Encapsulation of pectinase within polyacrylamide gel: characterization of its catalytic properties for continuous industrial uses.

Pectinase as a biocatalyst play a significant role in food and textile industries. In this study, the pectinase was immobilized by encapsulation within polyacrylamide gel to enhance its catalytic properties and ensure the reusability for continuous industrial processes. 9.5% acrylamide and 0.5% N, N'- methylenebisacrylamide concentration gave high percentage of pectinase immobilization yield within gel. The catalytic properties of immobilized pectinase was determined with comparison of soluble pectinase. The immobilization of pectinase within polyacrylamide gel didn't effect catalytic properties of pectinase and both the free and immobilized pectinase showed maximum pectinolytic activity at 45 °C and pH 10. The Michaelis-Menten kinetic behavior of pectinase was slightly changed after immobilization and immobilized pectinase showed somewhat higher Km and lower Vmax value as compared to soluble pectinase. Polyacrylamide gel encapsulation enhanced the thermal stability of pectinase and encapsulated pectinase showed higher thermal stability against various temperature ranging from ranging from 30 °C to 50 °C as compared free pectinase. Furthermore, the surface topography of polyacrylamide gel was analyzed using scanning electron microscopy and it was observed that the surface topography of polyacrylamide gel was changed after encapsulation. The encapsulation of pectinase within polyacrylamide gel enhanced the possibility of reutilization of pectinase in various industries and pectinase retained more than 50% of its initial activity even after seven batch of reactions.

Improvement of catalytic properties of starch hydrolyzing fungal amyloglucosidase: Utilization of agar-agar as an organic matrix for immobilization.

In this study, amyloglucosidase was immobilized within agar-agar through entrapment technique for the hydrolysis of soluble starch. Enzymatic activities of soluble and entrapped amyloglucosidase were compared using soluble starch as a substrate. Partially purified enzyme was immobilized and maximum immobilization yield (80%) was attained at 40 gL-1 of agar-agar. Enzyme catalysis reaction time shifted from 5.0 min to 10 min after immobilization. Similarly, a five-degree shift in temperature (60 °C-65 °C) and a 0.5 unit increase in pH (pH-5.0 to pH-5.5) were also observed. Substrate saturation kinetics revealed that Km of entrapped amyloglucosidase increased from 1.41 mg ml-1 (soluble enzyme) to 3.39 mg ml-1 (immobilized enzyme) whereas, Vmax decreased from 947 kU mg-1 (soluble enzyme) to 698 kU mg-1 (immobilized enzyme). Entrapped amyloglucosidase also exhibited significant catalytic performance during thermal and storage stability when compared with soluble enzyme. Reusability of entrapped amyloglucosidase for hydrolysis of soluble starch demonstrated its recycling efficiency up to six cycles which is an exceptional characteristic for continuous bioprocessing of soluble starch into glucose.

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.

Xylan deterioration approach: Purification and catalytic behavior optimization of a novel ß-1,4-d-xylanohydrolase from Geobacillus stearothermophilus KIBGE-IB29.

The ß-1,4-d-xylanohydrolase is an industry valuable catalytic protein and used to synthesize xylooligosaccharides and xylose. In the current study, ß-1,4-d-xylanohydrolase from Geobacillus stearothermophilus KIBGE-IB29 was partially purified up to 9.5-fold with a recovery yield of 52%. It exhibited optimal catalytic activity at pH-7.0 and 50 °C within 5 min. Almost 50% activity retained at pH-4.0 to 9.0 however, 70% activity observed within the range of 40 °C to 70 °C. The ß-1,4-d-xylanohydrolase showed a significant hydrolytic pattern with 48.7 kDa molecular mass. It was found that the enzymatic activity improved up to 160% with 1.0 mM ethanol. Moreover, the activity of enzyme drastically increased up to 2.3 and 1.5 fold when incubated with Tween 80 and Triton X-100 (1.0 mM), respectively. The ß-1,4-d-xylanohydrolase also retained 72% activity at -80 °C after 180 days. Such a remarkable biochemical properties of ß-1,4-d-xylanohydrolase make it possible to forecast its potential use in textile and food industries.

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.

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.

Role of two polysaccharide matrices on activity, stability and recycling efficiency of immobilized fungal amyloglucosidase of GH15 family.

