Induced Formation of Lipase by Geotrichum candidum Link

It was recognized that Geotrichum candidum Link which was selected as the efficient lipase producer formed lipase only in the presence of substrate or its relating compounds such as oils or fatty acids in a cultivation medium. From the experimental results obtained by the cultivation of the microorganism and also by using of washed cells, it seemed that lipase was formed inducibly. It is likely that the produced lipase is localized around the cell wall and membrane and it is released from the cells after a certain period from the inducible synthesis.     

Purification,Crystallization and Some Properties of Lipase from Geotrichum candidum Link

Lipase (EC 3.1.1.3) of Geotrichum candidum Link was purified by means of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, gel-filtration on Sephadex G–100 and Sephadex G–200, and was finally crystallized in concentrated aqueous solution. It was confirmed that the crystallized preparation was homogeneous electrophoretically and ultracentrifugally.

It was estimated with the crystalline enzyme that the sedimentation constant (s₂₀, w) was 4.0, the isoelectric point was pH 4.33, and the molecular weight was 53,000~55,000. From the result of amino acid analysis, none of sulfur containing amino acid was detected in the enzyme. It was also recognized that the crystalline preparation contained about 7% of the carbohydrate and very small amount of lipid. It was characterized that the lipase was the most active at pH 5.6~7.0 on olive oil, at 40°C and was stable in the range of pH 4.2 to 9.8 at 30°C for 24 hr, and was stable below 55°C for 15 min.     

Some Properties of the Lipase of Geotrichum candidum Evaluated by a Fluorimetric Assay Technique

The oleic acid ester of 4-methylumbelliferone was found to be a suitable substrate for the fluorimetric assay of the lipase of Geotrichum candidum . This substrate spontaneously formed an emulsion in the assay mixture and had a K _m of 10·3 μM. The fluorimetric method was at least a thousand times more sensitive than the conventional potentiometric method. The lower limit of sensitivity of the assay was 1 nmole fatty acid released per min. Optimum conditions for detection of lipase in the culture filtrate of G. candidum were devised. The influence of pH, Ca²⁺ and several other factors were investigated.     

多拷贝毕赤酵母重组菌表达GCL摇瓶优化和高密度发酵

目的:构建高效表达白地霉脂肪酶的毕赤酵母重组菌株,并对筛选得到的菌株进行摇瓶发酵条件优化和分批补料高密度发酵工艺研究。方法:将诱导型表达载体pPIC9K-gcl电转化至毕赤酵母GS115。通过橄榄油-罗丹明B平板和摇瓶发酵筛选高脂肪酶活力的重组菌株,运用基于TaqMan探针的实时荧光定量PCR 法确定其拷贝数,并对菌株进行摇瓶发酵条件优化。在此基础上,研究重组菌在3L 发酵罐中的高密度发酵工艺。结果:筛选得到一株具有3 个白地霉脂肪酶基因拷贝的菌株GS115/pPIC9K-gcl 78#,初始酶活力为220 U/ml。当摇瓶发酵条件为甲醇诱导96 h,每24 h甲醇添加量1 %,接种量2 %,培养基初始pH 7.0,500 ml摇瓶装液量50 ml,甲醇诱导温度25℃ 时酶活力达735 U/ml。3L 发酵罐高密度发酵176.5 h,酶活力达到3360 U/ml,总蛋白含量达到4.30 g/L,且发酵过程中细胞活性一直保持在96 % 以上。结论:基因拷贝数与重组菌株的产酶水平呈正相关,摇瓶优化可显著提高重组菌株的产酶能力,为白地霉脂肪酶的工业化生产奠定了技术基础。     

