Expanded bed adsorption of an alkaline lipase from Pseudomona cepacia

An extracellular lipase was isolated from Pseudomona cepacia by expanded bed adsorption on an Amberlite 410 ion-exchange resin. Enzyme characterization and hydrodynamic study of a chromatography column were done. Enzyme purification was done at three condition of expanded bed height (H): at one and half (6 cm), at two (8 cm) and at three (12 cm) times the fixed bed height (H₀ = 4 cm). The results showed that the experimental data was fitted to the Richardson and Zaki equation, and the comparison between the experimental and calculated terminal velocities showed low relative error. In enzyme purification for better condition, a purification factor of about 80 times was found at 6 cm of expanded bed height, or 1.5 times of expansion degree. Purified lipase had an optimal pH and a temperature of 8 and 37 °C, respectively.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

A novel surfactant-stable alkaline serine-protease from a newly isolated Bacillus mojavensis A21. Purification and characterization

An extracellular bleach stable protease producing strain was isolated from marine water sample and identified as Bacillus mojavensis A21 on the basis of the 16S rRNA gene sequencing and biochemical properties. The A21 alkaline protease was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration and CM-Sepharose ion exchange chromatography, with a 6.43-fold increase in specific activity and 16.56% recovery. The molecular weight of the purified enzyme was estimated to be 20 kDa by SDS-PAGE and gel filtration. The enzyme was highly active over a wide range of pH from 7.0 to 13.0, with an optimum at pH 8.5. The relative activities at pH 11.0 and 12.0 were about 80 and 71.7% of that obtained at pH 8.5. The enzyme was extremely stable in the pH range of 7.0–12.0. It exhibited maximal activity at 60 °C. The thermostability of the enzyme was significantly increased by the addition of CaCl₂. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease.The N-terminal amino acid sequence of the first 20 amino acids of the purified protease was DINGGGATLPQKLYQTSGVL. B. mojavensis A21 protease showed low homology with bacterial peptidases, suggesting that the enzyme is a new protease.The alkaline protease showed high stability towards anionic (0.1% SDS) and non-ionic (1 and 5% Tween 80 and 1% Triton X-100) surfactants. In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 79 and 70% of its initial activity after 1 h incubation in the presence of 1% H₂O₂ and 0.1% sodium perborate, respectively. The enzyme showed excellent stability with a wide range of commercial solid and liquid detergents at 30 and 40 °C. Considering its promising properties, B. mojavensis A21 may find potential application in laundry detergents.     

Degradation of chitin and production of bioactive materials by bioconversion of squid pens

Serratia marcescens TKU011, a protease- and chitosanase-producing bacterium, the optimized condition for protease and chitosanase production was found after the media were heated at 121 °C for 120 min and the culture was shaken at 25 °C for 5 days in 100 mL of medium containing 1% squid pen powder (SPP) (w/v), 0.1% K₂HPO₄, and 0.05% MgSO₄. An extracellular metalloprotease with novel properties of solvent stable, and alkaline was purified from the culture supernatant of S. marcescens TKU011 with squid pen wastes as the sole carbon/nitrogen source. The enzyme was a monomeric protease with a molecular mass of 48–50 kDa by SDS–PAGE and gel filtration chromatography. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU011 protease were 8, 50 °C, pH 5–11, and <40 °C, respectively. Besides protease and chitosanase, with this method, deproteinization of squid pen for β-chitin, the production of peptide and reducing sugar may be useful for biological applications.     

Characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli for enzyme replacement therapy of Morquio A disease

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Currently, specific therapies are not available for MPS IVA patients. In this study, a biologically active recombinant GALNS enzyme (rGALNS) produced in Escherichia coli was purified through a two-step chromatography process. The effect of temperature and pH on purified rGALNS stability was evaluated, as well as the stability in human serum. Finally, the uptake of rGALNS by HEK 293 cells and MPS IVA fibroblasts was evaluated. The use of a semi-continuous process allowed the production of an active extracellular rGALNS, which was used for protein purification. The purified rGALNS showed a specific activity of 0.29 U mg^?1 and a production yield of 0.78 mg L^?1. The rGALNS presented an optimal pH of 5.5 and was stable for 8 days at 4 °C. In human serum it was stable for up to 6 h. rGALNS was not taken up by the cultured cells, suggesting that N-linked oligosaccharides are not necessary for the production of an active enzyme or enzyme stability but for the cell uptake of protein. This study shows the first characterization of rGALNS produced by E. coli, and provides important information about purification, stability, and glycosylations effect for this type of enzymes.     

