Nitrophenyl Palmitate Research Articles

Thermo-Switchable Enzyme@Metal-Organic Framework for Selective Biocatalysis and Biosensing.

The stimulus-responsive regulation of enzyme catalytic activity and selectivity provides a new opportunity to extend the functionality and efficiency of immobilized enzymes. This work aims to design and synthesize a thermo-switchable enzyme@MOF for size-selective biocatalysis and biosensing through the immobilization of Candida rugosa lipase (CRL) within ZIF-8 functionalized with thermally responsive polymer, poly(N-isopropylacrylamide) (PNIPAM) (CRL@ZIF-8-PNIPAM). Unlike free CRL, which does not demonstrate substrate selectivity, we can reversibly tune the pore size of the ZIF-8-PNIPAM nanostructures (open pores or blocked pores) through temperature stimulus and subsequently modulate the substrate selectivity of CRL@ZIF-8-PNIPAM. CRL@ZIF-8-PNIPAM had the highest hydrolytic activity for small molecules (12 mM p-nitrophenol/mg protein/min, 4-nitrophenyl butyrate (p-NP Be)) and the lowest hydrolytic activity for large molecules (0.16 mM p-nitrophenol/mg protein/min, 4-nitrophenyl palmitate (p-NP P)). In addition, CRL@ZIF-8-PNIPAM demonstrated thermo-switchable behavior for large molecules (p-NP P). The p-NP P hydrolytic activity of CRL@ZIF-8-PNIPAM was significantly lower at 40 °C (blocked pores) than at 27 °C (open pores). However, the transition of blocked pores and open pores is a gradual process that resulted in a delay in the "thermo-switchable" catalytic behavior of CRL@ZIF-8-PNIPAM during thermal cycling. CRL@ZIF-8-PNIPAM was also successfully used for the fabrication of electrochemical biosensors for the selective biosensing of pesticides with different molecular sizes.

Simultaneous production of lipase and PHB using methanotrophic organism Methylosinus trichosporium OB3b

Methanotrophs based processes can help in reduction of greenhouse gases generated from various anthropogenic and natural activities. Present study demonstrates versatility of methanotrophic organisms for production of natural and biodegradable material like polyhydroxybutyrate (PHB) along with lipase as industrially important enzyme. Lipase A gene from Bacillus subtillis 168 was expressed in methanotroph M. trichosporium for simultaneous production of PHB and lipase. Lipase produced from methanotrophs showed enzyme activity up to 82.03 U/ mg of protein and 14.08 U/mg of protein on 4-nitrophenyl laurate and 4-nitrophenyl palmitate respectively. We also demonstrated simultaneous production of lipase and PHB with 191.41 mg/L PHB and 71.53 U/mg of protein lipase activity. This is the first report on co-production of PHB with value added product in a methanotrophic system.

Explore or exploit? A model-based screening strategy for PETase secretion by Corynebacterium glutamicum.

Extracellular production of target proteins simplifies downstream processing due to obsolete cell disruption. However, optimal combinations of a heterologous protein, suitable signal peptide, and secretion host can currently not be predicted, resulting in large strain libraries that need to be tested. On the experimental side, this challenge can be tackled by miniaturization, parallelization, and automation, which provide high-throughput screening data. These data need to be condensed into a candidate ranking for decision-making to focus bioprocess development on the most promising candidates. We screened for Bacillus subtilis signal peptides mediating Sec secretion of two polyethylene terephthalate degrading enzymes (PETases), leaf-branch compost cutinase (LCC) and polyester hydrolasemutants, by Corynebacterium glutamicum. We developed a fully automated screening process and constructed an accompanying Bayesian statistical modeling framework, which we applied in screenings for highest activity in 4-nitrophenyl palmitate degradation. In contrast to classical evaluation methods, batch effects and biological errors are taken into account and their uncertainty is quantified. Within only two rounds of screening, the most suitable signal peptide was identified for each PETase. Results from LCC secretion in microliter-scale cultivation were shown to be scalable to laboratory-scale bioreactors. This work demonstrates an experiment-modeling loop that can accelerate early-stage screening in a way that experimental capacities are focused to the most promising strain candidates. Combined with high-throughput cloning, this paves the way for using large strain libraries of several hundreds of strains in a Design-Build-Test-Learn approach.

