Synthesis of thermo-sensitive copolymer with affinity butyl ligand and its application in lipase purification

In this study, thermo-sensitive N-alkyl substituted polyacrylamide polymer P_NNB was synthesized by using N-hydroxymethyl acrylamide(NHAM), N-isopropyl acrylamide (NIPA) and butyl acrylate (BA) as monomers, and its low critical solution temperature (LCST) was controlled to be 28 °C. The recovery of the thermo-sensitive polymer was over 98%. Butanol as a hydrophobic ligand was covalently attached onto polymer P_NNB and butyl ligand density was 80 μmol g^?1 polymer. The affinity polymer was used for purification of lipase from crude material. Optimized condition was pH 7.0, 35 °C adsorption temperature, 120 min adsorption time and 0.5 mg ml^?1 initial concentration of lipase. The adsorption isotherm accords with a typical Langmuir isotherm. The maximum adsorption capacity (Q_m) of the affinity polymer for lipase was 24.8 mg g^?1polymer. The affinity copolymer could be recycled by temperature-inducing precipitation and there was only about 6% loss of adsorption capacity after five recyclings. Specific activity of lipase was improved from 14 IU mg^?1 to 506 IU mg^?1 protein, and its recovery achieved 82%. The affinity polymer is suitable for the purification of target proteins from the crude material with large volume and dilute solution.     

Preparation of a recyclable novel thermoresponsive affinity copolymer and its application towards ε-polylysine purification

Thermo-responsive polymers have great potential for industrial applications of bio-separation and purification. However, the poor affinity and low recovery of thermoresponsive copolymers are the main factors limiting their large-scale application. In this paper, a recyclable thermoresponsive affinity copolymer (P_NHM-IDA-Ni²⁺) was prepared by immobilizing nickel ions (Ni²⁺) on copolymer P_NHM, and P_NHM-IDA-Ni²⁺ was applied to the purification of ε-polylysine. The lower critical solution temperature (LCST) of P_NHM and P_NHM-IDA-Ni²⁺ were 31.0 °C and 34.0 °C, respectively. Additionally, the recoveries of both copolymers were over 95.0%. The main parameters, such as pH were investigated to optimize the adsorption conditions. In addition, the Langmuir and Freundlich adsorption models were used to predict the maximum adsorption capacity of ε-polylysine. The results of the affinity precipitation demonstrated that the maximum adsorption capacity was 42.9 mg/g, and the adsorption was considered to follow the mono-layer model. The thermodynamic parameters (ΔG⁰, ΔH⁰ and ΔS⁰) of the ε-polylysine adsorption indicated that the adsorption was spontaneous and exothermic. The maximum elution recovery (93.5%) was achieved at pH 5.0 with 0.2 M imidazole. The results of tricine-SDS-PAGE and HPLC (Purity: increased from 84% to 99%) showed that the ε-polylysine was well separated from the crude extract by using the affinity copolymer P_NHM-IDA-Ni²⁺.     

Preliminary application of light-pH sensitive recycling aqueous two-phase systems to purification of lipase

One key problem of aqueous two-phase systems (ATPS) is that phase-forming polymers could not be recycled efficiently. This results in high cost and environmental pollution. In this study, we introduced novel aqueous two-phase systems which are composed by pH-sensitive polymer P_ADB and light-sensitive polymer P_NNC. P_NNC is enriched in the top phase while P_ADB is found in the bottom phase. And recoveries of two-phase-forming polymers can both reach over 96%. This aqueous two-phase system was used for purification of lipase from its crude material. The influences of various process parameters such as concentration of the phase-forming polymer, system pH, different types and concentrations of neutral salts on partitioning of lipase are evaluated. It has been found that partition coefficient of pure lipase could reach 0.061 under optimized conditions. Lipase from crude material was purified with 83.7% recovery and a purification factor of approximately 18 folds.     

Direct purification of Burkholderia Pseudomallei lipase from fermentation broth using aqueous two-phase systems

An aqueous two-phase purification process was employed for the recovery of Burkholderia pseudomallei lipase from fermentation broth. The partition behavior of B. pseudomallei lipase was investigated with various parameters such as phase composition, tie-line length (TLL), volume ratio (V_R), sample loading, system pH, and addition of neutral salts. Optimum conditions for the purification of lipase were obtained in polyethylene glycol (PEG) 6000-potassium phosphate system using TLL of 42.2% (w/w), with VR of 2.70, and 1% (w/w) NaCl addition at pH 7 for 20% (w/w) crude load. Based on this system, the purification factor of lipase was enhanced to 12.42 fold, with a high yield of 93%. Hence, the simplicity and effectiveness of aqueous two-phase systems (ATPS) in the purification of lipase were proven in this study.     

