黑曲霉F044脂肪酶的分离纯化及酶学性质研究

黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAESepharoseFastFlow阴离子交换层析和SephadexG-75凝胶过滤层析得到电泳纯的脂肪酶,纯化倍数为73·71倍,活性回收率为34%。对纯化脂肪酶性质研究表明:该脂肪酶分子量约为35~40kD,水解橄榄油的最适温度和最适pH分别为45℃和7·0,在60℃以下和pH2·0~9·0之间有很好的稳定性。该脂肪酶的水解活性对Ca2 表现明显的依赖性,而Mn2 、Fe2 和Zn2 对脂肪酶则有显著的抑制作用。在最适条件下水解pNPP的Km和Vmax分别为7·37mmol/L和25·91μmol/(min·mg)。其N-端的15个氨基酸序列为Ser(Glu/His)-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。     

Effects of dietary saturated and polyunsaturated fat on lipoprotein lipase and hepatic triglyceride lipase activity

The effects of saturated and polyunsaturated dietary fat on the lipolytic activity of post-heparin plasma, lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were studied in the rat. The lipolytic activity was studied from 0 to 60 min using labelled chylomicrons as the substrate. Triacylglycerol hydrolysis rate was higher for the plasma of rats fed high fat diets (14% fat by weight). Chylomicrons of rats fed saturated or unsaturated fats were hydrolyzed at the same rate within the first 15 min but afterwards hydrolysis of chylomicrons of rats fed saturated fat was slower. The activities of LPL and HTGL were increased by high fat diets. Unsaturated fat increased more LPL activity than saturated fat conversely, HTGL activity was enhanced more by saturated fat than by unsaturated fat.     

Effect of sodium taurodeoxycholate, CaCl2 and albumin on the action of pancreatic lipase on droplets of trioleoylglycerol and the release of lipolytic products into aqueous media

1. Effects of various substances on the activity of pancreatic lipase and on the release of lipolytic products into aqueous media were studied with droplets of trioleoylglycerol suspended from a membrane filter at the top of a flow-through chamber. The droplets were perifused for 7 min with a commercial preparation of pancreatic lipase in 0.15 M NaCl solution at pH 6.5 and then perifused for 60 min with lipase-free media, either 0.15 M NaCl at pH 6.5 or basal medium at pH 7.4 (70 mM sodium barbital) containing different additives. 2. About 6% of the trioleoylglycerol in droplets was hydrolyzed during the perifusion with lipase. Another 15% was hydrolyzed in 30 min, but none thereafter, when the droplets were perifused with 0.15 M NaCl alone. The rate of hydrolysis was doubled and prolonged when droplets were perifused with basal medium at pH 7.4. Lipolytic products formed at pH 7.4 were 62% oleic acid, 20% monooleoylglycerol and 18% dioleoylglycerol, yet only 4% of the lipolytic products were released into the perifusate. 3. Sodium taurodeoxycholate (TDC) (17 mM ) added to basal medium increased 18 x the amount of lipolytic products released into the perifusate but increased lipolysis only 13%. The molar ratio of oleic acid to monooleoylglycerol in the perifusate was 5.7 during the first 30 min and 4.0 during the last 30 min. 4. Ca2+ (3.3 mM) added to basal medium increased lipolysis 87% but did not affect the amount (4%) of lipolytic products released into the perifusing medium. 5. TDC and Ca2+ added to basal medium produced the largest increase in lipolysis, with 59% of trioleoylglycerol hydrolyzed in 15 min and 91% in 60 min. The amount of lipolytic products released into the perifusing medium, however, was not increased above that released into medium containing TDC alone. 6. Serum albumin (0.6 mM) and Ca2+ added to basal medium increased 14 x the amount of lipolytic products released into the perifusate without affecting the basal lipolytic rate. Albumin, however, suppressed by 40% the stimulatory effect of Ca2+ on pancreatic lipase activity.     

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.     

Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity Exploratories

Microbial metagenomes derived from soils are rich sources for the discovery of novel genes and biocatalysts. Fourteen environmental plasmid and seven fosmid libraries obtained from 10 German forest soils (A horizons) and six grassland soils (A and B horizons) were screened for genes conferring lipolytic activity. The libraries comprised approximately 29.3 Gb of cloned soil DNA. Partial activity-based screening of the constructed libraries resulted in the identification of 37 unique lipolytic clones. The amino acid sequences of the 37 corresponding lipolytic gene products shared 29-90% identity to other lipolytic enzymes, which were mainly uncharacterized or derived from uncultured microorganisms. Multiple sequence alignments and phylogenetic tree analysis revealed that 35 of the predicted proteins were new members of known families of lipolytic enzymes. The remaining two gene products represent two putatively new families. In addition, sequence analysis indicated that two genes encode true lipases, whereas the other genes encode esterases. The determination of substrate specificity and chain-length selectivity using different triacylglycerides and p-nitrophenyl esters of fatty acids as substrates supported the classification of the esterases.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Cloning, expression, and characterization of a cold-adapted lipase gene from an antarctic deep-sea psychrotrophic bacterium, Psychrobacter sp 7195

A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at 30 degrees C, and was unstable at temperatures higher than 30 degrees C, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24 h incubation at 4 degrees C. The addition of Ca2+ and Mg2+ enhanced the enzyme activity of LipA1, whereas the Cd2, Zn2+, Co2+, Fe3+, Hg2+, Fe2+, Rb2+, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate (C14 acyl groups).     

