted in the current study utilizing a nontoxic and biocompatible matrix known as chitosan. Chitosan hydrogel microspheres were synthesized using chitosan which exhibited noteworthy physical and mechanical strength. After treatment with glutaraldehyde, chitosan hydrogel microspheres were used for immobilization of dextranase. The kinetic characteristics of immobilized dextranase were compared with that of the soluble enzyme. A shift in optimum pH and temperature from 7.0 to 7.5 and 50 to 60 °C was observed after immobilization, respectively. Recycling efficiency, thermal stability, and activation energy distinctly improved after immobilization, whereas anchoring of substrate at the active site of the soluble dextranase exhibited an increase in K m with no change in V max after crosslinking. This technique involves the reduction in the size of carrier molecules (microspheres) that provide a larger surface area for improved immobilization efficiency. Therefore, it is concluded that increased stability and reusability of this immobilized biocatalyst makes it a promising aspirant for the utilization at commercial level.

Hyper-production of levansucrase from Zymomonas mobilis KIBGEIB14 using submerged fermentation technique.

The industrial utilization of enzymes requires the high yield of enzyme production for the synthesis of polymers by microorganisms. Therefore, it is necessary to optimize different production parameters of levansucrase in order to increase its industrial applications. Zymomonas mobilis KIBGE-IB14 was considered as a promising candidate for the large scale production of levan among wide range of microorganisms. The current investigation is aimed to optimize the production parameters of levansucrase by Z. mobilis KIBGE-IB14 isolated from molasses. The results indicated that bacterial growth as well as enzyme production was greatly influenced by both physical and chemical conditions. It was revealed that high enzyme titers were achieved at 30°C with pH 6.5 after 24 hours of incubation in a modified medium. Moreover, the enzyme exhibited its induction in the presence of sucrose used as a substrate. Thus, the present study demonstrated that newly isolated Z. mobilis KIBGE-IB14 can be used as a plausible producer of levansucrase for industrial applications.

Influence of different metals on the activation and inhibition of α-amylase from thermophilic Bacillus firmus KIBGE-IB28.

Thermophilic Bacillus firmus KIBGE-IB28 produced extracellular α-amylase at temperature 70°C. Enzyme was partially purified by ammonium sulfate precipitation with 42.80 fold purification and specific activity of 1889.6 U/mg. Effects of various metals on enzyme activity were determined and it was found that enzyme activity boosted significantly in presence of Ca(2+), K(2+), Ba(2+), Co(2+) and Ni(2+) whereas Zn(2+), Mg(2+), Na(2+) and Cu(2+) were found inhibitory at concentration 10mM.

Calcium alginate matrix increases the stability and recycling capability of immobilized endo-ß-1,4-xylanase from Geobacillus stearothermophilus KIBGE-IB29.

Exploration of microbial pool from extremely diversified ecosystem is significantly important for various industrial applications. Bacterial communities from extreme habitats including volcanic vents, hot springs, and industrial sectors are eagerly explored for the isolation of thermophiles. Geobacillus stearothermophilus KIBGE-IB29, isolated from blast furnace site of a steel processing industry, is capable of producing thermostable endo-ß-1,4-xylanase. In the current study, this enzyme was immobilized within calcium alginate beads using entrapment technique. Amalgamation of sodium alginate (40.0 gL(-1)) and calcium chloride (0.4 M) was used for the formation of immobilized beads. It was observed that temperature (50 °C) and pH (7.0) optima of immobilized enzyme remained same, but enzyme-substrate reaction time increased from 5.0 to 30.0 min as compared to free enzyme. Diffusion limit of high molecular weight xylan (corncob) caused a decline in V max of immobilized enzyme from 4773 to 203.7 U min(-1), whereas K m value increased from 0.5074 to 0.5722 mg ml(-1) with reference to free enzyme. Immobilized endo-ß-1,4-xylanase showed its stability even at high temperatures as compared to free enzyme and retained 18 and 9 % residual activity at 70 and 80 °C, respectively. Immobilized enzyme also exhibited sufficient recycling efficiency up to five reaction cycles which indicated that this enzyme can be a plausible candidate in paper and pulp industry.

Continuous degradation of maltose: improvement in stability and catalytic properties of maltase (α-glucosidase) through immobilization using agar-agar gel as a support.

Maltose degrading enzyme was immobilized within agar-agar support via entrapment method due to its industrial utilization. The maximum immobilization efficiency (82.77%) was achieved using 4.0% agar-agar keeping the diameter of bead up to 3.0 mm. The matrix entrapment showed maximum catalytic activity at pH 7.0 and temperature 65 °C. Substrate saturation kinetics showed that the K m of immobilized enzyme increased from 1.717 to 2.117 mM ml(-1) where as Vmax decreased from 8,411 to 7,450 U ml(-1 )min(-1) as compared to free enzyme. The immobilization significantly increased the stability of maltase against various temperatures and immobilized maltase retain 100% of its original activity after 2 h at 50 °C, whereas the free maltase only showed 60% residual activity under same condition. The reusability of entrapped maltase showed activity up to 12 cycles and retained 50% of activity even after 5th cycle. Storage stability of agar entrapped maltase retain 73% of its initial activity even after 2 months when stored at 30 °C while free enzyme showed only 37% activity at same storage conditions.

