Purity Fold Research Articles

Sustainable production and preparative purification of thermostable alkaline α-amylase by Bacillus simplex (ON754233) employing natural deep eutectic solvent-based extractive fermentation

Using PEG-based deep eutectic solvents (PDES), the current study proposes extractive fermentation as a sustainable process integration for the production and purification of α-amylase from Bacillus simplex (ON754233). Glucose: PEG 400 outperformed five PDES in terms of tie lie length (58) and slope value (1.23) against sodium sulphatt. Apple cider pomace was used as a low-cost, sustainable carbon source to produce-amylase, with a maximum enzyme production of 2200.13 U/mL. PDES concentration (20% w/v), salt (12.75 w/v), and apple waste (2.75 g/mL) were all optimized using response surface methodology. When scaled upto 3 L benchtop bioreactor, extractive fermentation was proved to be better technology with maximum recovery of 92.4% with highest partition coefficient (3.59). The partially purified enzyme was further purified using a Sephadex G 100 followed by DEAE-Sephadex anion exchange chromatography with a purity fold of 33. The enzyme was found to be thermostable at the temperature (60 °C), remains alkaline (pH 8), and the activity was stimulated in the presence of Mg2+ ions. With SDS PAGE electrophoresis, the molecular weight was found to be around 140 kDa. Finally, the enzyme kinetics parameters were evaluated with observed Km (0.00396 mM) and Vmax (37.87 U/mL). Thus scaling up extractive fermentation entails increasing production capacity with improved extraction efficiency using green solvents.

Sustainable eutectic mixture-based ultrasound assisted multifaceted valorisation of pineapple waste for bromelain and bioethanol production

A large number of pineapple residues like peels, leaves and stems generated during processing remains underutilized. The current investigation provides one-pot task-specific valorisation of these residues by sequential extraction of bromelain and saccharification of cellulose residue for bioethanol production. Employing natural deep eutectic solvents (NADES) prepared by mixing ammonium salts with imidazole and glycerol has guaranteed sustainable extraction of bromelain. Ultrasound-assisted liquid phase microextraction (UA-LPME) was performed and NADES with moderate density (TBC:I:G/sodium sulphate) was observed to have a higher bromelain yield and purity. Response Surface Methodology-based optimization of UA-LPME was carried out and solid: liquid ratio (25 ml/g), ultrasound time (17.5 min) and ultrasound temperature (45 °C) were observed to be influential variables resulting in an optimal bromelain yield of (87%). Sequential fractionation of the enzyme concentrate with gel filtration chromatography and anion exchange chromatography has resulted in a corresponding purity fold of 89.27. The cellulose-rich residue remaining after bromelain extraction was enzymatically saccharified (Cellic CTec2) for bioethanol production (12.7 g/L). This novel approach guarantees sustainable valorisation of pineapple waste using NADES as a sustainable extraction medium.

Switchable deep eutectic solvent driven micellar extractive fermentation of ultrapure fibrin digesting enzyme from Bacillus subtilis

Fibrinolytic protease (FLP) is a therapeutic enzyme used in the treatment of thrombolytic diseases. The present study proposed the concept of pH-driven swappable micellar two-phase extraction for the concurrent production and purification of FLP from Bacillus subtilis at cloud point extraction. Extractive fermentation was carried out with a pH swap mechanism and FLP was extracted to the top phase by surfactant deep eutectic solvents (SDES). Shrimp waste was chosen as a sustainable low-cost substrate that yielded a maximum protease of 185 U/mg. Six SDESs were synthesized with nonionic surfactants as hydrogen bond donors and quaternary ammonium salts as hydrogen bond acceptors and their association was confirmed by H1 NMR. Thermophysical investigation of the synthetic SDES was accomplished as a function of temperature. Response surface methodology for extractive fermentation was performed with the concentration of SADES (35% w/v), Na2SO4 (15% w/v) and pH (6.3) as variables and the enzyme activity (248 IU/mg) as a response. Furthermore, purification using gel filtration chromatography was used to quantify the amount of enzyme obtained in the extraction phase (849 IU/ml). After final purification with an anion exchange column, the maximum purity fold (22.32) with enzyme activity (1172 IU/ml) was achieved. The in-vitro fibrinolytic activity has been confirmed using a fibrin plate assay.

