Lipases have been widely applied in a variety of industrial fields,such as food,pharmaceuticals,biofuels,and biotechnology.Recent years have witnessed a great interest in modifying lipids for the production of triacyl...Lipases have been widely applied in a variety of industrial fields,such as food,pharmaceuticals,biofuels,and biotechnology.Recent years have witnessed a great interest in modifying lipids for the production of triacylglycerols enriched with n-3 polyunsaturated fatty acids(PUFAs).Here,a novel salt-tolerant,organic solvent-stable,and bile salt-activated lipase was purified from golden pompano(Trachinotus ovatus)viscera,which was named as golden pompano lipase(GPL).GPL had a specific activity of 57.2U mg^(-1)with an estimated molecular weight of 14 k Da,exhibited optimal activity at 40℃a nd pH 8.0,and showed K_(m)and V_(max)of 40.16μmol L^(-1)and 769.23μmol L^(-1)min^(-1),respectively.GPL activity was enhanced by Mn^(2+)and sodium deoxycholate.It was active in organic solvents,including methanol,ethanol,chloroform,and hexane.GPL also showed a good salinity tolerance of up to 1 mol L^(-1).n-3PUFA enrichment in the glyceride fraction of golden pompano oil was performed by GPL-catalyzed hydrolysis and yielded a total PUFA concentration of 56.99%.EPA,DHA,and DPA were enriched by 10.4-,3.2-,and 1.8-fold of their initial levels,respectively.This study recognized the industrial applicability of GPL to prepare enriched C_(20-22)n-3 PUFA.展开更多
Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl ...Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.展开更多
Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To imp...Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly(acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine)s(PANCMPCs) were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile(PAN) nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%(molar fraction) in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.展开更多
Solid-state fermentation (SSF) holds tremendous potentials for the production of industrially significant enzymes. The present study describes the production of lipase by a novel rumen bacterium, Pseudomonas sp. strai...Solid-state fermentation (SSF) holds tremendous potentials for the production of industrially significant enzymes. The present study describes the production of lipase by a novel rumen bacterium, Pseudomonas sp. strain BUP6 on agro-industrial residues. Pseudomonas sp. strain BUP6 showed higher lipase production when grown in Basal salt medium (BSM) supplemented with oil cakes. Initially, five different oil cakes (obtained after extracting oil from coconut, groundnut, cotton seed, gingelly or soybean) were screened to find out the most suitable substrate-cum-inducer for the production of lipase. Among them, groundnut cake supported the maximum production of lipase (107.44 U/gds). Box-Behnken Design (BBD), followed by response surface methodology (RSM) was employed to optimize the culture parameters for maximizing the production of lipase. Using the software Minitab 14, four different parameters like temperature, pH, moisture content and incubation time were selected for the statistical optimization, which resulted in 0.7 fold increase (i.e., 180.75 U/gds) in production of lipase under the optimum culture conditions (temperature 28°C, pH 5.9, moisture 33% and incubation 2 d). Thus, this study signifies the importance of SSF for the production of industrially-significant lipase using agro-industrial residues as solid support.展开更多
In-silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues;Val, Met, Leu, Ile, Trp and Phe into the oxyanion Q114 of T1 lipase. We hypothesized that the oxyani...In-silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues;Val, Met, Leu, Ile, Trp and Phe into the oxyanion Q114 of T1 lipase. We hypothesized that the oxyanion Q114, involved in substrate binding is also associated with modulation of conformational stability and in conferring specific enzyme attributes. The insilico investigations accurately predicted the quality of the protein packing in some of the variants. Our study found by altering the hydrophobicity of the oxyanion 114, remarkably altered enzyme conformational stability and catalytic attributes. Substitution with Leu resulted improvements in four out of the six tested characteristics. The hydrophobic Leu might have improved local structure folding and increased hydrophobic interactions with other residues in the vicinity of the mutation. The Met variant showed higher activity over the wild-type in hydrolyzing a wider range of natural oils. The bulky amino acids, Phe and Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability and inferior enzyme characteristics. We have successfully illustrated that a single point residue changes at oxyanion 114 could result in a myriad of enzyme attributes, which implied there was some interplay between hydrophobicity and conformation for lipase catalytic functions.展开更多
Lipase-catalyzed stereoselective resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate(cis-(±)-1)is an attractive route for the synthesis of(S,S)-2,8-diazobicyclo[4.3.0]nonane,an important chir...Lipase-catalyzed stereoselective resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate(cis-(±)-1)is an attractive route for the synthesis of(S,S)-2,8-diazobicyclo[4.3.0]nonane,an important chiral intermediate of the fluoroquinolone antibiotic,moxifloxacin.In our previous study,a lipase from Sporisorium reilianum(SRL)was identified to possess excellent thermostability and pH stability.However,the low enzymatic activity of the SRL is a challenge that must be addressed.A rational design was initially employed for SRL tailoring according to the engineered Candida antarctica lipase B(CALB),resulting in a beneficial variant called SRL-I194N/V195L.Subsequently,two key amino acid residues in loop 6,L145 and L154,which might modulate the lid conformation between open and closed,were identified.A tetra-site variant,SRL-I194N/V195L/L145V/L154G(V13),with a significantly enhanced activity of 87.8 U∙mg^(−1) was obtained;this value was 2195-fold higher than that of wild-type SRL.Variant V13 was used to prepare optically pure(2S,3R)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate((2S,3R)-1),resolving 1 mol∙L^(−1) cis-(±)-1 with a conversion of 49.9%in 2 h and absolute stereoselectivity(E>200).Excellent stability with a half-life of 92.5 h was also observed at 50℃.Overall,the study findings reveal a lipase with high activity toward cis-(±)-1 at an industrial level and may offer a general strategy for enhancing the enzyme activity of other lipases and other classes of enzymes with a lid moiety.展开更多