Plasmid borne BAC-IB17: Localization of a potential antibacterial positive marker (Bac+) encoded broad inhibitory spectrum bacteriocin.

Production of antimicrobial compounds is considered as ubiquitous anti-competitor strategy in bacterial ecosystem. Bacteriocins are heterogeneous; highly specific and efficient anti-competitor agents and the gene responsible for the production of bacteriocins mostly exist in an autosomal state and associated with plasmids. BAC-IB17 is a broad spectrum bacteriocin and its production was observed at different stages of the growth cycle from Bacillus subtilis KIBGE-IB17. Growth kinetics of B. subtilis KIBGE-IB17 along with the production of BAC-IB17 showed that it exhibited secondary metabolite kinetics. Plasmid curing technique revealed that the gene responsible for the bacteriocinogenecity in B. subtilis KIBGE-IB17 was located on the plasmid of the bacterium. Overlay method also demonstrated the plasmid-mediated bacteriocinogenesis of the isolated colonies. With the advancement in genomics and proteomics, the plasmid borne BAC-IB17 can play a significant role in the transfer of bacteriocinogenic factor to other incompetent cells and also in the maintenance of plasmid in bacterial population.

Role of nutrients and environmental conditions for the production of dextransucrase from L. mesenteroides KIBGE-IB26.

The bacterial strains capable of producing dextransucrase enzyme were isolated from different fruits and vegetables sources. In primary screening, five strains were selected on the basis dextransucrase production and among them L. mesenteroides KIBGE- IB26 isolated from bottle gourd (Lagenaria Vulgaris) was selected for further studies. For the enhancement of enzyme production, different physicochemical parameters were optimized. Maximum production of dextransucrase was achieved after 06 hrs using sucrose (20.0 g/l) as a substrate at 25°C. Maximum dextransucrase production was achieved when medium pH was kept 7.5 before sterilization. In addition, medium was also supplemented with CaCl2 and K2HPO4 and maximum enzyme production was achieved at 0.0025 g/dl calcium chloride and 2.0 g/dl K2HPO4with enzyme activity of 87 DSU/ml/hr. Production of dextransucrase in shorter period of time makes this strain an attractive candidate for commercial production of dextransucrase.

Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process.

BACKGROUND: Cassava starch is considered as a potential source for the commercial production of bioethanol because of its availability and low market price. It can be used as a basic source to support large-scale biological production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to produce free glucose which can be utilized to produce bioethanol by microbial fermentation. RESULTS: In the present study, several filamentous fungi were screened for production of amylases and among them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase, amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt precipitation, fold purification of α-amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific activity of 9.2 kUmg⁻¹ and 393 kUmg⁻¹, respectively. Concentrated amylolytic enzyme mixture was incorporated in cassava starch slurry to give maximum glucose formation (40.0 gL⁻¹), which was further fermented using Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave 53.0% yield. CONCLUSION: An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production.

Enhanced production of maltase (α-glucosidase) from newly isolated strain of Bacillus licheniformis KIBGE-IB4.

Maltase (alpha-glucosidase) hydrolyzes α-(1→4) glucosidic bond of maltose into two glucose molecules. It is widely used in various foods, beverages and also in textile and biofuel industries. During current study, various physicochemical parameters for maltase production from newly isolated strain of Bacillus licheniformis KIBGE-IB4 were optimized using one-factor-at-a-time methodology. It was found that Bacillus licheniformis KIBGE-IB4 produced maximum maltase at 37°C, pH-7.0 after 48 hours using wheat starch (2.5%) as carbon source along with peptone (1.0%), yeast extract (0.2%) and meat extract (0.4%) as nitrogen sources in fermentation medium. It is concluded that the optimization of various medium ingredients and conditions increases maltase production upto 6.74 fold from B. licheniformis KIBGE-IB4 as compared to previously reported media and this strain could be used for the commercial production of maltase.

Optimization of growth conditions for the isolation of dextran producing Leuconostoc spp. from indigenous food sources.

Leuconostoc are known to produce dextran, which have great commercial importance in chemical, medical and food industry. The present study is an attempt to select the best medium for the isolation of indigenous dextran producing Leuconostoc, measuring their enzyme activities for dextransucrase, production of dextran and identification of dextran producing Leuconostoc CMG706, CMG707, CMG710 and CMG713. Since, dextran producing Leuconostoc produce slimy colonies, twenty-four slime producing bacterial strains were isolated from different food sources, fruits and vegetables. Three different isolation medium were evaluated for the isolation of Leuconostoc only and the best one was found to be one containing sucrose and sodium azide. Further, all slime producing bacterial strains were screened for enzyme activity of dextransucrase, which is responsible for dextran production. Four bacterial strains CMG706, CMG707, CMG710 and CMG713 giving high enzyme activities were selected for dextran production and identified.

Isolation and characterization of different strains of Bacillus licheniformis for the production of commercially significant enzymes.