High catalytic performances of Pseudomonas fluorescens lipase adsorbed on a new type of cyclodextrin-based nanosponges

Lipases are water-soluble enzymes that catalyze the hydrolysis of triacylglycerols (in aqueous media) or trans-esterification reactions (in microaqueous media) and are involved in a number of industrial applications. As a limit to lipase application is represented by the need for interfacial activation, the search for suitable solid supports able to fulfill this requirement is always ongoing. In the present work, we report the preliminary characterization of a system obtained by adsorbing Pseudomonas fluorescens lipase on a newly synthesized cyclodextrin-based carbonate nanosponge (CD–NS–1:4). The activity and structural stability of lipase adsorbed on this new support were evaluated by checking the effect of temperature, pH changes and organic solvents (methanol) on the enzyme structure and function, which were compared with those of the free enzyme in solution. Our data show that the non-covalent interaction of Ps. fluorescens lipase with CD–NS–1:4 results in enzyme structural and functional stabilization, as it was still active after 66 days of incubation at T ～ 18 °C. Stabilization with respect to T, pH and the presence of organic solvent was observed as well as, unlike the solubilized enzyme, the adsorbed lipase was active at T > 40 °C, at pH 5 and after 24-h incubation with 70% (v/v) methanol (13% residual activity).     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Bioreactor leaching of uranium from a low grade Indian silicate ore

Bio-leaching studies were carried out in a 2 L bioreactor- BIOSTAT-B^® equipped with a PLC based controller at 20–40% (w/v) pulp density using enriched culture of A.ferrooxidans for Turamdih uranium ore (Jharkhand, India). With the enriched culture of A.ferrooxidans adapted on Fe(II) at pH 2.0, 35 °C and 20% (w/v) pulp density, a 98.3% uranium recovery was recorded in 14 days. The leaching of uranium in the bioreactor improved the dissolution rate by reducing the time from 40 days in shake flask as per our earlier studies to 14 days. While investigating the importance of biogenic Fe(III) in the bio-leaching process a maximum recovery of 84.7% U₃O₈ was observed at pH 2.0 and 20% (w/v) pulp density in 10 h as compared to the uranium leaching of 38.3% in the control experiments. On raising the pulp density to 30%, uranium bio-recovery increased to 87.6% in 10 h at pH 2.0 with <76 μm size material. This showed a distinct advantage because of better mixing of slurry in the bioreactor with auto-controlled conditions that improved the kinetics.     

Effects of galactomannan as carbon source on production of α-galactosidase by Thermomyces lanuginosus: Fermentation,purification and partial characterisation

Thermophilic fungus Thermomyces lanuginosus CBS 395.62/b strain is able to grow and synthesise extracellular α-galactosidase in media containing galactomannan such as locust bean gum (LBG) or guar gum (GG). Production of extracellular α-galactosidase was enhanced from 1.2 U/mL to 4–6 U/mL meaning about 3–5 times increase by optimisation of medium composition. This enzyme was purified to homogeneity by partial precipitation with 2-propanol and different liquid chromatographical steps. The developed purification protocol yielded 22% of enzyme activity with 900 purified fold. Molecular mass of the purified α-galactosidase enzyme was estimated to be 53 kDa. Maximal catalytic activity of the enzyme was obtained in the acidic pH range between pH 4.6 and 4.8 and in the temperature range 60–66 °C. More than 95% of enzyme activity was remaining after 1-day incubation at 70 °C and on pH in the range from 4.0 to 7.0. The enzyme activity was significantly stimulated by Mg²⁺, Mn²⁺ and K⁺ ions, while considerably inhibited by the presence of Ca²⁺, Ag⁺ and Hg²⁺.     

Calcium dependent,alkaline detergent-stable trypsin from the viscera of Goby (Zosterisessor ophiocephalus): Purification and characterization

An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was purified to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE. The enzyme had an estimated molecular weight of 23.2 kDa.The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH 7.0 and 11.0. The optimum temperature for enzyme activity was 60 °C, and the activity and stability of trypsin was highly dependent on the presence of calcium ion. At 60 °C, Ca²⁺ (5 mM) stimulated the protease activity by 220%. The trypsin kinetic constants, K_m and k_cat, were 0.312 mM and 2.03 s^?1.The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents.     