The use of enzymatic route for production of biofuels is growing up due the mild reaction conditions that this method provides, as well as reducing SOx emission. To reduce costs, it’s necessary to immobilize the enzy...The use of enzymatic route for production of biofuels is growing up due the mild reaction conditions that this method provides, as well as reducing SOx emission. To reduce costs, it’s necessary to immobilize the enzyme, making possible to use it continuously as biocatalyst. The aim of this work was to measure the influence of the mass of support and pH used for immobilization of commercial lipase from Candida rugosa acquired by Sigma laboratory. The immobilization method chosen was adsorption on mesoporous and hydrophobic support MCM 41, this has been treated with nitric acid 10% v/v to remove any organic residue. Then, 20 ml of enzymatic solution in phosphate buffer (pH 6.0, 7.0 and 8.0;50 mM) and 1 g/L was placed under constant stirring with 0.30 and 0.45 g of support. Aliquots were taken from the reaction medium and analyzed by spectrophotometry at 10 minutes intervals. A volume of 0.2 ml of supernatant was put with 1.8 ml of substrate p-NFL at 0.18 g/L, and the absorbance at 410 nm was analyzed. In four cases there was a sharp reduction of supernatant’s activity at first 10 minutes, that ratifies the big affinity of the enzyme for the support and the negative influence of pH about the activity. Using the calibration curve, it was possible to calculate the final activity of each immobilization batch. This work suggests the occurrence of diffusional effects, which means that the enzyme mobility was restricted due the excessive amount of support, and then, it lost a part of accessibility to substrate, reflecting in not expressive activity values, and changing the state of ionization of the components of the system.展开更多
Alkane-based biodiesel is considered the next generation of biodiesel owing to its potential environmental benefits and the fact that it exhibits much higher specific caloric values than traditional biodiesel.However,...Alkane-based biodiesel is considered the next generation of biodiesel owing to its potential environmental benefits and the fact that it exhibits much higher specific caloric values than traditional biodiesel.However,the formidable obstacle impeding the commercialization of this cutting-edge fuel alternative lies in the cost associated with its production.In this study,an engineered strain Escherichia coli(E.coli)showcasing harmonized coexpression of a lipase(from Thermomyces lanuginosus lipase,TLL)and a fatty acid photodecarboxylase(from Chlorella variabilis,CvFAP)was first constructed to transform triglycerides into alkanes.The potential of E.coli BL21(DE3)/pRSFDuet-1-TLL-CvFAP for alkane synthesis was evaluated with tripalmitin as a model substrate under various process conditions.Following a comprehensive examination of the reaction parameters,the scope of the biotransformation was expanded to‘real’substrates(vegetable oils).The results showed that bioderived oils can be transformed into alkanes with high yields(0.80-10.20 mmol·L^(-1))under mild conditions(35℃,pH 8.0,and 36 h)and blue light illumination.The selected processes were performed on an increased lab scale(up to 100 ml)with up to 24.77 mmol·L^(-1) tripalmitin,leading to a yield of 18.89 mmol·L^(-1) pentadecane.With the employment of a method for efficiently producing alkanes under mild conditions and a simple procedure to isolate alkanes from the reaction system,the utilization of sustainable biomass as a fundamental feedstock emerges as the primary solution to lower the cost of alkane-based biodiesel.Thus,this study proposes a readily implementable and highly effective approach for alkane-based biodiesel production.展开更多
A strain of psychrophilic bacterium, 2-5-10-1, which produces low-temperature lipase, is isolated from the deep sea of Prydz Bay in Southern Ocean. The highest lipase secretion of this strain is observed at 5 degreesC...A strain of psychrophilic bacterium, 2-5-10-1, which produces low-temperature lipase, is isolated from the deep sea of Prydz Bay in Southern Ocean. The highest lipase secretion of this strain is observed at 5 degreesC and this temperature is also for optimal growth. Tween 80 and olive oil enhance secretion of lipase. The optimal temperature and pH for lipase activity are 35 degreesC and 7.5 degreesC respectively. At 0degreesC, the lipase still has 37% relative enzyme activity. The lipase shows high thermolability, more than 50% activity lost after incubation at 60 degreesC for 15 min. EDTA has no effect on lipase activity, indicating the lipase activity is independent of divalent cation. In contrast, the lipase activity is inhibited drastically by Cu2+ and Zn2+.展开更多
A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 (Pseudomonas sp.) is described, whereby a lipase gene (lip) was clon...A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 (Pseudomonas sp.) is described, whereby a lipase gene (lip) was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.展开更多
Fusarium graminearum(sexual stage:Gibberella zeae)is the causative agent of Fusarium Head Blight(FHB),which is one of the most destructive plant disease of cereals,accounting for high grain yield losses,especially for...Fusarium graminearum(sexual stage:Gibberella zeae)is the causative agent of Fusarium Head Blight(FHB),which is one of the most destructive plant disease of cereals,accounting for high grain yield losses,especially for wheat and maize.Like other fungal pathogens,several extracellular enzymes secreted by G.zeae are known to be involved in host infection.Among these secreted lipases,G.zeae lipase(GZEL),which is encoded by the FGL1 gene,was demonstrated to be crucial to G.zeae pathogenicity.However,the precise mechanism of GZEL remains unclear due to a lack of detailed structural information.In this study,we report the crystal structure of GZEL at the atomic level.The structure of GZEL displays distinct structural differences compared to reported homologues and indicates a unique“double lock”enzymatic mechanism.To gain insight into substrate/inhibitor recognition,we proposed a model of GZEL in complex with substrate and the lipase inhibitor ebelactone B(based on the reported structures of GZEL homologues),which defines possible substrate binding sites within the catalytic cleft and suggests an“anti sn-l”binding mode.These results pave the way to elucidating the mechanism of GZEL and thus provide clues for the design of anti-FHB inhibitors.展开更多