Evidence that pancreatic lipase is responsible for the hydrolysis of cutin,a biopolyester present in mammalian diet,and the role of bile salt and colipase in this hydrolysis

The enzyme, which catalyzes hydrolysis of cutin, an insoluble biopolyester of hydroxy and epoxy fatty acids, was purified from porcine pancreas. With three different purification methods, previously used for the purification of pancreatic lipase, it is shown that cutin hydrolase is pancreatic lipase. This enzyme released oligomers and all types of monomers from the polymer with a pH optimum around 7.5. Taurodeoxycholate inhibited cutin hydrolysis by lipase and colipase reversed this inhibition. Evidence is presented which suggests that bile salt stabilizes the enzyme at the surface of the insoluble substrate and that the interaction of the polymer surface with the lipase-colipase-bile salt system is similar to that previously observed with triglycerides. Diethyl-p-nitrophenyl phosphate inhibited cutin hydrolysis by lipase but the hydrolysis was insensitive to diisopropyl fluorophosphate.     

Inhibition of hepatic lipase by m-aminophenylboronate application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases

Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K₁ value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h⁻¹ mg⁻¹ protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h⁻¹ mg⁻¹ The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.     

Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

Cationic polyelectrolytes-lipases complexes formation as tool for recovery of these enzymes from their natural sources

In the present paper the formation of complexes between positively charge polyelectrolyte (polyethyleneimine and chitosan) and Candida rugosa lipase from a crude extract and porcine lipase from pancreas commercial homogenate preparations were analyzed. The solubility of lipases-cationic polyelectrolytes formation was dependent on: polyelectrolyte densities electrical charge, polyelectrolyte and enzyme concentrations and salts present in the solution. The lipase was recovered from the non-soluble complex by adding of NaCl at a given pH. Although the polyelectrolytes did not affect lipase biological activity, both of them produced good enzyme recovery (>90%); however, purification factors were low. This methodology appears to be a good previous prepurification and concentration method, using, low-cost polymers, allows the design of a purification method where the protein of interest is present in a large volume with respect to the small amount of polyelectrolyte added.     

Lipase from Candida paralipolytica

The lipase from Candida paralipolytica was purified, as judged by disc electrophoresis. The purification was about 132 fold, based on protein, with a recovery of 32% from the acetone precipitate of the cultivated broth.

After purification, modification of the enzyme was performed by dialyzing its solution against 1 m sodium chloride in acetate buffer at room temperature and by separating the modified enzyme from an unknown substance(s) with a Sephadex G–75 column.

The optimum pH for lipolysis of the purified lipase was 8.0, while that of the modified one was 7.0. Sodium taurocholate was required essentially by the purified enzyme, but not by the modified one. The purified lipase was stable below 37°C and in the pH range from 3.5 to 9.0 at 5°C.     

Kinetics of lipase recovery from the aqueous phase of biodiesel production by macroporous resin adsorption and reuse of the adsorbed lipase for biodiesel preparation

A commercial macroporous resin (D3520) was screened for lipase recovery by adsorption from the aqueous phase of biodiesel production. The influences of several factors on the adsorption kinetics were investigated. It was found that the kinetic behavior of lipase adsorption by macroporous resin could be well described by pseudo-first-order model. Temperature had no significant effects on lipase adsorption, while resin-to-protein ratio (R) significantly affected both rate constant (k₁) and equilibrium adsorption capacity (Q_e). No lipase was adsorbed when mixing (shaking) was not performed; however, protein recovery reached 98% after the adsorption was conducted at 200 rpm for 5 h in a shaker. The presence of methanol and glycerol showed significant negative influence on lipase adsorption kinetics. Particularly, increasing glycerol concentration could dramatically decrease k₁ but not impact Q_e. Biodiesel was found to dramatically decrease Q_e even present at a concentration as low as 0.02%, while k₁ was found to increase with biodiesel concentration. The adsorbed lipase showed a relatively stable catalytic activity in tert-butanol system, but poor stability in solvent-free system when used for biodiesel preparation. Oil and biodiesel were also found to adsorb onto resin during transesterification in solvent-free system. Therefore, the resin had to be washed by anhydrous methanol before re-used for lipase recovery.     