Radiation facilitated-immobilized lipase (RaFIL) mini biocatalytic reactor based on epoxy decorated PP tubes: Rapid colorimetric estimation of pesticide Chlorpyrifos

Polypropylene (PP) based ultracentrifuge tubes (PPt) have been internally surface functionalized with epoxy groups via 60Co gamma radiation mediated simultaneous grafting of monomer 2,3-Epoxypropyl methacrylate (PEPMA-g-PPt). Radiation dose, monomer concentration and solvent polarity are optimized to achieve grafting yield of ∼62.0 g.m−2. Enzyme Lipase from Candida rugosa has been immobilized inside the tubes via a simple, room temperature covalent coupling reaction (immobilization yield ∼73.3%). Immobilized and free lipase have been assayed spectrophotometrically by monitoring the hydrolysis of substrate 4-Nitrophenyl palmitate (PNPP) to Paranitrophenol (PNP) (λmax = 400 nm). The Radiation facilitated-immobilized Lipase system (RaFIL) demonstrates an operational temperature range enhancement from 50 °C (free lipase) to 70 °C, and a broader pH tolerance window with optimum pH increasing from 7.4 (free lipase) to 8.0, and ∼20% more activity as compared to the free enzyme at pH 10.0. The system retains its activity for >10 cycles over 30 days, with attrition losses of <15% after seven cycles. RaFIL has been successfully demonstrated to detect pesticide Chlorpyrifos within a linear range of 0–500 µg.ml−1 with LOD of 60.4 µg.ml−1 (R2=0.975). The system has potential applications as a facile, low cost biocatalytic technique for rapid, onsite detection of harmful pesticides in affected areas.

Evaluation of the Impact of Esterases and Lipases from the Circulatory System against Substrates of Different Lipophilicity.

Esterases and lipases can process amphiphilic esters used as drugs and prodrugs and impact their pharmacokinetics and biodistribution. These hydrolases can also process ester components of drug delivery systems (DDSs), thus triggering DDSs destabilization with premature cargo release. In this study we tested and optimized assays that allowed us to quantify and compare individual esterase contributions to the degradation of substrates of increased lipophilicity and to establish limitations in terms of substrates that can be processed by a specific esterase/lipase. We have studied the impact of carbonic anhydrase; phospholipases A1, A2, C and D; lipoprotein lipase; and standard lipase on the hydrolysis of 4-nitrophenyl acetate, 4-nitrophenyl palmitate, DGGR and POPC liposomes, drawing structure–property relationships. We found that the enzymatic activity of these proteins was highly dependent on the lipophilicity of the substrate used to assess them, as expected. The activity observed for classical esterases was diminished when lipophilicity of the substrate increased, while activity observed for lipases generally increased, following the interfacial activation model, and was highly dependent on the type of lipase and its structure. The assays developed allowed us to determine the most sensitive methods for quantifying enzymatic activity against substrates of particular types and lipophilicity.

Novel alkaline phosphatase/lipase-responsive composite hydrogel guar gum/pyruvic acid sodium modified by Zn2+ for mold and yeast biochemical signal exhibition