The production of extra cellular lipase in Solid State Fermentation (SSF) using Yarrowia lipolytica NCIM 3589 with Palm Kernal cake (Elaeis guineensis) has been studied. Different parameters such as incubation time, i...The production of extra cellular lipase in Solid State Fermentation (SSF) using Yarrowia lipolytica NCIM 3589 with Palm Kernal cake (Elaeis guineensis) has been studied. Different parameters such as incubation time, inoculum level, initial moisture content, carbon level and nitrogen level of the medium were optimized. Screening of various process variables has been accomplished with the help of Plackett-Burman design. The maximum lipase activity of 18.58 units per gram of dry fermented substrate (U/gds) was observed with the substrate of Palm Kernal cake in four days of fermentation.展开更多
A novel lipase gene(lip4346)encoding a primary translation product with 176 amino acids was screened from the genome fine mapping of the macroalgae-associated bacterial strain Microbulbifer sp.YNDZ01.Macroalgae were c...A novel lipase gene(lip4346)encoding a primary translation product with 176 amino acids was screened from the genome fine mapping of the macroalgae-associated bacterial strain Microbulbifer sp.YNDZ01.Macroalgae were collected from the coast of the Halmahera Island of Indonesia.The lip4346 gene was cloned and heterologously expressed in Escherichia coli.The purified recombinant Lip4346 protein had a molecular mass of 19 k Da,a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and a maximum enzyme activity of 31.2 U m L-1.The optimal temperature and pH for the lipase activity of Lip4346 were 70℃and 10.0,respectively.Lip4346 was tolerant with a number of organic solvents and detergents,and was active toward triacylglycerols and p-nitrophenyl esters with short-and medium-chain lengths.The unique characteristics of Lip4346 indicate that it is a promising nonaqueous biocatalyst for industrial applications.展开更多
Although herbal medicines(HMs)are widely used in the prevention and treatment of obesity and obesity-associated disorders,the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly...Although herbal medicines(HMs)are widely used in the prevention and treatment of obesity and obesity-associated disorders,the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood.Recently,we assessed the inhibitory potentials of several HMs against human pancreatic lipase(hPL,a key therapeutic target for human obesity),among which the root-extract of Rhodiola crenulata(ERC)showed the most potent anti-hPL activity.In this study,we adopted an integrated strategy,involving bioactivity-guided fractionation techniques,chemical profiling,and biochemical assays,to identify the key anti-hPL constituents in ERC.Nine ERC fractions(retention time=12.5e35 min),obtained using reverse-phase liquid chromatography,showed strong anti-hPL activity,while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS/MS).Among the identified ERC constituents,1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(PGG)and catechin gallate(CG)showed the most potent anti-hPL activity,with pIC50 values of 7.59±0.03 and 7.68±0.23,respectively.Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner,with inhibition constant(Ki)values of 0.012 and 0.082 mM,respectively.Collectively,our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC,as well as to elucidate their anti-hPL mechanisms.These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.展开更多
Obesity is usually considered as an overweight or excess body fat, leading to increased health problems. Obesity is a major risk factor for a number of serious diseases. Decreasing dietary fat absorption, through inhi...Obesity is usually considered as an overweight or excess body fat, leading to increased health problems. Obesity is a major risk factor for a number of serious diseases. Decreasing dietary fat absorption, through inhibition of pancreatic lipase activity, has been reported to be one of the most effective ways for managing obesity. The present study was aimed at investigating lipase inhibitors from edible plants. A lipase inhibitor was isolated from n-hexane and ethyl acetate extracts of the ripe fruits of Solanum stramonifolium Jacq. by column chromatography and identified by spectral analysis. Its structure was elucidated as (22R)-3β-benzoyloxy-22-hydroxy-4α-methyl-5α-stigmast-7-en-6-one or carpesterol (1). Carpesterol exhibited moderate lipase inhibition activity with IC50 value of 56.0 μg/mL while orlistat, a well- know pancreatic lipase inhibitor, had IC50 value of 3.5 ng/mL. Moreover, the kinetic properties of carpesterol on pancreatic lipase were evaluated. Carpesterol is a competitive inhibitor and exhibited antagonistic interaction when combined with orlistat on lipase inhibition activity.展开更多
The purification of a lipase isoenzyme from an Aspergillus niger lipase A is reported in this manuscript. Purification was carried out in a simple adsorption step, in which the lipase was offered at low ionic strength...The purification of a lipase isoenzyme from an Aspergillus niger lipase A is reported in this manuscript. Purification was carried out in a simple adsorption step, in which the lipase was offered at low ionic strength to the commercially available C8 modified magnetic particles, MaKProt C8. When the isoenzyme was desorbed with a 0.2% solution of Triton X-100, the SDS-PAGE gel showed a single pure band with a molecular weight of 35 KDa. The purified fraction showed 66.75-fold purification compared with the crude extract. The pure fraction was characterized along with the crude extract and the lipase adsorbed on the MaKProt C8. The purified and the adsorbed lipase showed better activity for the tested substrates (p-nitrophenyl acetate, decanoate, myristate and palmitate) than the crude extract, the preferred substrates being myristate (26.7 μmol·min-1·mg-1) and decanoate (17.4 μmol·min-1·mg-1), respectively. The temperature and pH profiles showed no change for the three enzymes, the optimum temperature being 37°C and the best pH 7.0.展开更多