Intracellular lipase activities in heart and skeletal muscle homogenates. The absence of trierucin cleavage by the heart: a possible biochemical basis for erucic acid lipidosis.

Rat heart and skeletal muscle homogenates were compared for their intracellular lipolytic activity towards a series of saturated and unsaturated triglycerides from trilaurin (C12:0) to trierucin (C22:1). It is shown that for all triglycerides esterified with fatty acids from C12 to C18, lipolytic activity in heart homogenates was higher than in skeletal muscle homogenates. For these triglycerides there was no relationship between the fatty acid chain length and the lipolytic activity. In both homogenates cleavage of unsaturated triglycerides was higher than cleavage of the homologous saturated triglyceride. Lipolysis of tri-delta-11-eicosenoin (C20:1) was similar in both homogenates but much lower than lypolysis of other triglycerides. Although cleavage of trierucin (C22:1) was very low in skeletal muscle homogenates, it was undetectable in heart homogenates, even when enzyme concentration was increased. A mixture of triglycerides did not show preferential hydrolysis of any simple triglyceride. Trierucin was the only triglyceride that did not complete for lipolytic activity and only with heart homogenates, which shows that that lipase(s) do not cleave trierucin. The absence of lipolytic activity towards trierucin in heart homogenates could explain the selective accumulation of erucic acid-rich triglycerides in hearts of animals fed a diet with a high erucic acid content.     

Recombinant Candida rugosa lipase 2 from Pichia pastoris: immobilization and use as biocatalyst in a stereoselective reaction

The characterization of the recombinant Candida rugosa Lip2 (r-Lip2) isoenzyme obtained from fed-batch cultures of Pichia pastoris under PAOX promoter was carried out, determining the optimal pH and temperature as well as their catalytic performance in both hydrolysis and synthesis reactions comparing with purified native Lip2 (n-Lip2) previously determined. The substrate specificity of r-Lip2 in hydrolysis reactions was determined with a series of triacylglycerols and p-nitrophenyl esters of variable acyl chain length. r-Lip2 showed the maximum specificity for both substrates towards medium-chain esters (C-8), similar behavior was observed with n-Lip2. However, significant differences were observed towards unsaturated substrates (triolein) or short-chain esters. A statistical design applied to study the effect of pH and temperature on lipase stability shown that r-Lip2, like n-Lip2, was more sensitive to pH than temperature changes. Nevertheless, the overall stability of soluble r-Lip2 was lower than soluble n-Lip2. The stability of r-lip2 was significantly improved by immobilization onto EP100, an excellent support for lipases with yields around 95% for offered lipolytic activity lower than 600 AU/mL. Finally, immobilized r-Lip2 was tested in the resolution of ibuprofen in isooctane by means of enantioselective esterification using 1-butanol as esterifying agent. r-Lip2 showed a better performance in terms of enantiomeric excess (74%) and enatiomeric factor (96%) than n-Lip2 (56 and 80%, respectively) for the same conversion (40%). Thus, r-Lip2 should be considered a good and pure biocatalyst, easy to produce and with a remaining activity of ca. 90% after one reaction cycle when immobilized on EP100.     

Quantification of intra- and extra-cellular thermophilic lipase/esterase production by Thermus sp.

Four Thermus strains produced lipolytic activity when grown in liquid medium for 30 h at 70 degrees C. The highest total lipase/esterase activity (57 U l(-1)) was in Thermus aquaticus YT-1, followed by Thermus thermophilus HB27 and HB8 (33 and 25 U l(-1), respectively), and finally by Thermus sp. (16 U l(-1)). Extra-cellular activity was detected in T. aquaticus YT-1 and T. thermophilus HB27 (33 and 17 U l(-1)). All enzymes were stable at 80 degrees C over 30 min, and their activity towards fatty acid esters increased as substrate chain-length diminished (i.e. hydrolysis rate was up to 6-fold higher on p-nitrophenyl caproate than on laurate).     

The hydrolysis of triacylglycerol and diacylglycerol by a rat brain microsomal lipase with an acidic pH optimum.

Lipase activity towards triacylglycerol and diacylglycerol was measured at pH 4.8 using a microsomal preparation from rat brain as the enzyme source. The optimal pH for the hydrolysis of triacylglycerol was 4.8, with only minor lipolytic activity in the alkaline pH range. Diacylglycerol was the major product of triacylglycerol hydrolysis, with only little monoacylglycerol being formed. When diacylglycerol was the starting substrate it was hydrolyzed at a rate 10-fold greater than triacylglycerol, and the product was monoacylglycerol. The enzyme showed positional specificity for the fatty acid moieties located at the primary positions of sn-glycerol. 1,3-Diacylglycerol was hydrolyzed at greater than twice the rate of the corresponding 1,2(2,3)-isomer.     