Zn2+-guar gum/pyruvic acid sodium (named as Zn-GG/PAS) composite hydrogel with chromogenic substrates 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP-2Na) and 4-nitrophenyl palmitate (PNP-PAL) was synthesized by incorporating Zn2+ into guar gum/pyruvic acid sodium composite hydrogel and used for mold and yeast biochemical signal fluorescence and visible exhibition. Zn-GG/PAS exhibited excellent water holding capacity (95%) with a final concentration of Zn2+ of 0.1 g/L. When the concentration of Zn2+ was 0.1 g/L, the cohesiveness and springiness were 0.14 g and 2.9 g, respectively. After GG/PAS with composite ratio of 8/2 (w/w) was added to Zn-GG/PAS, the elasticity modulus and viscous modulus remained relatively stable with the scanning range of viscosity. The cross-linked network of Zn-GG/PAS hydrogel provided an excellent encapsulation efficiency of 94% until 120 h. Compared with the national standard GB 4789.15–2016, the alkaline phosphatase/lipase-responsive Zn-GG/PAS hydrogel had no significant difference in terms of mold and yeast colony count. This work provides a convenient and effective method for fabricating composite hydrogel with alkaline phosphatase and lipase stimulate responsive that has promising application to sensitive exhibition of mold and yeast biochemical signals in microbiological detection.

Three-Dimensional Chiral Supramolecular Microenvironment Strategy for Enhanced Biocatalysis.

How the three-dimensional (3D) chiral environment affects the biocatalysis remains an important issue, thereby inspiring the development of a microenvironment that highly mimics the natural features of enzyme to guarantee enhanced biocatalysis. In this study, two gelators bearing d/l-phenylalanine as chiral centers are designed to construct the 3D chiral catalytic microenvironment for enhancing the biocatalysis of lipase. Such a microenvironment is programmed through chiral transmission of chirality from molecular chirality to achiral polymers. It shows that the chirality of the microenvironment evidently influences the catalytic efficiency of immobilized lipase inside the system, and the 3D microenvironment constructed by right-handed helical nanostructures can enhance the catalytic activity of lipase inside as high as 10-fold for catalyzing 4-nitrophenyl palmitate (NPP) to 4-nitrophenol (NP) and 1.4-fold for catalyzing lipids to triglycerides (TGs) in 3T3-L1 cells than that of the achiral microenvironment. Moreover, the 3D chiral microenvironment has the merits of good catalytic efficiency, high storage stability, and efficient recyclability. This strategy of designing a 3D chiral microenvironment suitable for biocatalysis will overcome the present limitations of enzymatic immobilization in traditional materials and enhance the understanding of biocatalysis.

Identification and Heterologous Production of a Lipase from Geobacillus kaustophilus DSM 7263T and Tailoring Its N-Terminal by a His-Tag Epitope

Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50°C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. Km and kcat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22mM and 417.1min-1, respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.

ZIF-8/Lipase Coated Tapered Optical Fiber Biosensor for the Detection of Triacylglycerides

A novel method for the detection of triacylglycerides was reported with a pure, stable, label-free enzyme functionalized optical fiber detector. This method has been accomplished by immobilizing Zeolitic imidazolate framework-8 (ZIF-8) packaged lipase on the tapered optical fiber. The evanescent field is generated by tapering the optical fiber birefringent interferometer (BFI). Usage of ZIF-8/lipase as a biosensing film on optical fiber has not previously been reported. 4-nitrophenyl palmitate (4-NP) is used to determine the stability of the lipase encapsulated in ZIF-8. Tapered BFI works at a refractive index (RI), which matches the turning point to provide high sensitivity. The wavelength shift has a quadratic polynomial response relationship with the concentration of triacylglycerol, showing enhanced sensitivity and bio-selectivity. LOD (Limit of detection) of $0.17~\mu \text{M}$ and 0.23 nM is obtained with diameters of $10~\mu \text{m}$ and $7~\mu \text{m}$ , respectively. This sensor can be applied to triacylglycerides detection, requiring a low volume of blood for each sample.

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p- nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p &lt;0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0 seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.