Sustainable valorization of papaya peels for thrombolytic cysteine protease isolation by ultrasound assisted disruptive liquid phase microextraction with task specific switchable natural deep eutectic solvents

The present investigation deals with task-specific purification of thrombolytic cysteine protease from Carica papaya peels using switchable eutectic solvent-based ultrasound-assisted dispersive liquid-phase microextraction. Four switchable polarity natural deep eutectic solvents were synthesized and their thermo-physical profile was recorded as a function of temperature. Ultrasound-assisted liquid phase microextraction was performed yielding 33.2 mg/g of papaya protease. Kinetic and thermodynamic modeling of the extraction process signifies that first-order rate laws describe the extraction process with an optimal time of 15 min. Ultrapure fractions were obtained from copper immobilized metal affinity chromatography resulting in a purity fold of 48.6 and specific enzyme activity of 112.5. The thrombolytic activity of the resulting ultrapure protease fraction was determined by the blood clot digestion assay yielding 4126 U/mg of thrombolytic papain.

Sustainable valorization of meat processing wastewater with synergetic eutectic mixture based purification of R-Phycoerythrin from porphyrium cruentium

Wastewater effluent from meat processing industries are enriched with nutrients but remain underutilized. Therefore, an efficient method was administered by employing this resource for the cultivation of Porphyreum cruentium. The cultured biomass was exposed to a one-step disruption and selective purification method using a protic deep eutectic solvent to obtain a commercially significant pigment, R-Phycoerythrin (R-PE). Six protic deep eutectic solvents (pDES) were synthesized and their thermophysical activity determined. A synergy between microwave and cavitation (MACE-DLPME) has been achieved for the effective recovery of these membrane proteins. The addition of pDES provides the selective medium for the concentration of R-PE. Optimization of the MACE-DLPME method yields 95.9% (w/w) of R-PE. Preparative size exclusion chromatography resulted in (28 µg/g) of R-PE. Further ultra-purification by anion exchange chromatography enhances the purity fold of R-PE to 125. The resulting ultrapure fraction exhibits enhanced anti-platelet activity (1.56 mg/g ascorbic acid equivalent).

One-pot simultaneous production and sustainable purification of fibrinolytic protease from Bacillus cereus using natural deep eutectic solvents

The present study report for the first time on the one-pot production and purification of fibrinolytic protease from Bacillus cereus by extractive fermentation using natural deep eutectic solvents (NADES). Cheese whey was chosen as a sustainable low-cost production alternative yielding a significantly high amount of protease (185.7 U/mg). Five natural deep eutectic solvents with menthol as hydrogen bond donor and sugar molecules as corresponding hydrogen bond acceptors were synthesized and their association was confirmed with H1 NMR. Thermophysical investigation of the synthetic NADES was accomplished as a function of temperature to define their extraction ability. Response surface methodology based optimization of concentration of NADES (77.5% w/w), Na2SO4 (14% w/v) and cheese whey (1% w/w) were accomplished for extractive fermentation. Further, preparative purification using size exclusion chromatography was used to quantify the amount of enzyme obtained in the extraction phase (190 U/ml). On subsequent purification with an anion exchange column, the maximum purity fold (21.2) with enzyme activity (2,607.8 U/ml) was attained. The optimal pH (8.0), temperature (50 °C) were determined and the in-vitro fibrinolytic activity has been confirmed using a fibrin plate assay.

Proteomic analysis reveals the damaging role of low redox laccase from Yersinia enterocolitica strain 8081 in the midgut of Helicoverpa armigera.