Current study deals with immobilization of amyloglucosidase using two different strategies (entrapment and covalent binding). Chitosan beads were prepared using neutralization method while alginate beads were synthesized by simple gelation. Results of this study showed that percent recovery of amyloglucosidase after covalent binding was 85% however in case of entrapment it was 66%. Immobilization was optimized by standardizing various conditions including concentrations of polysaccharide (alginate: 4%; chitosan: 3%), divalent ions (0.2M) and glutaraldehyde (5%). Slight shift in catalytic efficiency of soluble amyloglucosidase in terms of reaction time, pH and temperature was also noticed after immobilization. Activation energy decreased after immobilization due to which stability of amyloglucosidase increased for longer time period as compared to soluble enzyme. Results of recycling studies showed that covalently bound amyloglucosidase retained more enzymatic activity even after 15 cycles as compared to the entrapped enzyme that lost its activity within 10 cycles.

Immobilization of pectin depolymerising polygalacturonase using different polymers.

Polygalacturonase catalyses the hydrolysis of pectin substances and widely has been used in food and textile industries. In current study, different polymers such as calcium alginate beads, polyacrylamide gel and agar-agar matrix were screened for the immobilization of polygalacturonase through entrapment technique. Polyacrylamide gel was found to be most promising one and gave maximum (89%) immobilization yield as compared to agar-agar (80%) and calcium alginate beads (46%). The polymers increased the reaction time of polygalacturonase and polymers entrapped polygalacturonases showed maximum pectinolytic activity after 10 min of reaction as compared to free polygalacturonase which performed maximum activity after 5.0 min of reaction time. The temperature of polygalacturonase for maximum enzymatic activity was increased from 45°C to 50°C and 55°C when it was immobilized within agar-agar and calcium alginate beads, respectively. The optimum pH (pH 10) of polygalacturonase was remained same when it was immobilized within polyacrylamide gel and calcium alginate beads, but changed from pH 10 to pH 9.0 after entrapment within agar-agar. Thermal stability of polygalacturonase was improved after immobilization and immobilized polygalacturonases showed higher tolerance against different temperatures as compared to free enzyme. Polymers entrapped polygalacturonases showed good reusability and retained more than 80% of their initial activity during 2nd cycles.

NMR analysis of the binding mode of two fungal endo-ß-1,4-mannanases from GH5 and GH26 families.

The enzymatic digestion of the main components of lignocellulosic biomass, including plant cell wall mannans, constitutes a fundamental step in the renewable biofuel production, with great potential benefit in the industrial field. Despite several reports of X-ray structures of glycoside hydrolases, how polysaccharides are specifically recognized and accommodated in the enzymes binding site still remains a pivotal matter of research. Within this frame, NMR spectroscopic techniques provide key binding information, complementing and/or enhancing the structural view by X-ray crystallography. Here we provide deep insights into the binding mode of two endo-ß-1,4 mannanases from the coprophilous ascomycete Podospora anserina, PaMan26A and PaMan5A, involved in the hydrolysis of plant cell wall mannans and heteromannans. The investigation at a molecular level of the interaction between the wild-type enzymes and inactive mutants with manno-oligosaccharides, revealed a different mode of action among the two glycoside hydrolases most likely due to the presence of the additional and peculiar -4 subsite in the PaMan26A binding pocket.

Agar-agar entrapment increases the stability of endo-ß-1,4-xylanase for repeated biodegradation of xylan.

Microbial xylanases, specially endo-ß-1,4-xylanase catalyzes the hydrolysis of xylan, is considered one of the most significant hydrolases. It has numerous applications but most extensively is utilized in paper and pulp industry as a bio-bleaching agent. Immobilization technique is comprehensively studied with the expectation of modifying and improving enzyme stability and characteristics for commercial purposes. Currently, matrix entrapment technique is applied to immobilize endo-ß-1,4-xylanase within agar-agar gel beads produced by Geobacillus stearothermophilus KIBGE-IB29. Maximal enzyme immobilization yield was achieved at 2.5% of agar-agar concentration. Optimized conditions demonstrated an increase in the optimal reaction time from 05 min to 30 min and incubation temperature from 50 °C to 60 °C with reference to free enzyme whereas; no effect was observed for optimum pH. Entrapment technique uniquely changed the kinetic parameters of immobilized endo-ß-1,4-xylanase (Km: 0.5074 mg min(-1) to 0.5230 mg min(-1) and Vmax: 4773 U min(-1) to 968 U min(-1)) as compared to free enzyme. However, immobilized enzyme displayed broad thermal stability and retained 79.0% of its initial activity at 80 °C up to 30 min whereas; free enzyme completely lost its activity at this temperature. With respect to economic feasibility, the immobilized enzyme showed impressive recycling efficiency up to six reaction cycles.