A maize hydrolase with activity against maize insect and fungal pests

A gene annotated as a chitinase-like protein that occurred in a quantitative trait locus for fungal maize ear rot resistance was cloned from a maize inbred with Fusarium proliferatum/verticillioides resistance. The protein sequence was similar to type III chitinases and homologous proteins without chitinase activity generally referred to as narbonins or chitolectins. The gene was expressed in Pichia pastoris, and the resulting protein was purified and examined for different types of activity. The yeast-produced protein did not hydrolyze chitin substrates, or 4-nitrophenyl sugars, but did hydrolyze 4-nitrophenyl acetate and 4-nitrophenyl palmitate. The protein also significantly retarded the growth of first instar fall armyworms (Spodptera fruigiperda) and significantly enhanced the activity of miconazole nitrate toward F. graminearum. The gene was biolistically introduced into maize BMS callus and the transgene was detectible by PCR. Some transformant lines significantly reduced corn earworm (Helicoverpa zea), fall armyworm, F. graminearum, F. proliferaturm and F. verticillioides growth compared to GUS transformed control callus. When transformed callus protein extracts were separated by native polyacrylamide gel electrophoresis, a band of 4-nitrophenyl acetate activity was found in bioactive callus that was not present in GUS control callus. Although the true target of the hydrolase is not known, it does appear to play a role in resistance to pests of maize and appears to be a previously unreported type of resistance mechanism.

A novel sensitive visual count card for detection of hygiene bio-indicator—molds and yeasts in contaminated food

The study developed a novel, sensitive and visual count card for detection of molds and yeasts in food. A series of concentrations of ethanol and four cations for molds and yeasts growth and coloration were evaluated. Furthermore, cold water gel texture of count card was also determined under the conditions. These results demonstrated that the effects of 8 mL L−1 ethanol, 0.2 g L−1 CuSO4, 0.2 g L−1 CaCl2 and 0.1 g L−1 ZnCl2 promoted the growth and coloration of molds and yeasts, especially inhibited fungal hyphae spreading to eliminate the omissions by mutual hyphae coverage. The gels texture was satisfactory for molds and yeasts growth. Two kinds of non-diffused chromogenic agents in different concentrations for molds and yeasts were also evaluated. The compound chromogenic agents (5-bromo-4-chloro-3-indolyl phosphate and nitrophenyl palmitate) provided more sensitive and accurate count card for detection of molds and yeasts. Artificial contamination method was used to evaluate the performance of detecting count card of molds and yeasts. Compared with national standard agar plate method (for 5–7 days), the count card (for 36 h) reduced detection time greatly.

Secreted Hydrolytic and Haemolytic Activities of Malassezia Clinical Strains.

Malassezia yeasts are opportunistic pathogens associated with a number of skin diseases in animals and humans. The free fatty acids released through these organisms' lipase and phospholipase activities trigger inflammation in the host; thus, these lipase and phospholipase activities are widely recognised as some of the most important factors in Malassezia pathogenesis. In this study, we sought to investigate and examine the relationship between these secreted hydrolytic activities and haemolytic activity in newly isolated Malassezia clinical strains. This characterisation was expected to elucidate pathogenicity of this fungus. We isolated 35 clinical strains of Malassezia spp.; the most frequently isolated species were M. sympodialis and M. furfur. Next, we analysed the hydrolytic activities of all of these clinical isolates; all of these strains (except for one M. dermatis isolate) showed detectable lipase and phospholipase activities against 4-nitrophenyl palmitate and L-α-phosphatidylcholine, dipalmitoyl, respectively. Most of the M. globosa isolates showed higher lipase activities than isolates of other Malassezia species. In terms of phospholipase activity, no significant difference was observed among species of Malassezia, although one isolate of M. globosa showed considerably higher phospholipase activity than the others. All tested strains also exhibited haemolytic activity, both as determined using 5% (v/v) sheep blood agar (halo assay) and by quantitative assay. Although all tested strains showed detectable haemolytic activity, we did not observe an apparent correlation between the secreted lipase and phospholipase activities and haemolytic activity. We infer that the haemolytic activities of Malassezia spp. are mediated by non-enzymatic factor(s) that are present in the secreted samples.