Production of Sulfur Flavors by Ten Strains of Geotrichum candidum

Ten strains of Geotrichum candidum were studied on a liquid cheese model medium for the production of sulfur compounds which contribute to the aroma of cheeses. The volatile components produced by each cultured strain were extracted by dynamic headspace extractions, separated and quantified by gas chromatography (GC), and identified by GC-mass spectrometry. It was shown that four strains of this microorganism produced significant quantities of S-methyl thioacetate, S-methyl thiopropionate, S-methyl thiobutanoate, S-methyl thioisobutanoate, S-methyl thioisovalerate, and S-methyl thiohexanoate. This is the first example of the production of these compounds by a fungus. In addition, dimethyldisulfide, dimethyltrisulfide, dimethylsulfide, and methanethiol, which are more commonly associated with the development of cheese flavor in bacterial cultures, were also produced by G. candidum in various yields, depending on the strain selected. The potential application of these strains in cultured microbial associations to produce modified cheeses with more desirable organoleptic properties is discussed.     

Production of sulfur flavors by ten strains of Geotrichum candidum

Ten strains of Geotrichum candidum were studied on a liquid cheese model medium for the production of sulfur compounds which contribute to the aroma of cheeses. The volatile components produced by each cultured strain were extracted by dynamic headspace extractions, separated and quantified by gas chromatography (GC), and identified by GC-mass spectrometry. It was shown that four strains of this microorganism produced significant quantities of S-methyl thioacetate, S-methyl thiopropionate, S-methyl thiobutanoate, S-methyl thioisobutanoate, S-methyl thioisovalerate, and S-methyl thiohexanoate. This is the first example of the production of these compounds by a fungus. In addition, dimethyldisulfide, dimethyltrisulfide, dimethylsulfide, and methanethiol, which are more commonly associated with the development of cheese flavor in bacterial cultures, were also produced by G. candidum in various yields, depending on the strain selected. The potential application of these strains in cultured microbial associations to produce modified cheeses with more desirable organoleptic properties is discussed.     

白地霉脂肪酶在毕赤酵母表面展示

将白地霉脂肪酶基因N端与酿酒酵母FLO絮凝结构域序列融合,构建成脂肪酶毕赤酵母表面展示载体并转化毕赤酵母GS115。免疫荧光检测证实脂肪酶已展示于毕赤酵母细胞表面。甲醇诱导96 h后展示酶活性达到81 U/g干细胞,酶的热稳定性较游离酶有较大提高,50℃孵育4 h后酶活仍保持初始酶活70%以上。     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

产低温脂肪酶菌株Geotrichum candidum ch-3的选育、发酵条件及酶学性质研究

从新疆昌吉市油脂化工厂的含油冻土中筛选到一株产低温脂肪酶的菌株ch-3,形态鉴定及18SrDNA序列分析表明该菌株为白地霉,命名为Geotrichum candidum ch-3。我们对其生长及产酶情况和酶性质做了初步研究。该菌株的最适生长温度是20℃。玉米浆、豆油可显著促进脂肪酶的产生。粗酶液经双水相萃取和Sephadex G-75凝胶过滤分离纯化后进行酶学性质的研究。该脂肪酶最适作用温度为35℃,在0℃可保持66%的相对酶活性,最适pH值为7.0,对热较敏感,50℃处理20min,剩余酶活为55%,Fe2＋、Cu2＋、Mn2＋以及Ca2＋对酶有较强的激活作用。     

Le Geotrichum candidum Link,cause possible de graves enterites chez les pintadeaux

The ‘transmissible enteritis’ of Guinea fowl chick, which appears in less than 12 weeks birds, is characterized by a yellowish diarrhea and a general dehydratation, a swelling of caecums, a large mortality. The mycological analysis of food points out that it is not a classical mycotoxicosis. On the other hand, the isolation ofGeotrichum candidum from the intestine of dead animals let presume that this fungus plays a role in the disease. There is no transmission by egg. The dungs, the litters and the drink water can be origin of contamination. Experimental infections with cultures ofGeotrichum are actually unsuccessful.     