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix.

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme-substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in Vmax of immobilized enzyme from 8411.0 to 4919.0 U ml-1 min-1 whereas, Km apparently increased from 1.71 to 3.17 mM ml-1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.

Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic Bacillus sp.

Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic Bacillus sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca2+ and Mn2+ enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn2+, K+, Ba2 +, Co2 +, Hg2+ and Cu2+ significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride) completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80) and other commercial detergents (SDS, Triton X-100).

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.

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.

Partial purification and some properties of alpha-amylase from Bacillus subtilis KIBGE-HAS.

An extracellular alpha-amylase from Bacillus subtilis KIBGE-HAS was partially purified by ultrafiltration and ammonium sulphate precipitation with 19.2-fold purification and specific activity of 4195 U/mg. The enzyme showed relatively high thermostability and retained 62% of its activity when kept at 70 degrees C for 15 min. alpha-Amylase was highly stable at -18 degrees C and loss of activity was very low during stability study. Metal ions like Mn2+ Ca2+, Co2+, K+, Mg2+, and Fe3+ activated the enzyme, while Hg2+ Ba2+, Cu2+, Na+ and Al3+ strongly inhibited the activity. The a-amylase was highly stable in various surfactants and detergents. In the presence of surfactants such as SDS and Triton X-100 the enzyme activity was found 2.9 and 1.8-fold higher respectively than control. The non-ionic detergents (Tween 20 and Tween 80) exhibited slightly inhibitory effect on the enzyme activity.

Production & characterization of a unique dextran from an indigenous Leuconostoc mesenteroides CMG713.

On the basis of high enzyme activity a newly isolated strain of L. mesenteroides CMG713 was selected for dextran production. For maximum yield of dextran, effects of various parameters such as pH, temperature, sucrose concentration and incubation period were studied. L. mesenteroides CMG713 produced maximum dextran after 20 hours of incubation at 30 masculineC with 15% sucrose at pH 7.0. The molecular mass distribution of dextran produced by this strain showed that its molecular mass was about 2.0 million Da. Dextran analysis by (13)C-NMR spectrometry showed no signals corresponding to any other linkages except alpha-(1-->6) glycosidic linkage in the main chain, which has not been reported before. Physico-chemical properties of this unique dextran were also studied. These optimised conditions could be used for the commercial production of this unique high molecular weight dextran, which have significant industrial perspectives.

Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4.

Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35 degrees C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25 degrees C with respect to time.

Estimation of total and direct serum bilirubin using modified micro assay method.

Estimation of total and direct bilirubin in serum plays an important role in differential diagnosis of hyperbilirubinemia. Several direct spectrophotometric methods are commercially available for total and direct bilirubin estimation in which the amount of the sample (serum) varies from 200 ml to 800 ml. It is difficult to collect such amount of serum from infants, as neonatal jaundice is the most common problem in this age group. To overcome this problem modified micro assay method was developed using dimethylsulfoxide (DMSO). The amount of the serum sample is reduced from 100 ml to 20 ml per test for both total and direct bilirubin. A method comparison study was performed using 100 consecutive serum samples, by modified micro assay method and a reference Jendrassik-Grof method. Total bilirubin in these human serum samples ranged from 0.4-15.0 mg/dl and direct bilirubin ranged from 0.05-12.0 mg/dl. The results conclude that modified micro assay method had significant correlation with r-value of 0.99989 for total serum bilirubin and with r-value of 0.99971 for direct serum bilirubin. Linearity of the method is 20 mg/dl and 15 mg/dl for total and direct bilirubin, respectively. Monoreagent used during the assay is stable for 24 hours at 2-8 degrees C while the kit is stable for one year at 2-8 degrees C. In conclusion this micro assay method is rapid, reliable, simple and accurate for the estimation of total and direct bilirubin with small serum quantities. It is equally reliable for manual; semi automated and automated chemistry analyzers.