Purification and characterization of a novel fungal alpha-glucosidase from Mortierella alliacea with high starch-hydrolytic activity

The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable alpha-amylase activity. Here, a alpha-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal alpha-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55 degrees C and in a pH range of 5.0-6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-alpha-D-glucoside was optimal at pH 5.0 and a temperature range of 45-50 degrees C.     

Improved production of various polyunsaturated fatty acids through filamentous fungus Mortierella alpina breeding

Studies on the application of functional lipids such as polyunsaturated fatty acids (PUFAs) have proceeded in various fields regarding health and dietary requirements in a search for novel and rich sources. Filamentous fungus Mortierella alpina 1S-4 produces triacylglycerols rich in arachidonic acid, ones reaching 20 g/L and containing 30–70% arachidonic acid as to the total fatty acids. Mutants derived from M. alpina 1S-4, defective in Δ5 and Δ6 desaturases, accumulate triacylglycerols rich in unique PUFAs, i.e., dihomo-γ-linolenic acid and Mead acid, respectively. Furthermore, various mutants derived from M. alpina 1S-4 have led to the production of oils containing n−1, n−3, n−4, n−6, n−7, and n−9 PUFAs. A variety of genes encoding fatty acid desaturases and elongases involved in PUFA biosynthesis in M. alpina 1S-4 has been isolated and characterized. Molecular breeding of M. alpina strains by means of manipulation of these genes facilitates improvement of PUFA productivity and elucidation of the functions of enzymes involved in PUFA biosynthesis.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea

We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity.     

Genome characterization of the oleaginous fungus Mortierella alpina

Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids.     

Unusual fatty acid composition of cerebrosides from the filamentous soil fungus Mortierella alpina

The cerebrosides produced by the soil filamentous fungus Mortierella alpina strain KG-1/95 account for about 13% of the total polar lipids extractable from lyophilised cells with chloroform/methanol mixtures. By means of 1H NMR and (13)C NMR spectroscopy, matrix-assisted laser-desorption ionisation mass spectrometry, and chemical degradation experiment, they have been shown to be 1-O-beta-D-glucopyranosyl-2-N-(2'-D-hydroxyalkanoyl)-9-methylsphinga-4(E),8(E)-dienines, the fatty acid composition of which is unusual and consists of 2-hydroxytridecanoic (4%), 2-hydroxytetradecanoic (60%), 2-hydroxypentadecanoic (20%), and 2-hydroxyhexadecanoic (16%) acids.     

Delta6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus. Gene cloning and its heterologous expression in a fungus, Aspergillus

A DNA fragment was cloned from the fungal strain, Mortierella alpina 1S-4 (which is used industrially to produce arachidonic acid), after PCR amplification with oligonucleotide primers designed based on the sequence information for delta6-desaturase genes (from borage and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (delta9, delta12-18:2) to gamma-linolenic acid (delta6, delta9, delta12-18:3). This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 457 amino acids from a M. calpina 1S-4 library. The predicted amino-acid sequence showed similarity to those of the above delta6-desaturases, and contained a cytochrome b5-like domain at the N-terminus, being different from the yeast delta9-desaturase which has the corresponding domain at the C-terminus. The full-length cDNA clone was expressed under the control of the amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) to the level of 25.2% of the total fatty acids. These findings revealed that the recombinant product has delta6-desaturase activity. The Mortierella delta6-desaturase is the first to be reported in fungi.     

Gene cloning and functional analysis of a second delta 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus

We demonstrated that Mortierella alpina 1S-4 has two delta 6-desaturases, which are involved in the desaturation of linoleic acid to gamma-linolenic acid. For one of the two delta 6-desaturases, designated as delta 6I, gene cloning and its heterologous expression in a fungus, Aspergillus oryzae, has previously been reported. In addition, we indicated in this paper that there is an isozyme of the two delta 6-desaturases, designated as delta 6II, in M. alpina 1S-4. The predicted amino acid sequences of the Mortierella delta 6-desaturases were similar to those of ones from other organisms, i.e. borage and Caenorhabditis elegans, and had a cytochrome b5-like domain at the N-terminus, being different from the yeast delta 9-desaturase, which has the corresponding domain at the C-terminus. The full-length delta 6II cDNA was expressed in A. oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) up to 37% of the total fatty acids. The analysis of real-time quantitative PCR (RTQ-PCR) showed that the quantity of delta 6I RNA was 2.4-, 9-, and 17-fold higher than that of delta 6II RNA on 2, 3, and 4 days in M. alpina 1S-4, respectively. M. alpina 1S-4 is the first fungus to be confirmed to have two functional delta 6-desaturase genes.     