Bioconversion of glyceryl trinitrate into mononitrates by Geotrichum candidum

Glyceryl trinitrate (GTN) 2 mM was quantitatively converted into its 1 and 2 mononitrate derivatives by Geotrichum candidum, with consumption of the nitrite ions produced. The conversion proceeded at a rate independent of the addition of either organic carbon or organic nitrogen sources. Eight batches of nitrate ester, which were added every 24 hours, were successfully converted as far as during the bioconversion process GTN concentration did not exceed 2 mM. When those limiting conditions were not observed, dramatic toxicity of GTN was noticed.     

Ribosomal DNA polymorphisms in the yeast Geotrichum candidum

The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution.     

Purification and Characterization of Anti-Listeria Compounds Produced by Geotrichum candidum

Geotrichum candidum can produce and excrete compounds that inhibit Listeria monocytogenes. These were purified by ultrafiltration, centrifugal partition chromatography, thin-layer chromatography, gel filtration, and high-pressure liquid chromatography, and analyzed by liquid chromatography-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation. Two inhibitors were identified: d-3-phenyllactic acid and d-3-indollactic acid.

Contamination by Listeria has become a problem over the past 20 years in many parts of the world. The ubiquitous nature of Listeria monocytogenes, its capacity to multiply at refrigeration temperatures, its thermal tolerance (11), and its resistance to relatively low pH (it can multiply at pH 5.3 and 4°C and at pH 4.39 and 30°C) (5), together with its tolerance of high salt concentrations (4, 18), make controlling this potentially pathogenic microorganism in food products difficult. This bacterium has been incriminated in several cases of food poisoning (2, 10, 19). At risk are the immunodepressed, the old, pregnant women, fetuses, and newborn babies. Several groups have worked on biological control. As a result, many bacteriocins, which inhibit the growth of L. monocytogenes, have been isolated, purified, and characterized (12, 13, 16, 18). We have worked with Geotrichum candidum, a yeast-like member of the natural milk flora that is used as a maturing agent for soft and hard cheeses. In an extensive study carried out in 1984 (7), the interactions between G. candidum and the microflora in cheeses were examined. G. candidum inhibited the growth of gram-negative bacteria, gram-positive bacteria, and fungi (6). We recently showed (3) that G. candidum inhibits the growth of L. monocytogenes on both solid and liquid media (a bacteriostatic effect). The inhibitors are stable over a wide pH range and can be heated to 120°C for 20 min. The present report describes the purification and characterization of compounds responsible for this antibacterial action.Microorganisms, culture conditions, and detection of inhibitory activity.The strain of G. candidum used came from the collection of the Caen University Food Microbiology Laboratory, Caen, France (UCMA G91) and was initially isolated from a cheese, Pont l’Evêque. One percent of a preculture (optical density at 620 nm [Milton Roy Spectronic 301; Bioblock Scientific, Illkirch, France] of 0.7 [10⁷ arthrospores or hyphae/ml]) of G. candidum was grown in a fermentor (20 liters; Biolafitte type PI) in 15 liters of Trypticase soy broth (30 g/liter; Biomerieux, Marcy l’Etoile, France) with yeast extract (6 g/liter; AES, Combourg, France) (TSBYE) buffered to pH 6.3 with 0.1 M citrate-0.2 M phosphate. The culture was stirred at 300 rpm for 64 h at 25°C under a pressure of 0.2 bar and was then filtered through a 1,000-Da cut-off membrane by tangential ultrafiltration (Sartorius, Palaiseau, France) under a pressure of 2 bars. The resulting ultrafiltrate was sterilized by passage through a capsule (Sartorius) containing 0.45-μm- and 0.2-μm-pore-size membranes.The inhibition of L. monocytogenes was checked at each purification step by the agar diffusion well assay (3). Antimicrobial activity was estimated by measuring the diameter of the inhibitory halo on two right-angle axes (average of two plates). The strain of L. monocytogenes (UCMA L205) (serovar 1/2a; Centre National de Référence