Earlier, we have found that the enteropathogenic Yersinia enterocolitica have evolved the survival mechanisms that regulate the expression of laccase-encoding genes in the gut. The present study aims to characterize the purified recombinant laccase from Y. enterocolitica strain 8081 biovar 1B and understand its effect on the midgut of cotton bollworm, Helicoverpa armigera (Hübner) larvae. The recombinant laccase protein showed high purity fold and low molecular mass (~ 43kDa). H. armigera larvae fed with laccase protein showed a significant decrease in body weight and damage in the midgut. Further, transmission electron microscopy (TEM) studies revealed the negative effect of laccase protein on trachea, malpighian tubules, and villi of the insect. The proteome comparison between control and laccase-fed larvae of cotton bollworm showed significant expression of proteolytic enzymes, oxidoreductases, cytoskeletal proteins, ribosomal proteins; and proteins for citrate (TCA cycle) cycle, glycolysis, stress response, cell redox homeostasis, xenobiotic degradation, and insect defence. Moreover, it also resulted in the reduction of antioxidants, increased melanization (insect innate immune response), and enhanced free radical generation. All these data collectively suggest that H. armigera (Hübner) larvae can be used to study the effect of microbes and their metabolites on the host physiology, anatomy, and survival.

Cost effective characterization process and molecular dynamic simulation of detergent compatible alkaline protease from Bacillus pumilus strain MP27

Abstract A psychrothermotolerant alkaline protease isolated from Bacillus pumilus MP27 with a molecular mass ∼53 kDa was isolated from Southern ocean water samples. It was partially purified by single step TPP with purity fold of 16.65. The enzyme was found to be widely stable within a range of temperature and pH, maintaining 52.25% of its activity at 50 °C and 92% at pH 12. The enzyme exhibited an exceptional activity along with tested detergents, showing 98% stability with SDS (10 mg/ml) and 99% stability with Tide detergent (7 mg/ml). Further, the alkaline protease gene of 1152 bp was successfully cloned in pGEM-T Easy vector in E. coli DH5α. The gene sequence was further translated, modeled and molecular dynamic simulation was performed. The modeled protein was highly unstable during the first 5 ns and therefore could not able to form bonds with the ligand after 1 ns of simulation.

Characterization of Genetically Engineered Linamarase (β-glucosidase) from Saccharomyces cerevisiae

The characterization parameters of genetically engineered linamarase (β-glucosidase) from Saccharomyces cerevisiae due to action of the enzyme on linamarin as influenced by degree of purification, pH and temperature were investigated. Commercial native linamarase (CNLIN) was used as control. Linamarase genes (chromosomal DNA) and plasmids (circular DNA) isolated from bitter cassava and yeast respectively were restricted and ligated to produce recombinant genes (r-DNA). The r-DNA were introduced into the nucleus of CaCl2 induced competent Saccharomyces cerevisiae cells which transformed into strains capable of producing genetically engineered linamarase (GELIN). Recombinant S. cerevisiae cells at the stationary phase of growth were recovered, homogenized and centrifuged to obtain crude extracts designated as GELIN0. Carboxy methyl cellulose, diethyl amino-ethyl-sephadex and diethyl amino-ethyl-cellulose were used to purify the crude extracts resulting in GELIN1, GELIN2 and GELIN3, respectively. The physical characterization parameters of the enzyme extracts such as impurity levels, molecular weights (Mwt), number of isoenzyme, sulphur amino acids (methionine and cysteine) and the electrical charges were evaluated using standard methods. The ability of the enzyme extracts and a commercial native linamarase (CNLIN) to hydrolyse cyanogenic glucosides was challenged using linamarin (cassava) as substrates for characterization of activity kinetic profiles such as optimum pH (pHopt), temperature (Topt), total activity, specific activity, purity fold, yield and efficiency ratio. The results indicated that the genetically engineered linamarase(β-glucosidase) consisted of 3 isoenzyme forms. Purification conferred different ionic charges of zero to GELIN0, unit positive charge GELIN1, and unit negative charge to GELIN2 and GELIN3 respectively. Ranges for other parameters were Mwt (22,000-26,000 Daltons), insoluble protein impurity (0.4 -3.5 mg/100g sample) and purity fold (11.5 -1.0) for GELIN3 - GELIN0). Methionine and cystiene varied from 2.0 to 2.6% and 3.0 to 20% respectively (CNLIN - GELIN3). The native commercial enzyme (CNLIN) acted only at pH 6.8 on linamarin with pHopt and Topt of 6.8 and 35 oC respectively. The wide pH tolerance and specific activity towards linamarin degradation suggest a possible use of the genetically engineered linamarase from S. cerevisiae in detoxification of cassava for increased production exportation of cassava-based food products.