The aim of the present work was to isolate <em>Bacillus</em> spp. With high lipase activity;to characterize the isolates using both biochemical and molecular methods;to produce lipase using <em>Bacil...The aim of the present work was to isolate <em>Bacillus</em> spp. With high lipase activity;to characterize the isolates using both biochemical and molecular methods;to produce lipase using <em>Bacillus</em> isolates and to study the biochemical and biophysical characteristics of the produced lipase. Sixty five <em>Bacillus</em> isolates were isolated from soil 20 isolates from guar field soil (G), 15 isolates from Abusabein field soil (B), 15 isolates from sun flower field soil (S) and 15 isolates from oil effluent (O). Lipase producing isolates were screened;a Chromogenic plate’s method was used. Enzyme activity was quantitatively assayed. Lipase production under submerged fermentation (SMF) conditions using a production medium that contained metal salts, Tween-20 and olive oil as substrate at different period 24, 48, 72 and 96 h, the optimum pH, temperature for lipase activity was determinated and kinetics as well. The isolates showed the highest lipase activity which was identified as Bacillus sp. The optimum pH, temperature, thermostability and kinetic of the produced enzymes were found in three isolates G14, O1 and B10 with the highest enzyme activity and best stability. The isolates G14, O1 and B10 revealed the highest lipase activity of 63.4, 41.2 and 28.3 U/ml, respectively. The results showed optimum pH of the lipase activity from isolates G14, O1 and B10 8.0, 6.0 and 6.0 and the optimum temperature 40, 60 and 75<span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;">˚</span></span></span></span>C, respectively. Lipase enzymes from isolates O1 and B10 were found to be more thermostable after incubation time for 120 min at 90<span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;">˚</span></span></span></span>C. The V<sub>max</sub> and K<sub>m</sub> values of lipase for isolates G14, OI and B10 were 17.6, 135 and 24.4 μmole<span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;">∙</span></span></span></span>min<span style="white-space:nowrap;"><sup><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;">−</span></span></span></span></sup></span><sup>1</sup> and 1.3, 1.6 and 0.681 mM, respectively. According to these results <em>Bacillus</em> spp. with high lipase activity and thermostability can be used to promote food, pharmaceuticals, paper, detergents agrochemicals industries and pollution control in Sudan.展开更多
Lipase from Candida sp.99-125 catalyzed ring-opening polymerization of e-caprolactone in the presence of 6-mercapto-1-hexanol was presented as a new metal-free approach for direct synthesis of welldefined thiol-termin...Lipase from Candida sp.99-125 catalyzed ring-opening polymerization of e-caprolactone in the presence of 6-mercapto-1-hexanol was presented as a new metal-free approach for direct synthesis of welldefined thiol-terminated poly(e-caprolactone).Remarkably,high chemoselectivity of lipase from Candida sp.99-125 toward hydroxyl and thiol was exhibited and quantitative thiol fidelity over 90% was achieved.The tedious protecting/deprotecting steps for thiol and metal residue were avoided.The polymerizations with around 70% monomer conversion were conducted in bulk and toluene at relative low temperature of 40 ℃.Number–average molecular weight of resulted polymers ranged from 3000 to4700 Da by changing the feed ratio between monomer and initiator.The structures of obtained thiolterminated poly(e-caprolactone) were demonstrated by combining NMR and SEC analyses.展开更多
The aim of this research work was to explore psychrotrophic microbes from soil sample of NyAlesund, Svalbard, arctic region and to investigate their potential use as an effective tool for industrial application. A nov...The aim of this research work was to explore psychrotrophic microbes from soil sample of NyAlesund, Svalbard, arctic region and to investigate their potential use as an effective tool for industrial application. A novel psychrotrophic bacterial strain showed good growth on minimal medium containing lipid as the only carbon source. Microbiological characterisation of the isolate showed that it was a gram negative rod. The strain was tested for the production of extracellular lipase enzyme. The enzymes were partially purified by 90% saturated ammonium sulfate and dialysis for desalting. The bacterium was identified as Pseudomonas sp ADT3 by 16S rRNA amplification and sequencing which had been deposited in the NCBI GenBank with accession number JX914667. Phylogenetic tree was also constructed with MEGA5 software and showed the highest level of sequence similarity with Pseudomonas sp. HC3-13 strain. The microorganism had a growth optimum at pH 8.0 and temperature 22°C. Optimization of different parameters e.g. temperature, pH, incubation time, cofactors etc. was performed for the extracellular lipase activity. The hydrolytic activity of the enzyme was enhanced 5 times by Pb2+ but strongly inhibited by heavy metals Hg2+ as well as EDTA and β-mercaptoethanol. For the molecular weight estimation of enzyme SDS-PAGE was done which showed an inducible band of approximately 13.9 KDa. Activity staining and mass spectrometry techniques were also performed.展开更多
A lipase from Sporisorium reilianum SRZ2(SRL)with 73%amino acid sequence identity to Candida antarctica lipase B(CALB)was cloned and overexpressed in Pichia pastoris.The recombinant SRL showed a preference for short-c...A lipase from Sporisorium reilianum SRZ2(SRL)with 73%amino acid sequence identity to Candida antarctica lipase B(CALB)was cloned and overexpressed in Pichia pastoris.The recombinant SRL showed a preference for short-chain p-nitrophenyl esters.It achieved maximum activity at pH 8.0 and 65°C for p-nitrophenyl hexanoate(C6)with Km and kcar/Km values of 0.14 mmol·L-1 and 1712 min 1.mmol·L-1 at 30℃,respec-tively.SRL displayed excellent thermostability and pH stability,retaining more than 79%of its initial activity after incubation at 60℃ for 72h and 75%atpH 3to 11 for 72 h.It also maintained most of its activity in the presence of inhibitors and detergents except sodium dodecyl sulfate,and it tolerated organic solvents.SRL was covalently immobilized and successfully used for ethyl hexanoate synthesis in cyclohexane or in a solvent-free system with a high conversion yield(>95%).Furthermore,high con-version yield was also achieved for the synthesis of various short-chain flavor esters when high substrate concentra-tions of2 mol.LI were applied.This study indicated that a.CALB-type lipase from S.reiliamum SRZ2 showed great potential in organic ester synthesis.展开更多
基金This work was supported by the National Key R&D Programs of China(No.2018YFD0901103)the Program of the Hainan Association for Science and Technology Plans to Youth R&D Innovation(No.QCXM202003)the Hainan Provincial Natural Science Foundation of China(No.2019RC093).