Immobilization and characterisation of a lipase from a new source, Bacillus sp. ITP-001

A new source of lipase from Bacillus sp. ITP-001 was immobilized by physical adsorption on the polymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. The support and immobilized lipase were characterised, compared to the lyophilised lipase, with regard to the specific surface area, adsorption–desorption isotherms, pore volume (Vp) and size (dp) by nitrogen adsorption, differential scanning calorimetry, thermogravimetric analysis, chemical composition analysis, Fourier transform infrared spectroscopy and biochemical properties. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0, whereas the optimum pH for the free enzyme was at pH 7.0; the optimum temperature of activity was 80 and 37 °C for the free and immobilized enzyme, respectively. The inactivation rate constant for the immobilized enzyme at 37 °C was 0.0038 h^?1 and the half-life was 182.41 h. The kinetic parameters obtained for the immobilized enzyme gave a Michaelis–Menten constant (K _m) of 49.10 mM and a maximum reaction velocity (V _max) of 205.03 U/g. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that it could be reused for 10 successive cycles, duration of each cycle (1 h), maintaining 33 % of the initial activity.     

Gelatin blends with alginate: gels for lipase immobilization and purification

Blends of natural polysaccharide sodium alginate (5%) with gelatin (3%) cross-linked with glutaraldehyde provide beads with excellent compressive strength (8 x 10(4) Pa) and regular structure on treatment with calcium chloride. Lipases from porcine pancreas, Pseudomonas cepacia, and Candida rugosa were immobilized in such a blend with excellent efficiency. The immobilized enzymes were stable and were reused several times without significant loss of enzyme activity both in aqueous and reverse micellar media. The beads were functionalized with succinic anhydride to obtain beads with extra carboxylic acid groups. These functionalized beads were then successfully used for 7.4-fold purification of crude porcine pancreatic lipase in a simple operation of protein binding at pH 5 and release at pH 8.5.     

Carotenol fatty acid esters: easy substrates for digestive enzymes?

To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied. Digestions were carried out in phosphate buffer at pH 7.4 and 37 °C. As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; β-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; β-cryptoxanthin esters) as well as retinyl palmitate. These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase. As reference enzyme a yeast lipase from Candida rugosa was applied. A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut. Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed. The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (U/mg solid). Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility.     

Selective separation and purification of two lipases from chromobacterium viscosum using AOT reversed micelles

Selective separation and purification of two lipases form Chromobacterium viscosum were carried out by liquid-liquid extraction using a reversed micellar system. Optimum parameters for extraction were determined using a 250 mM AOT micellar solution in isooctane. Complete separation of the two lipases was achieved at pH 6.0 with a 50mM potassium phosphate buffer solution containing 50 mM KCI. By adding 2.5% by volume of ethanol to the lipase-loaded micellar solution, 85% of the extracted lipase could be recovered in a new aqueous phase, 50 mM K(2)HPO(4) with 50 mM KCl, at pH 9.0. Lipase A was purified 2.6-fold with a recovery of 86%, and lipase B by 1.5-fold with a recovery of 76%.     

Partial Optimization of the 5-Terminal Codon Increased a Recombination Porcine Pancreatic Lipase (opPPL) Expression in Pichia pastoris

Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P.pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), K_m (0.041 mM), and V_max (2.008 µmol.mg protein ⁻¹.min⁻¹) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg.L⁻¹ vs 36 mg.L⁻¹) and total enzyme activity increased about 5 folds (1900 IU.L⁻¹ vs 367 IU.L⁻¹) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.     

Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications

Abstract

The recovery of activity of lipases immobilized onto a photo-crosslinked polymer network was 76.0% and 41.0% for entrapment and adsorption methods, respectively. Both entrapped and adsorbed immobilized enzymes were very stable, retaining more than 60% of their activity over the range of temperatures studied. Immobilization by either method protected their relative activities nearly 96% at 70°C. The optimum pH was 8.0 for immobilized enzymes and 6.0 for the free enzyme at 40°C, while the relative activities after storage at 0–4°C for 30 days were 98% and 75% using entrapment and adsorption methods, respectively. These results indicated that lipase immobilized by entrapment and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Also, it can be safely stated that, photo-crosslinked polymer network can be used as alternative supports for immobilization of lipase for enzymatic polymerization reactions. In the ring-opening polymerization of ?-caprolactone, polymerization rates were clearly affected as monomer conversions were 58% and 49% and the highest molecular weights (M_n) obtained were 7890 and 5600 gmol^{? 1} for entrapment and adsorption methods, respectively.     

Preparation of a novel hydrophobic affinity cryogel for adsorption of lipase and its utilization as a chromatographic adsorbent for fast protein liquid chromatography

In this study, we have prepared a hydrophobic cryogel for the chromatographic separation of lipase from its aqueous solutions including single protein and protein mixture and also Yarrowia lipolytica cell extract. N‐methacryloyl‐(l )‐phenylalanine methyl ester was used as a monomer to provide the hydrophobic character to the prepared cryogels. The highest adsorption capacity was observed at pH 5.0 at 0.5 mL min^?1 flow rate. The chromatographic separation of lipase was achieved from a binary mixture of lipase:bovine serum albumin (BSA) and lipase:lysozyme, and was also achieved from triple‐mixture of lipase:lysozyme:BSA by using fast protein liquid chromatography. Finally, lipase purification was performed from Yarrowia lipolytica cell extract used as a natural source. These studies have shown that the hydrophobic cryogel has good chromatographic performance for the separation and purification of lipase not only from aqueous solution, but also from cell extract as a natural source of lipase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:376–382, 2014     

Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography

Intracellular phospholipase A₂ was purified to homogenity from rat spleen supernatant by reverse-phase high-performance liquid chromatography with a trifluoroacetic acid-acetonitrile solvent system. The method simplified the purification procedure, which includes three consecutive chromatographic steps. The recovery of the enzyme activity was greater than 70% with an about 23,000-fold purification. The solvent system did not affect the catalytic properties of the enzyme. Phospholipases A₂ from rat spleen, human pancreatic juice, and porcine pancreas were eluted in that order from a column of octadecasilyl silica gel in a similar concentration range of acetonitrile. This result suggests that the phospholipases A₂ examined have similar hydrophobicities. This method may be applicable to the purification of phospholipases A₂ from other sources.     

Regioselective Enzymatic Hydrolysis of 3′,5′-DI-O-Acetylthymidine and Observation of a Surface Effect Due to Polystyrene

The selective enzymatic hydrolysis of 3′,5′-di-O-acetylthyidine (1) was studied. The lipases from porcine pancreas and Aspergillus niger, and pig liver esterase, all catalysed selective hydrolysis of the 5′O-acetyl group, but the lipase from Candida cylindracea catalysed selective hydrolysis of the 3′-O-acetyl group. Highest selectivity, leading to essentially pure 3′-O-acetylthymidine, was achieved using porcine pancreatic lipase in dilute solution at pH 7.5. Provision of an artificial interface in the form of polystyrene beads led to a significant increase in the rate of hydrolysis, accompanied by a marked fall in selectivity. Other changes in the hydrolysis conditions, such as raising the concentration of substrate or adding cosolvent, also led to a fall in selectivity.     

Anti-obesity effect of Dioscorea nipponica Makino with lipase-inhibitory activity in rodents

In the process of screening for pancreatic lipase inhibitors, which could be used as an anti-obesity measure, the methanol extract of Dioscorea nipponica Makino powder (DP) appeared to have potent inhibitory activity against porcine pancreatic lipase with an IC50 value of 5-10 microg/ml, where the enzyme activity was assayed by using 4-methylumbelliferyl oleate as a substrate. Further purification of active components present in the herb generated dioscin that belongs to the saponin family. Dioscin and its aglycone, diosgenin, both suppressed the time-dependent increase of blood triacylglycerol level when orally injected with corn oil to mice, suggesting their inhibitory potential against fat absorption. Sprague-Dawley rats fed on a high-fat diet containing 5% Dioscorea nipponica Makino and 40% beef tallow gained significantly less body weight and adipose tissue than control animals fed on a high-fat diet alone during an 8-week experimental period (P<0.05).