Concentration and purification of α-galactosidase from watermelon (Citrullus vulgaris) by three phase partitioning

Three-phase partitioning (TPP) was used to concentrate and purify α-galactosidase from watermelon (Citrullus vulgaris). The various process parameters required for efficient purification of α-galactosidase were optimized to get highest purity fold and yield. The best α-galactosidase yield (76.7%) with a nearly 2.7-fold purification was obtained in the interphase of the TPP system, which consisted of the crude extract to t-butanol ratio of 1:1 (v/v) in the presence of 50% (w/v) (NH4)2SO4 at pH 5.5. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis revealed a substantial level of purification of α-galactosidase from watermelon. The molecular weight of the enzyme was determined approximately as 45kDa. The purified enzyme was characterized with respect to its activity and stability. Various parameters (temperature, pH and substrate concentration) affecting to the enzyme activity and stability were studied. Optimum pH and temperature of α-galactosidase were determined as pH 6.0 and 60°C, respectively. The purified enzyme was also very stable at a temperature range of 4–50°C and a pH range of 4.0–6.5. The Km and Vmax values were calculated from Lineweaver–Burk plot as 0.14mM and 0.12U, respectively. The results indicated that, TPP is a simple, quick, economical and very attractive process for primary purification of α-galactosidases compared to conventional chromatographic protocols.

Purification and characterization of exo-β-agarase from an endophytic marine bacterium and its catalytic potential in bioconversion of red algal cell wall polysaccharides into galactans

An extracellular exo-β-agarase was characterized from an endophytic bacterial strain Pseudomonas sp. isolated from the red alga Gracilaria dura. The enzyme was purified to homogeneity with a recovery of 28.2% and a purity fold of 8.33. The purified enzyme was composed of single polypeptide with a molecular mass of about 66 kDa. The enzyme exhibited a maximum activity of 81.74 U mL−1 and a specific activity of 615.5 U mg−1. The optimal pH and temperature for its maximum activity were 9.0 and 35 °C respectively. The enzyme stabilized its activity in alkaline pH 7–11 and high salt concentration up to 4 mol dm−3. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose while the bacterium when incubated with G. dura biomass yielded galactose 20% on dry wt basis. The agarolytic ability of the former was further confirmed by release of protoplasts from G. dura tissue through digestion of cell wall polysaccharides. The bacterium investigated in this study could possibly be used for bioconversion of marine red algal polysaccharides into energy feedstock and the purified enzyme for preparation of compounds having pharmaceutical importance.