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas I. Studies on neutral glycerides

The substrate specificity of two cationic lipases with high phospholipase A₁ activity purified from guinea pig pancreas has been tested towards various neutral glycerides. Triolein hydrolysis proceeded in the absence of di- and monoolein accumulation. Optimal conditions for di- and monoolein hydrolysis included an alkaline pH (9–10), a substrate concentration of 10 mM, and the presence of sodium deoxycholate (12 and 24 mM, respectively). Pancreatic colipase (bovine) had no effect on the activity of the two lipases. The comparison between the rates of hydrolysis of various substrates revealed the following order of decreasing enzyme activity: diolein > 1(3)-monoolein > tributyrin = triacetin ⩾ triolein = 2-monoolein. No hydrolysis of p-nitrophenylacetate and cholesteryloleate could be detected. Using 1-[³H]palmitoyl-2-[¹⁴C]linoleoyl-sn-glycerol, both enzymes displayed a strong preference for the 1-position, leading to the accumulation of 2-[¹⁴C]linoleoyl-sn-glycerol. Identical activities were found for the two lipases. It is concluded that the two cationic lipases from guinea pig pancreas represent a unique group of lipolytic enzymes different from other previously described enzymes, including classical pancreatic lipase, gastric and lingual enzymes, mold lipases and carboxylesterhydrolase.     

Identification of a carboxylesterase-producing Rhodococcus soil isolate

Subtropical soil microbial isolates were screened for carbohydrate, tributyrin, or olive oil hydrolysis using agar plates supplemented with the corresponding substrates. A heterotrophic, aerobic, Gram-positive strain displaying activity on tributyrin was selected and further characterized. Analysis of the morphological and physiological traits of the strain placed it as a member of the genus Rhodococcus. Further 16S rDNA sequencing revealed a 99% identity to Rhodococcus erythropolis. The strain displayed lipolytic activity on fatty-acid-derivative substrates of short chain length, with cell extract fractions having highest activity, as confirmed by the presence, after zymogram analysis, of a ca. 60-kDa intracellular protein band with activity on 4-methylumbelliferone-butyrate substrate. The presence of such a lipolytic enzyme, similar to those found in other Gram-positive bacteria, indicates that the strain could be of interest for certain biotechnological applications, like the synthesis of pharmaceuticals or biocide detoxification.     

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent K_M was 6.5 mmol l^-1, with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l^-1 NaCl and on the presence of at least 0.1 mol l^-1 NaCl in the test mixture. Desoxycholate and up to 0.1 mol l^-1 CaCl₂ also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.     

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K _m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.     

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40°C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.     

The 44-kDa c-type cytochrome induced in Rhodobacter capsulatus during growth with dimethylsulphoxide as an electron acceptor is a cytochrome c peroxidase

Abstract The propionate (Pro), decanoate (Dec) and laurate (Lau) esters of 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline-3-acetic acid were assessed as substrates for lipase and esterase. On hydrolysis these substrates yield an intensely red coloured phenol which could be assayed at 505 nm. The Pro ester was an effective substrate for porcine esterase and was hydrolysed at a rate 20 times greater than the Lau and Dec esters. Conversely, Pseudomonas Lipase had a high activity towards the Lau and Dec esters, especially in the presence of bovine serum albumin, but little activity towards the Pro ester. The Dec and Lau were used to detect lipolytic activity in Pseudomonas strains associated with milk spoilage. For this purpose, the substrates were absorbed onto filter paper disks, which were placed over bacterial colonies growing on agar plates; activity was indicated by bright red colouration of discs within 2 h. Escherichia coli colonies hydrolysed the Pro but not the Lau or Dec esters.     

Alteration of substrate specificity of Galactomyces geotrichum BT107 lipase I on eicosapentaenoic acid-rich triglycerides

One cycle of random mutagenesis and screening of an expression mutant library in P. pastoris was used to isolate two variants of Galactomyces geotrichum BT107 lipase I showing a 2-fold reduction in their hydrolytic activity towards the homogeneous triglyceride of eicosapentaenoic acid (triEPA, C20:5 n-3) compared with the wild-type enzyme. Only one amino acid substitution on each variant was enough to decrease their activity on this polyunsaturated substrate. The activity of the enzyme for triglycerides containing oleic acid was scarcely affected. Results obtained after hydrolysis of commercial marine oil confirmed the low lipolytic activity of one of the variants toward glycerides containing EPA. The amino acid substitutions were located in the lid region pointing out the role of this structure on substrate selectivity. This is a good starting point to obtain enzyme variants than can be used to enrich oils in PUFAs fraction, mainly EPA, from fish oils.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc](2) and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K(m) and k(cat) values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4-G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2-G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1-G5, and then the high-M(r) intermediates (G3-G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.