Purification and characterization of an intracellular lipase from pleopods of whiteleg shrimp (Litopenaeus vannamei)

An intracellular lipase present in the whiteleg shrimp Litopenaeus vannamei was detected in pleopods. The lipase from pleopods was purified and characterized by biochemical and kinetic parameters. Purified intracellular lipase has a molecular mass of 196kDa, the polypeptide is assembled by two monomers, 95.26 and 63.36kDa. The enzyme lacks glycosylation, and it has an isoelectric point of 5.0. The enzyme showed the highest activity at a temperature range of 30-40°C at pH 8.0-10.0. Activity was completely inhibited by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate, suggesting that the intracellular lipase is a serine lipase. The lipase hydrolyzes short and long-chain triacylglycerides, as well as naphthol derivatives at comparable rates in contrast to other sources of lipases. Specific activity of 930U mg(-1) and 416.56U mg(-1) was measured using triolein and tristearin at pH 8.0 at 30°C as substrates, respectively. The lipase showed a K(M,app) of 41.03mM and k(cat)/K(M,app) ratio of 4.88 using MUF-butyrate as the substrate. The intracellular lipase described for shrimp has a potential role in hydrolysis of triacylglycerides stored as fat body, as has been shown in humans.     

Effect of temperature and glucose supply on the production of polyunsaturated fatty acids by the fungus Mortierella alpina CBS 343.66 in fermentor cultures

The fungus Mortierella alpina CBS 343.66 was grown in a fermentor at different pH, temperatures and supplies of carbon source (glucose) in order to optimize the culture conditions for rapid biomass and lipid production with a high proportion of polyunsaturated fatty acids, especially arachidonic acid (AA). Good growth and lipid production with 31% AA was obtained at pH 6.5 and 25°C. A temperature decrease to 18°C gave a significantly higher degree of polyunsaturated fatty acids. Eicosapentaenoic acid was not detected at 25°C, but was formed at 18°C at about 10%. The AA concentration but was similar for 18°C about 10%. The AA concentration was similar for 18°C and 25°C (around 30%): 18°C allowed relatively good growth and had a beneficial effect on the fungus morphology, i.e. pellets were formed. Best lipid production and a AA content of up to 33% was achieved at an excess of glucose (carbon source) and a deficit of ammonium chloride (nitrogen source). The percentage of AA of the total fatty acid composition was constant as ong as glucose was present. At glucose exhaustion, the proportion of AA increased to 57%. The increase in AA corresponded to a decrease in palmitic acid, stearic acid and oleic acid. Correspondence to: G. Molin     

Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe. Purification and characterization of intracellular beta-glucosidase

Intracellular beta-glucosidase was extracted from the mycelium of Th. aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity. Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM. With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C. beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins. Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).     

Kinetic analysis of oil biosynthesis by an arachidonic acid-producing fungus, Mortierella alpina 1S-4

 Kinetic analysis of arachidonic acid (AA)-oil biosynthesis by Mortierella alpina 1S-4 growing under lipid-accumulating (LN medium) and non-lipid-accumulating (HN medium) conditions was investigated and compared with industrial AA fermentation. Various kinetic parameters of these cultivation processes demonstrate a characteristic pattern of the lipogenesis in this fungus, where growth phase, phase of oil accumulation and phase of AA synthesis are distinct from each other. The fungus utilizing LN medium synthesized 32.3 g fatty acid 100 g⁻¹ glucose on the 4th day of cultivation and reached the maximum daily fatty acid accumulation (expressed as differential specific rate q _D(FA/B)) of 9.5%. Our results also indicate that a q _D(FA/B) value of about 2.5% might be critical for lipid overproduction in M. alpina. AA was rapidly incorporated into triacylglycerols (90% of total AA) at the later cultivation phase and overall AA yield was directly related to the total yield of fatty acid. Received: 10 December 1999 / Accepted: 13 February 2000     

Long-chain polyunsaturated fatty acids, endothelial lipase and atherosclerosis

Endothelial lipase (EL), a new member of the lipase gene family, was recently cloned and has been shown to have a significant role in modulating the concentrations of plasma high-density lipoprotein levels (HDL). EL is closely related to lipoprotein and hepatic lipases both in structure and function. It is primarily synthesized by endothelial cells, functions at the cell surface, and shows phospholipase A1 activity. Overexpression of EL decreases HDL cholesterol levels whereas blocking its action increases concentrations of HDL cholesterol. Pro-inflammatory cytokines suppress plasma HDL cholesterol concentrations by enhancing the activity of EL. On the other hand, physical exercise and fish oil (a rich source of eicosapentaenoic acid and docosahexaenoic acid) suppress the activity of EL and this, in turn, enhances the plasma concentrations of HDL cholesterol. Thus, EL plays a critical role in the regulation of plasma HDL cholesterol concentrations and thus modulates the development and progression of atherosclerosis. The expression and actions of EL in specific endothelial cells determines the initiation and progression of atherosclerosis locally explaining the patchy nature of atheroma seen, especially, in coronary arteries. Both HDL cholesterol and EPA and DHA enhance endothelial nitric oxide (eNO) and prostacyclin (PGI2) synthesis, which are known to prevent atherosclerosis. On the other hand, pro-inflammatory cytokines augment free radical generation, which are known to inactivate eNO and PGI2. Thus, interactions between EL, pro- and anti-inflammatory cytokines, polyunsaturated fatty acids, and the ability of endothelial cells to generate NO and PGI2 and neutralize the actions of free radicals may play a critical role in atherosclerosis.     