文摘Lipases have been widely applied in a variety of industrial fields,such as food,pharmaceuticals,biofuels,and biotechnology.Recent years have witnessed a great interest in modifying lipids for the production of triacylglycerols enriched with n-3 polyunsaturated fatty acids(PUFAs).Here,a novel salt-tolerant,organic solvent-stable,and bile salt-activated lipase was purified from golden pompano(Trachinotus ovatus)viscera,which was named as golden pompano lipase(GPL).GPL had a specific activity of 57.2U mg^(-1)with an estimated molecular weight of 14 k Da,exhibited optimal activity at 40℃a nd pH 8.0,and showed K_(m)and V_(max)of 40.16μmol L^(-1)and 769.23μmol L^(-1)min^(-1),respectively.GPL activity was enhanced by Mn^(2+)and sodium deoxycholate.It was active in organic solvents,including methanol,ethanol,chloroform,and hexane.GPL also showed a good salinity tolerance of up to 1 mol L^(-1).n-3PUFA enrichment in the glyceride fraction of golden pompano oil was performed by GPL-catalyzed hydrolysis and yielded a total PUFA concentration of 56.99%.EPA,DHA,and DPA were enriched by 10.4-,3.2-,and 1.8-fold of their initial levels,respectively.This study recognized the industrial applicability of GPL to prepare enriched C_(20-22)n-3 PUFA.
基金supported by the Marine S&T Fund of Shandong Province for the Pilot National Laboratory for Marine Science and Technology (Qingdao) (Nos. 2018 SDKJ0304-4-2, 2018SDKJ0303-1)the Central Public-Interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Sciences (Nos. 2017HY-XKQ01-01, 2016ZD0902, 2018GH10)+3 种基金the Central Public-Interest Scientific Institution Basal Research Fund, YSFRI, CAFS (No. 20603022018025)the Aoshan S&T Innovation Project from Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02-02-04)the Antarctic Marine Biological Resources Development and Utilization Project from the Ministry of Agriculture and Rural Affairs, People’s Republic of China (2017)the Financial Fund of the Ministry of Agriculture and Rural Affairs, People’s Republic of China (Nos. NFZX2018, FSTICE2019)
文摘Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.
基金Supported by the National Natural Science Foundation of China for Distinguished Young Scholars(No50625309)the National Postdoctoral Science Foundation of China(No20060400337)
文摘Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly(acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine)s(PANCMPCs) were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile(PAN) nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%(molar fraction) in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.
文摘Solid-state fermentation (SSF) holds tremendous potentials for the production of industrially significant enzymes. The present study describes the production of lipase by a novel rumen bacterium, Pseudomonas sp. strain BUP6 on agro-industrial residues. Pseudomonas sp. strain BUP6 showed higher lipase production when grown in Basal salt medium (BSM) supplemented with oil cakes. Initially, five different oil cakes (obtained after extracting oil from coconut, groundnut, cotton seed, gingelly or soybean) were screened to find out the most suitable substrate-cum-inducer for the production of lipase. Among them, groundnut cake supported the maximum production of lipase (107.44 U/gds). Box-Behnken Design (BBD), followed by response surface methodology (RSM) was employed to optimize the culture parameters for maximizing the production of lipase. Using the software Minitab 14, four different parameters like temperature, pH, moisture content and incubation time were selected for the statistical optimization, which resulted in 0.7 fold increase (i.e., 180.75 U/gds) in production of lipase under the optimum culture conditions (temperature 28°C, pH 5.9, moisture 33% and incubation 2 d). Thus, this study signifies the importance of SSF for the production of industrially-significant lipase using agro-industrial residues as solid support.
文摘In-silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues;Val, Met, Leu, Ile, Trp and Phe into the oxyanion Q114 of T1 lipase. We hypothesized that the oxyanion Q114, involved in substrate binding is also associated with modulation of conformational stability and in conferring specific enzyme attributes. The insilico investigations accurately predicted the quality of the protein packing in some of the variants. Our study found by altering the hydrophobicity of the oxyanion 114, remarkably altered enzyme conformational stability and catalytic attributes. Substitution with Leu resulted improvements in four out of the six tested characteristics. The hydrophobic Leu might have improved local structure folding and increased hydrophobic interactions with other residues in the vicinity of the mutation. The Met variant showed higher activity over the wild-type in hydrolyzing a wider range of natural oils. The bulky amino acids, Phe and Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability and inferior enzyme characteristics. We have successfully illustrated that a single point residue changes at oxyanion 114 could result in a myriad of enzyme attributes, which implied there was some interplay between hydrophobicity and conformation for lipase catalytic functions.
基金This work was financially supported by the National Key Research and Development Program of China(2021YFC2102100 and 2018YFA0901400)the Fundamental Research Funds for the Provincial Universities of Zhejiang(RF-C2019005)+1 种基金the National Natural Science Foundation of China(32000898)the Natural Science Foundation of Zhejiang Province(LQ21B060006).