Mycelium-bound lipase from a locally isolated strain of Aspergillus westerdijkiae

Abstract Lipases can hydrolyze ester bonds in triacylglycerols and can be used, for instance, in detergent industries and in bioremediation. They are also able to esterify in organic interface. A strain of Aspergillus westerdijkiae was isolated from used cooking oil. The fungus was cultivated at 25 °C, 100 rpm, for 3 days, in a liquid medium containing olive oil as carbon source. The obtained mycelium was acetone washed, freeze dried, and macerated. The average mycelium yield production was 0.115 g of dried mycelium/25 mL of liquid media. The average enzyme yield was 40,000 U and 52 U per g of dry mycelium, evaluated by the release of p -nitrophenol from p -nitrophenyl palmitate and by titration of fatty acids released from olive oil, respectively. The enzyme optimum temperature was 40 °C and it was stable at 50 °C for 30 min, presenting a T 50 of 61.62 °C. The enzyme remained stable after 6 h of incubation at 40 °C or 50 °C. The enzyme optimum pH was 7.0–8.0 and it remained active after incubation of the mycelia for 6 h at pHs 5.0–8.0. Several ions, EDTA, PMSF, and non-ionic detergents inhibited the enzyme, but SDS did not. The enzyme activity also could use other short and medium chain esters as substrates. In addition, the lipase was able to synthesize ethyl oleate from oleic acid and ethanol. In conclusion, the mycelium bound lipase of A. westerdijkiae was characterized and this naturally immobilized enzyme could be useful in the biodiesel industry.

Surface Modification of Fe(3)O(4)@SiO(2) Magnetic Nanoparticles for Immobilization of Lipase.

Magnetic Fe(3)O(4) nanoparticles were prepared through hydrothermal method and coated with silica on the surface to obtain Fe3O4@SiO2 core–shell nanoparticles. After modification with different functional groups including aldehyde, amine and diimide, the nanoparticles were used as carrier for covalent immobilization of lipase. The nanoparticles with aldehyde groups showed highest immobilization yield (52.8%) and efficiency (86.5%). And the immobilization conditions including pH, temperature and the concentration of enzyme were optimized. After immobilization, the K m of lipase was altered from 2.3 to 3.2 mM. The thermal stability and pH stability were enhanced by immobilization at the investigated conditions: pH 5.0–8.0 and temperature 30–70 °C. After 10 batches conversion of 4-Nitrophenyl palmitate into p-Nitrophenol, the immobilized lipase retained over 75% of the original activity. Compared with the commercial lipase Novozym435, the immobilized lipase showed better stability and higher catalytic efficiency. These results demonstrate that the immobilized lipase on the modified Fe3O4@SiO2 magnetic nanoparticles has enhanced stability and reusability, which make lipase of potential interest in a number of industrial applications.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07.

The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 °C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 °C and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

English

Lipases hydrolyze lipids to yield fatty acids and glycerol which are used as raw materials in industries. The lipase from raphia mesocarp was extracted, purified and characterized. Assay was carried out using p- nitrophenyl palmitate as substrate. The lipase was subjected to 80% ammonium sulphate precipitation, purified on ion exchange and gel filtration chromatography. Native and subunit molecular weights were determined on a gel filtration column and 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis respectively. Kinetic parameters (Km and Vmax), effect of temperature, pH, sensitivity to metal ions and substrate specificities of the lipase enzyme were studied. Specific activity of 17.40µmol/min/mg and purification fold of 2.68% were obtained.Native and subunitmolecular weightwas 35 kDa. The Km and Vmax values were0.01mM and 20.5µmol/min/ml respectively. The lipase enzyme was inhibited by all metallic salts used and enhanced by CaCl2. Optimal pH was 7.0 and temperature for maximal activity was 40C. The lipase hydrolyzed coconut oil at about twice the rate of palm kernel oil and palm oil, and at a higher rate than olive oil and Raphia oil. The study reveals that raphia mesocarp isa possible source of lipase which can be of industrial use. Key words: Lipase, raphia palm fruit, mesocarp