Purification and characterization of haloalkaline thermoactive, solvent stable and SDS-induced protease from Bacillus sp.: A potential additive for laundry detergents

An extracellular haloalkaline, thermoactive, solvent stable, SDS-induced serine protease was purified and characterized from an alkali-thermo tolerant strain Bacillus sp. SM2014 isolated from reverse osmosis reject. The enzyme was purified to homogeneity with recovery of 54.4% and purity fold of 64. The purified enzyme was composed of single polypeptide of molecular mass about 71kDa. The enzyme showed optimum activity at alkaline pH 10 and temperature 60°C. The km and Vmax for the enzyme was 0.57mg/ml and 445.23U/ml respectively. The enzyme showed novel catalytic ability at high pH (10), temperature (60°C) and salinity (3M). Moreover, the stability of enzyme in organic solvents (50% v/v) of logP⩾2 signified the prospective of this enzyme for peptide synthesis. The compatibility of the enzyme with surfactants and various detergent matrices together with wash performance test confirmed its potential applicability in laundry industry.

Purification of α-galactosidase from pepino ( Solanum muricatum) by three-phase partitioning

Three-phase partitioning (TPP) is a bioseparation technique used for the extraction, concentration and purification of biomolecules. The technique consists of simultaneous addition of a salt (generally ammonium sulfate) to the crude extract followed by the addition of an organic solvent (generally t-butanol). In the present study, α-galactosidase was purified from pepino ( Solanum muricatum) fruit by TPP. The influence of various process parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) on α-galactosidase partitioning is investigated to get highest purity fold and yield. The results showed that, 50% (w/v) ammonium sulfate saturation with 1:1.5 crude extract to t-butanol ratio at pH 5.25 gave 6.2-fold purification with 127% activity recovery of α-galactosidase. Characterization and determination of biochemical properties of the partitioned α-galactosidase were also aimed. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis showed considerable purification and the molecular weight of the enzyme was nearly found to be as 38 kDa. Various parameters (temperature, pH and substrate concentration) affecting to the enzyme activity and stability were studied. The optimum temperature and pH of the partitioned α-galactosidase were found to be 50 °C and pH 5.5, respectively. The enzyme was very stable at the temperature ranged from 37 to 45 °C and also in a pH range of 4.5–7.0. The kinetic constants; K m and V max were determined to be 0.37 mM and 0.46 U, respectively. The results indicated that, TPP technique is very attractive process for purification of pepino α-galactosidase and the characteristic properties of the enzyme partitioned by TPP make the enzyme good candidates for its several industrial applications.

An alkali-halotolerant cellulase from Bacillus flexus isolated from green seaweed Ulva lactuca

An extracellular alkali-halotolerant cellulase from the strain Bacillus flexus NT isolated from Ulva lactuca was purified to homogeneity with a recovery of 25.03% and purity fold of 22.31. The molecular weight of the enzyme was about 97 kDa and the V max and K m was 370.17 U/ml/min and 6.18 mg/ml respectively. The optimum pH and temperature for enzyme activity was 10 and 45 °C respectively. The enzymatic hydrolysis of the CMC was confirmed with GPC and GC-MS analysis. The stabilized activity of the enzyme even at high pH of 9.0–12.0 and residual activity of about 70% at salt concentration (NaCl 15%) revealed for its alkali-halotolerance nature. The metal ions Cd 2+ and Li 1+ were found as inducers while Cr 2+, Co 2+, Zn 2+ and metal chelator EDTA have significantly inhibited the enzyme activity. Enzyme activity was insensitive to ethanol and isopropanol while partially inhibited by acetone, cyclohexane and benzene.

Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato

Three-phase partitioning (TPP), a technique used in protein purification, was used to purify invertase from tomato ( Lycopersicon esculentum). The method consists of simultaneous addition of ammonium sulfate and t-butanol to the crude enzyme extract in order to obtain the three phases. Different parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) essential for the extraction and purification of invertase were optimized to get highest purity fold and yield. It was seen that, 50% (w/v) ammonium sulfate saturation with 1:1 (v/v) ratio of crude extract to t-butanol at pH 4.5 gave 8.6-fold purification with 190% activity recovery of invertase in a single step. Finally, the purified enzyme was also characterized and the general biochemical properties were determined. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was nearly found to be as 20 kDa. This work shows that, TPP is a simple, quick and economical technique for purification of invertases.