文摘Lipase-catalyzed stereoselective resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate(cis-(±)-1)is an attractive route for the synthesis of(S,S)-2,8-diazobicyclo[4.3.0]nonane,an important chiral intermediate of the fluoroquinolone antibiotic,moxifloxacin.In our previous study,a lipase from Sporisorium reilianum(SRL)was identified to possess excellent thermostability and pH stability.However,the low enzymatic activity of the SRL is a challenge that must be addressed.A rational design was initially employed for SRL tailoring according to the engineered Candida antarctica lipase B(CALB),resulting in a beneficial variant called SRL-I194N/V195L.Subsequently,two key amino acid residues in loop 6,L145 and L154,which might modulate the lid conformation between open and closed,were identified.A tetra-site variant,SRL-I194N/V195L/L145V/L154G(V13),with a significantly enhanced activity of 87.8 U∙mg^(−1) was obtained;this value was 2195-fold higher than that of wild-type SRL.Variant V13 was used to prepare optically pure(2S,3R)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate((2S,3R)-1),resolving 1 mol∙L^(−1) cis-(±)-1 with a conversion of 49.9%in 2 h and absolute stereoselectivity(E>200).Excellent stability with a half-life of 92.5 h was also observed at 50℃.Overall,the study findings reveal a lipase with high activity toward cis-(±)-1 at an industrial level and may offer a general strategy for enhancing the enzyme activity of other lipases and other classes of enzymes with a lid moiety.
文摘The use of enzymatic route for production of biofuels is growing up due the mild reaction conditions that this method provides, as well as reducing SOx emission. To reduce costs, it’s necessary to immobilize the enzyme, making possible to use it continuously as biocatalyst. The aim of this work was to measure the influence of the mass of support and pH used for immobilization of commercial lipase from Candida rugosa acquired by Sigma laboratory. The immobilization method chosen was adsorption on mesoporous and hydrophobic support MCM 41, this has been treated with nitric acid 10% v/v to remove any organic residue. Then, 20 ml of enzymatic solution in phosphate buffer (pH 6.0, 7.0 and 8.0;50 mM) and 1 g/L was placed under constant stirring with 0.30 and 0.45 g of support. Aliquots were taken from the reaction medium and analyzed by spectrophotometry at 10 minutes intervals. A volume of 0.2 ml of supernatant was put with 1.8 ml of substrate p-NFL at 0.18 g/L, and the absorbance at 410 nm was analyzed. In four cases there was a sharp reduction of supernatant’s activity at first 10 minutes, that ratifies the big affinity of the enzyme for the support and the negative influence of pH about the activity. Using the calibration curve, it was possible to calculate the final activity of each immobilization batch. This work suggests the occurrence of diffusional effects, which means that the enzyme mobility was restricted due the excessive amount of support, and then, it lost a part of accessibility to substrate, reflecting in not expressive activity values, and changing the state of ionization of the components of the system.
基金financially supported by the National Natural Science Foundation of China(42376097)Guangdong Basic and Applied Basic Research Foundation(2023A1515030226,2021A1515010829).
文摘Alkane-based biodiesel is considered the next generation of biodiesel owing to its potential environmental benefits and the fact that it exhibits much higher specific caloric values than traditional biodiesel.However,the formidable obstacle impeding the commercialization of this cutting-edge fuel alternative lies in the cost associated with its production.In this study,an engineered strain Escherichia coli(E.coli)showcasing harmonized coexpression of a lipase(from Thermomyces lanuginosus lipase,TLL)and a fatty acid photodecarboxylase(from Chlorella variabilis,CvFAP)was first constructed to transform triglycerides into alkanes.The potential of E.coli BL21(DE3)/pRSFDuet-1-TLL-CvFAP for alkane synthesis was evaluated with tripalmitin as a model substrate under various process conditions.Following a comprehensive examination of the reaction parameters,the scope of the biotransformation was expanded to‘real’substrates(vegetable oils).The results showed that bioderived oils can be transformed into alkanes with high yields(0.80-10.20 mmol·L^(-1))under mild conditions(35℃,pH 8.0,and 36 h)and blue light illumination.The selected processes were performed on an increased lab scale(up to 100 ml)with up to 24.77 mmol·L^(-1) tripalmitin,leading to a yield of 18.89 mmol·L^(-1) pentadecane.With the employment of a method for efficiently producing alkanes under mild conditions and a simple procedure to isolate alkanes from the reaction system,the utilization of sustainable biomass as a fundamental feedstock emerges as the primary solution to lower the cost of alkane-based biodiesel.Thus,this study proposes a readily implementable and highly effective approach for alkane-based biodiesel production.
文摘A strain of psychrophilic bacterium, 2-5-10-1, which produces low-temperature lipase, is isolated from the deep sea of Prydz Bay in Southern Ocean. The highest lipase secretion of this strain is observed at 5 degreesC and this temperature is also for optimal growth. Tween 80 and olive oil enhance secretion of lipase. The optimal temperature and pH for lipase activity are 35 degreesC and 7.5 degreesC respectively. At 0degreesC, the lipase still has 37% relative enzyme activity. The lipase shows high thermolability, more than 50% activity lost after incubation at 60 degreesC for 15 min. EDTA has no effect on lipase activity, indicating the lipase activity is independent of divalent cation. In contrast, the lipase activity is inhibited drastically by Cu2+ and Zn2+.
基金The article was a part of the research program financed by the Science and Technology Bureau of Hebei Province, China (06220106D)
文摘A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 (Pseudomonas sp.) is described, whereby a lipase gene (lip) was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.
基金supported by the National Programs for High Technology Research and Development Program of China(863 Program)(Grant No.2009ZX10004-304)the National Natural Science Foundation of China(NSFC)(Grant No.30870486)the National Major Project(Grant Nos.2009ZX09311-00,2006AA02A322).
文摘Fusarium graminearum(sexual stage:Gibberella zeae)is the causative agent of Fusarium Head Blight(FHB),which is one of the most destructive plant disease of cereals,accounting for high grain yield losses,especially for wheat and maize.Like other fungal pathogens,several extracellular enzymes secreted by G.zeae are known to be involved in host infection.Among these secreted lipases,G.zeae lipase(GZEL),which is encoded by the FGL1 gene,was demonstrated to be crucial to G.zeae pathogenicity.However,the precise mechanism of GZEL remains unclear due to a lack of detailed structural information.In this study,we report the crystal structure of GZEL at the atomic level.The structure of GZEL displays distinct structural differences compared to reported homologues and indicates a unique“double lock”enzymatic mechanism.To gain insight into substrate/inhibitor recognition,we proposed a model of GZEL in complex with substrate and the lipase inhibitor ebelactone B(based on the reported structures of GZEL homologues),which defines possible substrate binding sites within the catalytic cleft and suggests an“anti sn-l”binding mode.These results pave the way to elucidating the mechanism of GZEL and thus provide clues for the design of anti-FHB inhibitors.