Enzymatic hydrolysis in an aqueous organic two-phase system using centrifugal partition chromatography

Multi-phase reaction systems, mostly aqueous organic systems, are used in enzyme catalysis to convert hydrophobic substrates which are almost insoluble in aqueous media. In this study, a Centrifugal Partition Chromatograph is used as a compact device for enzymatic multi-phase reaction that combines efficient substrate supply to the aqueous phase and separation of both phases in one apparatus. A process design procedure to systematically select the aqueous and organic phase to achieve stable and efficient reaction rates and operation conditions in Centrifugal Partition Chromatography for efficient mixing and separation of the phases is presented. The procedure is applied to the hydrolysis of 4-nitrophenyl palmitate with a lipase derived from Candida rugosa. It was found that the hydrolysis rate of 4-nitrophenyl palmitate was two times higher in Centrifugal Partition Chromatography than in comparable stirred tank reactor experiments.

Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production.

Using enrichment procedures, a lipolytic strain was isolated from a stinky tofu brine and was identified as Bacillus amyloliquefaciens (named B. amyloliquefaciens Nsic-8) by morphological, physiological, biochemical tests and 16S rDNA sequence analysis. Meanwhile, the key enzyme gene (named lip BA) involved in ester metabolism was obtained from Nsic-8 with the assistance of homology analysis. The novel gene has an open reading frame of 645bp, and encodes a 214-amino-acid lipase (LipBA). The deduced amino acid sequence shows the highest identity with the lipase from B. amyloliquefaciens IT-45 (NCBI database) and belongs to the family of triacylglycerol lipase (EC 3.1.1.3). The lipase gene was expressed in Escherichia coli BL21(DE3) using plasmid pET-28a. The enzyme activity and specific activity were 250±16U/ml and 1750±153U/mg, respectively. The optimum pH and temperature of the recombinant enzyme were 9.0 and 40°C respectively. LipBA showed much higher stability under alkaline conditions and was stable at pH 7.0-11.0. The Km and Vmax values of purified LipBA using 4-nitrophenyl palmitate as the substrate were 1.04±0.06mM and 119.05±7.16μmol/(mlmin), respectively. After purification, recombinant lipase was immobilized with the optimal conditions (immobilization time 3h at 30°C, with 92% enzyme recovery) and the immobilized enzyme was applied in biodiesel production. This is the first report of the lipase activity and lipase gene obtained from B. amyloliquefaciens (including wild strain and recombinant strain) and the recombinant LipBA with the detailed enzymatic properties. Also the preliminary study of the transesterification shows the potential value in biodiesel production applications.

Investigation of Lipozyme TL IM-catalyzed transesterification using ultraviolet spectrophotometric assay

A novel ultraviolet (UV) spectrophotometric method for evaluating lipase transesterification activity in organic solvents is described. The model reaction was lipase-catalyzed transesterification between p -nitrophenyl palmitate (pNPP) and n -butanol in n -hexane at 30 °C. The conversion of pNPP to p -nitrophenol was monitored by measuring UV absorbance changes at 310 nm. Seven commercial lipases were tested using this method, and the results were similar to those obtained using gas chromatography. The UV spectrophotometric method was used to investigate the kinetics of the Lipozyme TL IM-catalyzed reaction of pNPP, and the correlation between synthetic activity and biocatalyst concentration was determined. The method was also used to investigate the Lipozyme TL IM-catalyzed transesterification properties, namely solvent effect, acyl acceptor specificity, alcohol tolerance, optimum reaction temperature, and thermostability. A modified p -nitrophenyl palmitate assay for lipase transesterification activity determination in organic solvents was described. On the basis of the method, the transesterification properties of Lipozyme TL IM were determined.