des Listeria, Nantes, France) and lysovar 1652 (Institut Pasteur, Paris, France) came from the laboratory collection and was isolated from milk. The initial lyophilized ultrafiltrate (900 mg/ml) gave a halo diameter of 36 ± 0.7 mm in the inhibition assay.Purification.Samples of ultrafiltrate (20 μl) were spotted on thin-layer chromatography (TLC) plates (silica gel, 10 by 5 cm, 0.25 mm thick, 60 F₂₅₄; Merck, Darmstadt, Germany), with 20 μl of TSBYE for controls, and eluted by vertical chromatography with a butanol-acetic acid-water (40:10:20 [vol/vol/vol]) solvent system. The bands were examined under UV light (254 nm) or after treatment with Ehrlich’s reagent. Four well-separated bands were found (R_fs, 0.11 ± 0.04; 0.41 ± 0.04; 0.7 ± 0.03; and 0.86 ± 0.03), but only the band with an R_f of 0.7 ± 0.03 differed from that of control preparations (TSBYE not containing G. candidum). The microbiological bioautography test (1) confirmed the presence of the inhibitor in the band with an R_f of 0.7. Lyophilized ultrafiltrate was subjected to centrifugal partition chromatography (Sanki 1000 Engineering Ltd.; EverSeiko, Tokyo, Japan) in butanol-acetic acid-water (40:10:50 [vol/vol/vol]). Partitioning was carried out under the following conditions: ascending mode, 1,200 rpm; flow rate, 3 ml/min; pressure, 40 bars. An aliquot of material (3 g) previously equilibrated with the solvent system was injected into the separatus via a 12-ml injection loop. Fractions (10 ml each) were collected and evaporated to dryness in a SpeedVac (Jouan RC 1022, Saint Herblain, France). The dried extracts of certain fractions were taken up in 800 μl of water and brought to pH 5.6 with 0.2 M NaOH. A total of 10 mg of pooled fractions with an R_f close to 0.7 and showing L. monocytogenes inhibitory activity (36 ± 0.7 mm for a solution of 38 mg/ml) was taken up in 250 μl of methanol-water (50:50 [vol/vol]) and automatically deposited (Camag Linomat) on a 10- by 20-cm TLC plate (silica gel, 0.25 mm thick, 60 F₂₅₄; Merck). The plate was developed with butanol-acetic acid-water (40:10:20 [vol/vol/vol]) and examined under UV light. Bands with an R_f of 0.7 were scraped off and placed in methanol. The silica was washed several times and removed by centrifugation and filtration through a 0.2-μm-pore-size filter. An aliquot (20 μl) was spotted on a small silica TLC plate to confirm elution of the solute by the methanol solvent. The purity of the band with an R_f of 0.7 was confirmed by high-pressure liquid chromatography (HPLC) coupled with a photodiode array detector at 206 and 222 nm (solvent A: 0.1% formic acid in water; solvent B: CH₃CN-H₂O [95:5] plus 0.1% formic acid) on a C₁₈ Grom-Sil ODS2 column (4.6 by 30 mm; particle size, 1.5 μm; Grom Analytic, Herrenberg, Germany) at the flow rate of 1 ml/min. Inhibitory activity was assessed as above. Preparative TLC indicated that the band with an R_f of 0.7 contained two components, one eluting at 4.5 min on HPLC (peak 1) and the other at 5.5 min (peak 2). The latter fraction gave an inhibitory halo of 36 ± 0.7 mm at a concentration of 20 mg/ml (a 45-fold purification over the ultrafiltrate). The material from preparative TLC (40 mg in 200 μl of methanol-water) was placed on a column of Sephadex LH20 (1 m by 1 cm; Pharmacia), and the column was eluted with methanol-water at 12 ml h⁻¹. Fractions (1 ml each) were collected and examined by HPLC to determine the material in each fraction. This final purification on Sephadex LH20 gave two peaks, with two-thirds of the eluate at peak 1 and one-third at peak 2 (Fig. ​(Fig.1).1). As the concentration for peak 2 was very low, only the inhibitory activity for peak 1 was assayed. A concentration of 20 mg/ml gave a halo diameter of 26 ± 0.7 mm. Open in a separate windowFIG. 1Reverse-phase liquid chromatography (HPLC) of inhibitory compounds of G. candidum after purification by centrifugal partition chromatography, preparative TLC, and Sephadex LH20 gel filtration. Column, C₁₈ Grom-Sil ODS2 column (4.6 by 30 mm; particle size, 1.5 μm; Grom Analytic). Eluent: solvent A (0.1% formic acid in water), solvent B (CH₃CN-H₂O [95:5] plus 0.1% formic acid). Flow rate, 1 ml/min. (a) Product 1 (detection at 206 nm); (b) product 2 (detection at 222 nm).