文摘The production of extra cellular lipase in Solid State Fermentation (SSF) using Yarrowia lipolytica NCIM 3589 with Palm Kernal cake (Elaeis guineensis) has been studied. Different parameters such as incubation time, inoculum level, initial moisture content, carbon level and nitrogen level of the medium were optimized. Screening of various process variables has been accomplished with the help of Plackett-Burman design. The maximum lipase activity of 18.58 units per gram of dry fermented substrate (U/gds) was observed with the substrate of Palm Kernal cake in four days of fermentation.
基金financially supported by the China Ocean Mineral Resources R&D Association project(No.DY 135-B2-11)the China-ASEAN Maritime Cooperation Fund。
文摘A novel lipase gene(lip4346)encoding a primary translation product with 176 amino acids was screened from the genome fine mapping of the macroalgae-associated bacterial strain Microbulbifer sp.YNDZ01.Macroalgae were collected from the coast of the Halmahera Island of Indonesia.The lip4346 gene was cloned and heterologously expressed in Escherichia coli.The purified recombinant Lip4346 protein had a molecular mass of 19 k Da,a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and a maximum enzyme activity of 31.2 U m L-1.The optimal temperature and pH for the lipase activity of Lip4346 were 70℃and 10.0,respectively.Lip4346 was tolerant with a number of organic solvents and detergents,and was active toward triacylglycerols and p-nitrophenyl esters with short-and medium-chain lengths.The unique characteristics of Lip4346 indicate that it is a promising nonaqueous biocatalyst for industrial applications.
基金supported by the National Natural Science Foundation of China(Grant Nos.:82160739,81922070,81973286,and 81973393)Sailing Special Project of Shanghai Rising-Star Program(Grant No.:22YF1441500)+6 种基金Program for Innovative Leading Talents of Qinghai Province(2018&2019)Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(Grant No.:ZYYCXTD-D-202004)Shanghai Science and Technology Innovation Action Plans(Grant Nos.:20S21901500 and 20S21900900)supported by the Shanghai Science and Technology CommitteeProject of the National Multidisciplinary Innovation Team of Traditional Chinese Medicine supported by the National Administration of Traditional Chinese MedicineKey R&D and Transformation Science and Technology Cooperation Project of Qinghai Province(Grant No.:2019-HZ-819)Basic Public Welfare Research Program of Zhejiang Province(Grant No.:LGF22H280012).
文摘Although herbal medicines(HMs)are widely used in the prevention and treatment of obesity and obesity-associated disorders,the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood.Recently,we assessed the inhibitory potentials of several HMs against human pancreatic lipase(hPL,a key therapeutic target for human obesity),among which the root-extract of Rhodiola crenulata(ERC)showed the most potent anti-hPL activity.In this study,we adopted an integrated strategy,involving bioactivity-guided fractionation techniques,chemical profiling,and biochemical assays,to identify the key anti-hPL constituents in ERC.Nine ERC fractions(retention time=12.5e35 min),obtained using reverse-phase liquid chromatography,showed strong anti-hPL activity,while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS/MS).Among the identified ERC constituents,1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(PGG)and catechin gallate(CG)showed the most potent anti-hPL activity,with pIC50 values of 7.59±0.03 and 7.68±0.23,respectively.Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner,with inhibition constant(Ki)values of 0.012 and 0.082 mM,respectively.Collectively,our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC,as well as to elucidate their anti-hPL mechanisms.These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.
文摘Obesity is usually considered as an overweight or excess body fat, leading to increased health problems. Obesity is a major risk factor for a number of serious diseases. Decreasing dietary fat absorption, through inhibition of pancreatic lipase activity, has been reported to be one of the most effective ways for managing obesity. The present study was aimed at investigating lipase inhibitors from edible plants. A lipase inhibitor was isolated from n-hexane and ethyl acetate extracts of the ripe fruits of Solanum stramonifolium Jacq. by column chromatography and identified by spectral analysis. Its structure was elucidated as (22R)-3β-benzoyloxy-22-hydroxy-4α-methyl-5α-stigmast-7-en-6-one or carpesterol (1). Carpesterol exhibited moderate lipase inhibition activity with IC50 value of 56.0 μg/mL while orlistat, a well- know pancreatic lipase inhibitor, had IC50 value of 3.5 ng/mL. Moreover, the kinetic properties of carpesterol on pancreatic lipase were evaluated. Carpesterol is a competitive inhibitor and exhibited antagonistic interaction when combined with orlistat on lipase inhibition activity.
文摘The purification of a lipase isoenzyme from an Aspergillus niger lipase A is reported in this manuscript. Purification was carried out in a simple adsorption step, in which the lipase was offered at low ionic strength to the commercially available C8 modified magnetic particles, MaKProt C8. When the isoenzyme was desorbed with a 0.2% solution of Triton X-100, the SDS-PAGE gel showed a single pure band with a molecular weight of 35 KDa. The purified fraction showed 66.75-fold purification compared with the crude extract. The pure fraction was characterized along with the crude extract and the lipase adsorbed on the MaKProt C8. The purified and the adsorbed lipase showed better activity for the tested substrates (p-nitrophenyl acetate, decanoate, myristate and palmitate) than the crude extract, the preferred substrates being myristate (26.7 μmol·min-1·mg-1) and decanoate (17.4 μmol·min-1·mg-1), respectively. The temperature and pH profiles showed no change for the three enzymes, the optimum temperature being 37°C and the best pH 7.0.