Utilization of highly specific enzymes for various industrial processes and applications has gained huge momentum in the field of white biotechnology. Selection of a strain by efficient plate-screening method for a specific purpose has also favored and boosted the isolation of several industrially feasible microorganisms and screening of a large number of microorganisms is an important step in selecting a potent culture for multipurpose usage. Five new bacterial isolates of Bacillus licheniformis were discovered from indigenous sources and characterized on the basis of phylogeny using 16S rDNA gene analysis. Studies on morphological and physiological characteristics showed that these isolates can easily be cultivated at different temperatures ranging from 30°C to 55°C with a wide pH values from 3.0 to 11.0 All these 05 isolates are salt tolerant and can grow even in the presences of high salt concentration ranging from 7.0 to 12.0%. All these predominant isolates of B. licheniformis strains showed significant capability of producing some of the major industrially important extracellular hydrolytic enzymes including α-amylase, glucoamylase, protease, pectinase and cellulase in varying titers. All these isolates hold great potential as commercial strains when provided with optimum fermentation conditions.

Expression of sucrose metabolizing enzymes in different sugarcane varieties under progressive heat stress.

Studying the thermal stress effect on sucrose-metabolizing enzymes in sugarcane is of great importance for understanding acclimation to thermal stress. In this study, two varieties, S2003-US-633 and SPF-238, were grown at three different temperatures ( ± 2°C): 30°C as a control, 45°C for various episodes of high temperature treatments and recovery conditions at 24, 48 and 72 hours. Data showed that reducing sugar content increased until the grand growth stage but sharply declined at the maturity stage in both cultivars. On the other hand, sucrose is enhanced only at the maturity stage. The expression of all invertase isozymes declined prominently; however, the expression of SPS was high at the maturity stage. Hence, the sucrose accumulation in mature cane was due to increased SPS activity while decreased invertase isozymes (vacuolar, cytoplasmic and cell wall) activities at maturity stage in both cultivars. Heat shock decreased the sucrose metabolizing enzymes, sucrose content and sugar recovery rate in both cultivars. In contrast, heat-shock treatments induced maximum proline, MDA, H2O2 and EC in both cultivars. Notably, this is the first report of diverse invertase isozyme molecular weight proteins, such as those with 67, 134 and 160 kDa, produced under heat stress, suggesting that these enzymes have varied activities at different developmental stages. Overall, S2003-US-633 performs better than the cultivar SPF-238 under heat stress conditions at all development stages, with increased sucrose content, enzyme expression, proline and sugar recovery rate. This work will provide a new avenue regarding sugarcane molecular breeding programs with respect to thermal stress.

Bacteriocin (BAC-IB17): screening, isolation and production from Bacillus subtilis KIBGE IB-17.

Bacteriocins are peptides produced by a variety of different microbes and have antimicrobial activity against closely related species. These antimicrobial agents are gaining more and more attention as an alternative therapeutics not only in pharmaceutical but also as a preservative in food industries. In this study several bacterial strains were isolated from soil and screened for bacteriocin production. Among them, one strain identified as Bacillus subtilis KIBGE IB-17 on the basis of taxonomic studies and confirmed by 16S rDNA analysis. This newly isolated strain showed antibacterial activity against several Gram positive and Gram negative bacteria. Different concentrations of tryptone, yeast extract and NaCl and physiochemical factors such as temperature, pH and incubation period were selected as variables for maximum production of bacteriocin by using agar well diffusion method and significant effects of variables were observed on the production of Bac-IB17. A newly designed modified TY medium showed maximum bacteriocin production containing 1.0% tryptone, 0.5% yeast extract and 0.5% NaCl. Maximum Bac-IB17 production was observed at 37° after 24 hours with initial medium pH 7.0. Bacillus subtilis KIBGE IB-17 is capable of producing a bacteriocin at a wide range of pH and temperature that makes it an ideal strain that can be used for the production of bacteriocin on industrial scale level. The identification and production of such bacteriocin like compound against a wide spectrum of microbial species is very important for food and pharmaceutical industry.

Purification and characterization of novel α-amylase from Bacillus subtilis KIBGE HAS.

Purification of extracellular α-amylase from Bacillus subtilis KIBGE HAS was carried out by ultrafiltration, ammonium sulfate precipitation and gel filtration chromatography. The enzyme was purified to homogeneity with 96.3-fold purification with specific activity of 13011 U/mg. The molecular weight of purified α-amylase was found to be 56,000 Da by SDS-PAGE. Characteristics of extracellular α-amylase showed that the enzyme had a Km and V (max) value of 2.68 mg/ml and 1773 U/ml, respectively. The optimum activity was observed at pH 7.5 in 0.1 M phosphate buffer at 50 °C. The amino acid composition of the enzyme showed that the enzyme is rich in neutral/non polar amino acids and less in acidic/polar and basic amino acids. The N-terminal protein sequence of 10 residues was found to be as Ser-Ser-Asn-Lys-Leu-Thr-Thr-Ser-Trp-Gly (S-S-N-K-L-T-T-S-W-G). Furthermore, the protein was not N-terminally blocked. The sequence of α-amylase from B. subtilis KIBGE HAS was a novel sequence and showed no homology to other reported α-amylases from Bacillus strain.