Characterization.

The pooled fractions were run on HPLC with a Grom-Sil ODS2 column coupled to a mass detector (Sciex Api III, triple quadrupole; Thornhill, Canada). Product 1, analyzed by desorption and chemical ionization, gave a signal at an m/z of 184 for (M⁺ NH₄)⁺ on desorption and chemical ionization and thus had a mass of 166. Product 2 was analyzed by ion spray and gave a signal at an m/z of 297 (M + 4 Na)⁺ for a mass of 205. Spectra were determined in a Nicolet model 60 SXR FT-IR. Samples were dissolved in dimethyl sulfoxide, and the ¹H and ¹³C resonances were measured in a Brucker spectrometer at 200 and 400 Hz, respectively. The purified material was taken up in methanol, and the isomeric form of the substance(s) inhibiting L. monocytogenes was determined in a Perkin-Elmer model 341 polarimeter. Two inhibitors were identified (Fig. ​(Fig.2);2); product 1 was 2-hydroxy-3-phenylpropanoic acid (phenyllactic acid, mass 166), and product 2 was 2-hydroxy-3-indolpropanoic acid (indollactic acid, mass 205). The rotation of polarized light showed that the phenyllactic acid produced by G. candidum was the d form. The spectrum properties of the isolated compounds are identical to those of authentic commercial compounds (Sigma Chemical Co., St. Louis, Mo.). d-Phenyllactic acid can be purchased from Aldrich (product no. 37 690-6), and dl-indollactic acid is available from Sigma (catalog no. I2875). Inhibitory activity with commercial compounds showed that dl-phenyllactic acid (Sigma catalog no. P7251) was a stronger inhibitor of Listeria than dl-indollactic acid (34 and 26 ± 0.7 mm for 187 mM, respectively) and that the d form of phenyllactic acid was more active (38 mm for 120 mM) than the l form (Aldrich 11, 306-9, 30 mm for 120 mM). The samples were taken up in methanol-water (50:50 [vol/vol]) and brought to pH 5.6 (Table ​(Table1).1). Open in a separate windowFIG. 2Structure of two inhibitory compounds of G. candidum characterized by LC-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation.TABLE 1Anti-Listeria activity of phenyllactic acid and indollactic acid^a

Effect of Lipid Materials on the Production of Lipase by Torulopsis ernobii

The yeast Torulopsis ernobii produced approximately 50 units of extracellular lipase (glycerol ester hydrolase, E.C. 3.1.1.3) per ml when grown in 30-liter fermentors at 33 C for 40 hr in a base medium at pH 5.0. The addition of fats, oils, triglycerides, or higher fatty acids to this medium at concentrations of 0.2 to 0.6% markedly increased production; twofold increases in yield were obtained when 0.2% olive oil or a mixture of 0.14% oleic acid and 0.04% palmitic acid was added. The production of lipase paralleled growth, and the role of lipid materials in augmenting lipase production appears to be related to cell growth.