Purification and characterization of a lipase from Pichia burtonii

An extracellular lipase from Pichia burtonii was purified to homogeneity by a combination of DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-100 gel filtration, and isoelectric focusing. The purified enzyme preparation showed a single protein band corresponding to a molecular mass of 51 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be 47 kDa on Superdex 200 gel filtration, suggesting that the enzyme was a monomeric protein. The pI was about 5.8. The optimum pH and temperature for the hydrolysis of olive oil were about 6.5 and 45°C respectively. Rapid loss of the enzyme activity was observed above 30°C in the absence of olive oil, but the addition of olive oil or trimethylolpropane diallyl ether greatly stabilized the enzyme. At 30°C, the enzyme hydrolysed Spans and Tweens as well as simple triglycerides of short- and middle-chain fatty acids. Although the enzyme cleaved all the ester bonds of triolein, it showed some preference for the outer ester bonds.     

Purification and characterization of a lipase from Staphylococcus aureus

An extracellular lipase from Staphylococcus aureus (strain FN 37) was purified to homogeneity. A cell-free culture broth was subjected to ammonium sulphate precipitation, and the lipase was isolated from the resuspended pellet by adsorption chromatography on octyl-Sepharose. The purification was 957-fold, and the recovery of the octyl-Sepharose chromatography was about 100%. The specific activity of the purified lipase was 546 mU of lipase activity per micrograms protein. The purity of the final product was documented by SDS-polyacrylamide gel electrophoresis in which a homogeneous protein band of 43 kDa was found. In gel chromatography on Sephadex G-200 the lipase eluted as a homogeneous peak with an apparent molecular mass of 110 kDa, suggesting that the lipase may exist as an oligomer in physiological media. Analysis of the amino-acid composition revealed a predominance of polar, non-charged amino acids, with serine accounting for 24 mol% of the amino-acid residues.     

Purification and characterization of hepatic lipase from Todarodes pacificus

Lipase was purified from squid (Todarodes pacificus) liver in an attempt to investigate the possibility of applying the enzyme for biotechnological applications. Crude extract of squid liver was initially fractionated by the batch type ion exchange chromatography. The fraction containing lipase activity was further purified with an octyl-Sepharose column. Finally, lipase was purified by eluting active protein from a non-dissociating polyacrylamide gel after zymographic analysis. Molecular weight of the purified enzyme was determined to be 27 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at a temperature range of 35-40 degrees C and at pH 8.0. The activity was almost completely inhibited at 1 mM concentration of Hg(2+) or Cu(2+) ion. Partial amino acid sequence of the enzyme was also determined.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta

A peroxidase oxidizing Mn²⁺ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn²⁺-mediated as well as Mn²⁺-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.     

Purification of lipase from Aspergillus niger and its characterization analysis

从黑曲霉发酵液中经硫酸铵分级沉淀,Phenyl-Sepharose疏水柱层析,DEAE-Sepharose 4B阴离子交换柱得到电泳纯的脂肪酶,纯化倍数达10倍,回收率50％.对脂肪酶的性质分析表明:该酶分子质量约为35 kDa,最适温度和最适pH分别为37℃和9.5,50℃以下和pH6.0～11.0之间保持稳定,属于碱性脂肪酶.Mg2+、Ca2+、Cu2+、Zn2+、Co2+、Mn2+对该酶有激活作用,而Al3+、Fe2+、Fe3+对酶有严重抑制作用.变性剂盐酸胍和脲对其未见显著影响,而SDS强烈抑制其酶活.用不同氨基酸修饰剂对酶进行修饰,其中NBS和PMSF强烈抑制该酶活性,NBSF和DTT在低浓度下对酶活影响不大,2,3-丁二酮在高浓度下影响其活性.外加稳定剂如NaCl、PEG、甘油、山梨醇、海藻酸钠,均可不同程度的延长脂肪酶的半衰期.在一定质量比条件下,该酶有良好的抗蛋白酶性质.