Optimization of fermentation conditions for the production of ethanol from sago starch by co-immobilized amyloglucosidase and cells of Zymomonas mobilis using response surface methodology

Statistical experimental design was used to optimize the conditions of simultaneous saccharification and fermentation (SSF), viz. temperature, pH and time of fermentation of ethanol from sago starch with co-immobilized amyloglucosidase (AMG) and Zymomonas mobilis MTCC 92 by submerged fermentation. Maximum ethanol concentration of 55.3 g/l was obtained using a starch concentration of 150 g/l. The optimum conditions were found to be a temperature of 32.4 °C, pH of 4.93 and time of fermentation of 17.24 h. Thus, by using SSF process with co-immobilized AMG and Z. mobilis cells MTCC 92, the central composite design (CCD) was found to be the most favourable strategy investigated with respect to ethanol production and enzyme recovery.     

Process engineering of cellulosic n-butanol production from corn-based biomass using Clostridium cellulovorans

The cellulolytic Clostridium cellulovorans has been engineered to produce n-butanol from low-value lignocellulosic biomass by consolidated bioprocessing (CBP). The objective of this study was to establish a robust cellulosic biobutanol production process using a metabolically engineered C. cellulovorans. First, various methods for the pretreatment of four different corn-based residues, including corn cob, corn husk, corn fiber, and corn bran, were investigated. The results showed that better cell growth and a higher concentration of n-butanol were produced from corn cob that was pretreated with sodium hydroxide. Second, the effects of different carbon sources (glucose, cellulose and corn cob), basal media and culture pH values on butanol production were evaluated in the fermentations performed in 2-L bioreactors to identify the optimal CBP conditions. Finally, the engineered C. cellulovorans produced butanol with final concentration >3 g/L, yield >0.14 g/g, and selectivity >3 g/g from pretreated corn cob at pH 6.5 in CBP. This study showed that the fermentation process engineering of C. cellulovorans enabled a high butanol production directly from agricultural residues.     

Characterization of a recombinant thermostable β-glucosidase from Putranjiva roxburghii expressed in Saccharomyces cerevisiae and its use for efficient biomass conversion

A β-glucosidase gene from Putranjiva roxburghii (PRGH1) was heterologously expressed in Saccharomyces cerevisiae to enable growth on cellobiose. The recombinant enzyme was secreted to the culture medium, purified and biochemically characterized. The enzyme is a glycoprotein with a molecular weight of ∼68 kDa and exhibited enzymatic activity with β‐linked aryl substrates like pNP-Fuc, pNP-Glc, pNP-Gal and pNP-Cel with catalytic efficiency in that order. Significant enzyme activity was observed for cellobiose, however the enzyme activity was decreased with increase in chain length of glycan substrates. Using cellobiose as substrate, the enzyme showed optimal activity at pH 5.0 and 65 °C. The enzyme was thermostable up to 75 °C for 60 min. The enzyme showed significant resistance towards both glucose and ethanol induced inhibition. The recombinant S. cerevisiae strain showed advantages in cell growth, glucose and bio-ethanol production over the native strain with cellobiose as sole carbon source. In simultaneous saccharification and fermentation (SSF) experiments, the recombinant strain was used for bio-ethanol production from two different cellulosic biomass sources. At the end of the SSF, we obtained 9.47 g L⁻¹ and 14.32 g L⁻¹ of bio-ethanol by using carboxymethyl cellulose and pre-treated rice straw respectively. This is first report where a β-glucosidase gene from plant origin has been expressed in S. cerevisiae and used in SSF.     

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

Production of endoxylanase with enhanced thermostability by a novel polyextremophilic Bacillus halodurans TSEV1 and its applicability in waste paper deinking

The screening and selection of the culture variables followed by optimization using statistical approaches led to a 23-fold enhancement in thermo-alkali-stable xylanase production by the polyextremophilic Bacillus halodurans TSEV1. The optimization of crucial parameters involved in the extraction of xylanase from the bacterial bran led to a high enzyme recovery. The purified xylanase produced in submerged fermentation (SmF) and solid state fermentation (SSF) was visualized as a single band on SDS-PAGE with a molecular mass of 40 kDa. The SSF-xylanase is optimally active at 78 °C and pH 9.0 and stable in the pH range between 7.0 and 12.0 with a T_1/2 of 65 min at 90 °C, which is higher than that of SmF-xylanase. The higher activation energy, enthalpy of deactivation (ΔH*), free energy change of deactivation (ΔG*) and T_1/2 of SSF-xylanase than these of SmF xylanase further confirmed higher thermostability of the former than the latter. The combination of commercial cellulase and TSEV1 xylanase was highly effective in deinking of waste paper at alkaline pH and elevated temperatures.     

Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media

Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (q_max) of Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate for the GFP was 1,42,860 FU ml⁻¹ and 18,000 FU ml⁻¹, respectively. The expanded bed column chromatography using Ni²⁺ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni²⁺ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.     

Application of seaweeds for the removal of lead from aqueous solution

Ten different seaweed species were compared on the basis of lead uptake at different pH conditions. The brown seaweed, Turbinaria conoides, exhibited maximum lead uptake (at pH 4.5) and hence was selected for further studies. Sorption isotherms, obtained at different pH (4–5) and temperature (25–35 °C) conditions were fitted using Langmuir and Sips models. According to the Langmuir model, the maximum lead uptake of 439.4 mg/g was obtained at optimum pH (4.5) and temperature (30 °C). The Sips model better described the sorption isotherms with high correlation coefficients at all conditions examined. Various thermodynamic parameters such as ΔG°, ΔH° and ΔS° were calculated indicating that the present system was a spontaneous and endothermic process. Through potentiometric titrations, number of binding sites (carboxyl groups) and pK₁ were determined as 4.1 mmol/g and 4.4, respectively. The influence of co-ions (Na⁺, K⁺, Mg²⁺ and Ca²⁺) on lead uptake was well pronounced in the case of divalent ions compared to monovalent ions. The solution of 0.1 M HCl successfully eluted all lead ions from lead-loaded T. conoides biomass. The regeneration experiments revealed that the alga could be successfully reused for five cycles without any loss in lead biosorption capacity. A glass column (2 cm i.d. and 35 cm height) was used to study the continuous lead biosorption performance of T. conoides. At 25 cm (bed height), 5 ml/min (flow rate) and 100 mg/l (initial lead concentration), T. conoides exhibited lead uptake of 220.1 mg/g. The column was successfully eluted using 0.1 M HCl, with elution efficiency of 99.7%.     

Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads

The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4 mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1 unit (pH 8.0) and 5 °C higher (45 °C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30 min of each reaction time, indicating stable reusability under aqueous medium.     

Purification and characterization of cellulase from a marine Bacillus sp. H1666: A potential agent for single step saccharification of seaweed biomass

In this study, 115 marine bacterial isolates were screened for cellulase enzymatic activity and enzyme with a molecular mass of 40 kDa was purified from culture supernatant of the marine bacterium Bacillus sp. H1666 using ion exchange and size exclusion chromatography method. Growth of bacterial strain H1666 with efficient cellulase enzyme production was observed on untreated wheat straw and rice bran. The biochemical properties of the extracted cellulase were studied and enzyme was found active over a range of pH 3–9. The optimum cellulase activity was observed at pH 7 and temperature 50 °C. The enzyme was also shown to be slightly thermo-stable with 40% residual activity at 60 °C for 4 h. The potential applicability of enzyme was tested on dried green seaweed (Ulva lactuca) and 450 mg/g increase in glucose yield was obtained after saccharification. MALDI TOF–TOF analysis of cellulase peptide fingerprint showed similarity to the sequence of the glycoside hydrolase family protein.     

Integrated biooxidation and acid dehydration process for monohydroxylation of aromatics

We examined the performance of an integrated biooxidation and acid dehydration process for aromatic monohydroxylation using the production of o-cresol from toluene as the model conversion. A toluene cis-glycol dehydrogenase gene (todD)-disrupted mutant of Pseudomonas putida T-57 was employed as the whole cell biocatalyst for oxidizing toluene to toluene cis-glycol (TCG). After bioconversion of toluene to TCG, o-cresol was produced by adding HCl to the culture medium, since it was found that TCG became unstable at a low pH and underwent spontaneous dehydration. When the todD-disrupted mutant cells were grown in a two-liquid-phase culture system with oleyl alcohol as the organic solvent, this integrated process produced 40 g l⁻¹ of o-cresol in the organic solvent phase at 30 h. The total concentration of o-cresol in the two-liquid-phase culture reached 6.6 g l⁻¹ at 30 h, which was approximately four-fold greater than that in the single-liquid-phase culture.