文摘The aim of the present work was to isolate <em>Bacillus</em> spp. With high lipase activity;to characterize the isolates using both biochemical and molecular methods;to produce lipase using <em>Bacillus</em> isolates and to study the biochemical and biophysical characteristics of the produced lipase. Sixty five <em>Bacillus</em> isolates were isolated from soil 20 isolates from guar field soil (G), 15 isolates from Abusabein field soil (B), 15 isolates from sun flower field soil (S) and 15 isolates from oil effluent (O). Lipase producing isolates were screened;a Chromogenic plate’s method was used. Enzyme activity was quantitatively assayed. Lipase production under submerged fermentation (SMF) conditions using a production medium that contained metal salts, Tween-20 and olive oil as substrate at different period 24, 48, 72 and 96 h, the optimum pH, temperature for lipase activity was determinated and kinetics as well. The isolates showed the highest lipase activity which was identified as Bacillus sp. The optimum pH, temperature, thermostability and kinetic of the produced enzymes were found in three isolates G14, O1 and B10 with the highest enzyme activity and best stability. The isolates G14, O1 and B10 revealed the highest lipase activity of 63.4, 41.2 and 28.3 U/ml, respectively. The results showed optimum pH of the lipase activity from isolates G14, O1 and B10 8.0, 6.0 and 6.0 and the optimum temperature 40, 60 and 75<span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;">˚</span></span></span></span>C, respectively. Lipase enzymes from isolates O1 and B10 were found to be more thermostable after incubation time for 120 min at 90<span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;">˚</span></span></span></span>C. The V<sub>max</sub> and K<sub>m</sub> values of lipase for isolates G14, OI and B10 were 17.6, 135 and 24.4 μmole<span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;">∙</span></span></span></span>min<span style="white-space:nowrap;"><sup><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;"><span style="white-space:nowrap;">−</span></span></span></span></sup></span><sup>1</sup> and 1.3, 1.6 and 0.681 mM, respectively. According to these results <em>Bacillus</em> spp. with high lipase activity and thermostability can be used to promote food, pharmaceuticals, paper, detergents agrochemicals industries and pollution control in Sudan.
基金supported by a grant from the National Basic Research Program of China (Nos.2012CB725204 and 2011CB710803)the National High Technology Research and Development Program of China (No.2014AA021201)+1 种基金Natural Science Foundation of China (No.81302632)the Priority Academic Program Development of Jiangsu Higher Education Institutions and China Postdoctoral Science Foundation (No.2014M551574)
文摘Lipase from Candida sp.99-125 catalyzed ring-opening polymerization of e-caprolactone in the presence of 6-mercapto-1-hexanol was presented as a new metal-free approach for direct synthesis of welldefined thiol-terminated poly(e-caprolactone).Remarkably,high chemoselectivity of lipase from Candida sp.99-125 toward hydroxyl and thiol was exhibited and quantitative thiol fidelity over 90% was achieved.The tedious protecting/deprotecting steps for thiol and metal residue were avoided.The polymerizations with around 70% monomer conversion were conducted in bulk and toluene at relative low temperature of 40 ℃.Number–average molecular weight of resulted polymers ranged from 3000 to4700 Da by changing the feed ratio between monomer and initiator.The structures of obtained thiolterminated poly(e-caprolactone) were demonstrated by combining NMR and SEC analyses.
文摘The aim of this research work was to explore psychrotrophic microbes from soil sample of NyAlesund, Svalbard, arctic region and to investigate their potential use as an effective tool for industrial application. A novel psychrotrophic bacterial strain showed good growth on minimal medium containing lipid as the only carbon source. Microbiological characterisation of the isolate showed that it was a gram negative rod. The strain was tested for the production of extracellular lipase enzyme. The enzymes were partially purified by 90% saturated ammonium sulfate and dialysis for desalting. The bacterium was identified as Pseudomonas sp ADT3 by 16S rRNA amplification and sequencing which had been deposited in the NCBI GenBank with accession number JX914667. Phylogenetic tree was also constructed with MEGA5 software and showed the highest level of sequence similarity with Pseudomonas sp. HC3-13 strain. The microorganism had a growth optimum at pH 8.0 and temperature 22°C. Optimization of different parameters e.g. temperature, pH, incubation time, cofactors etc. was performed for the extracellular lipase activity. The hydrolytic activity of the enzyme was enhanced 5 times by Pb2+ but strongly inhibited by heavy metals Hg2+ as well as EDTA and β-mercaptoethanol. For the molecular weight estimation of enzyme SDS-PAGE was done which showed an inducible band of approximately 13.9 KDa. Activity staining and mass spectrometry techniques were also performed.
文摘A lipase from Sporisorium reilianum SRZ2(SRL)with 73%amino acid sequence identity to Candida antarctica lipase B(CALB)was cloned and overexpressed in Pichia pastoris.The recombinant SRL showed a preference for short-chain p-nitrophenyl esters.It achieved maximum activity at pH 8.0 and 65°C for p-nitrophenyl hexanoate(C6)with Km and kcar/Km values of 0.14 mmol·L-1 and 1712 min 1.mmol·L-1 at 30℃,respec-tively.SRL displayed excellent thermostability and pH stability,retaining more than 79%of its initial activity after incubation at 60℃ for 72h and 75%atpH 3to 11 for 72 h.It also maintained most of its activity in the presence of inhibitors and detergents except sodium dodecyl sulfate,and it tolerated organic solvents.SRL was covalently immobilized and successfully used for ethyl hexanoate synthesis in cyclohexane or in a solvent-free system with a high conversion yield(>95%).Furthermore,high con-version yield was also achieved for the synthesis of various short-chain flavor esters when high substrate concentra-tions of2 mol.LI were applied.This study indicated that a.CALB-type lipase from S.reiliamum SRZ2 showed great potential in organic ester synthesis.