Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Substrate specificity of Geotrichum candidum lipase preparations

Summary Lipases with different fatty acid specificity were produced byGeotrichum candidum depending on growth condition. The hydrolysis of olive oil was inhibited by glycerol tributyrate and was dependent on Ca-ions for running at maximal rate.     

cDNA molecular cloning of Geotrichum candidum lipase

The cDNA clone of Geotrichum candidum (Geo.) lipase was isolated from the Geo. cDNA library by colony hybridization using 32P-labeled oligonucleotides corresponding to a partial amino acid sequence of this enzyme. The nucleotide sequence of the cDNA determined by the dideoxy chain terminating method included some partial amino acid sequences determined by Edman degradation, and the overall amino acid composition deduced from the cDNA coincided with that from amino acid analysis of this protein. The cloned cDNA coded a protein of 554 amino acids and a hydrophobic signal sequence of 19 amino acids. Geo. lipase contained the -Gly-X-Ser-X-Gly- sequence which is believed to form part of the interfacial lipid recognition site.     

白地霉Y162脂肪酶基因克隆及其在毕赤酵母中的高效表达

借助生物信息学,对已克隆的地霉属脂肪酶全长基因序列进行同源比对,根据保守序列设计引物,在基因组DNA和cDNA水平上,于国内首次克隆了Geotrichum candidum Y162脂肪酶基因.Gcandidum Y162脂肪酶基因全长1692bp,不含内含子,编码包括19个氨基酸信号肽在内的563个氨基酸.与NCBI GenBank中已报道的地霉属脂肪酶氨基酸序列有86%的一致性.将该基因克隆到pPIC9K表达载体上,转化毕赤酵母GS115,摇瓶发酵96h后毕赤酵母分泌表达55 U/mL重组脂肪酶,实现了脂肪酶的高效表达.酶学性质研究表明,该重组脂肪酶对C9位顺式双键的甘油酯具有明显的底物特异性;对甲醇、甘油等有机溶剂呈现耐受性;最适温度和最适pH分别为50℃和8.0,在pH6.0～10.0及60℃以下能保持60%以上的酶活力.底物特异性、有机溶剂、温度及pH耐受性赋予该重组酶良好工业应用潜力.     

cDNA cloning and characterization of Geotrichum candidum lipase II

Geotrichum candidum produces two extracellular lipases, I and II. A lipase II cDNA clone was isolated from a cDNA library by colony hybridization using the 32P-labeled fragment of lipase I cDNA isolated previously. The nucleotide sequence of lipase II cDNA determined by the dideoxy chain terminating method includes the N- and C-terminal amino acid sequences of lipase II, and the overall amino acid composition deduced from the cDNA coincides with that deduced on amino acid analysis of this protein. The cloned lipase II cDNA codes a protein of 544 amino acids and a part of the signal sequence of 13 amino acids. The peptide chain lengths of lipases I and II are the same, their overall identity being 84%. Furthermore, four Cys residues are completely conserved, which may participate in the formation of disulfide bridges. A homology search indicated that the G. candidum lipases and Candida cyclindracea lipase are homologous enzymes and that they are members of the cholinesterase family.     

单因子-响应面法优化白地霉Y162产脂肪酶条件

对白地霉Y162液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适碳源为橄榄油,氮源为黄豆粉和NH4Cl,无机盐为BaCl2和MgCl2。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的橄榄油、BaCl2和NH4Cl三个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,得出橄榄油、BaCl2和NH4Cl最佳浓度分别为2.35％,0.36％,1.35％。优化后液体发酵液中脂肪酶活力提高到31.85 U/mL,比初始酶活力14.16 U/mL提高了2.25倍,表明单因子-响应面结合法可显著优化白地霉Y162液体发酵产脂肪酶条件。     

白地霉的化学成分研究

白地霉(Geotrichum candidum Link)固体发酵培养物,经乙醇提取、柱层析分离得到了7个化合物.通过光谱分析,分别鉴定为亮氨酸(1)、尿嘧啶(2)、胸腺嘧啶(3)、焦儿茶酸(4)、4-羟基苯甲酸(5)、3,5-二羟基苯甲酸(6)和7,8-dimethylalloxazine(7).7个化合物均是首次从白地霉中得到.     

Low resolution crystal structure of lipase from Geotrichum candidum (ATCC34614)

Lipase from Geotrichum candidum (ATCC34614) is a glycerol ester hydrolase which has a molecular weight of 55,000 with about 7% carbohydrate, displaying a high affinity for triolein. The enzyme was crystallized from more than 2% protein solution without using any salt or organic solvent. The crystals were cross-linked by soaking in 0.37% glutaraldehyde solution (0.1 M acetate buffer solution, pH 5.6). The structure was determined by X-ray diffraction using the isomorphous replacement technique. Two heavy-atom derivatives [K2PtCl4 and UO2(CH3COO)2] were obtained by the soaking method. The electron density map calculated at 5 A resolution clearly showed the molecular boundary. A balsa wood model was made on the basis of the 6 A electron density map. The molecular has an ellipsoidal shape with dimensions of 70 A X 50 A X 50 A. Several columns of density corresponding to alpha-helix and a few clefts were found in the molecule. The active site is presumably located in the vicinity of one of the Pt sites in the Pt-derivative crystal, judging from the inactivation of the enzyme by K2PtCl4.     

白地霉ch-3脂肪酶Ⅰ基因在大肠杆菌中的表达

构建了白地霉脂肪酶Ⅰ的基因工程菌,为进一步进行蛋白质工程改造和脂肪酶应用奠定了基础。从新疆昌吉市油脂化工厂含油冻土中分离得到1株低温脂肪酶产生菌-白地霉ch-3。该菌发酵上清液中的脂肪酶最适作用温度为35℃,在0℃仍可保持66%的相对酶活力。应用PCR技术从白地霉ch-3基因组DNA中克隆得到脂肪酶Ⅰ基因lip1,将该基因与原核表达质粒载体pET-22b（＋）连接,构建重组质粒pETl-ip1,转化E.coliBL21（DE3）,酶切鉴定,筛选得到重组菌。十二烷基磺酸钠-聚丙希酰胺（SDS-PAGE）显示重组脂肪酶Ⅰ的相对分子质量约为5.8×10^4,酶活为2.73 U/mL,表明lip1基因的表达产物具有正常的生物学活性。白地霉ch-3脂肪酶Ⅰ基因lip1能够在大肠杆菌中有效地表达。     

beta-Glucosidases from fungus Geotrichum candidum]

beta-Glucosidases from Geotrichum candidum 3C cellulase preparation were separated from C1 enzymes and beta-1,4-glucanases by means of DEAE-Sephadex A-50 chromatography, gel filtration through P-150 Biogel and chromatography on CM-cellulose, and then were fractionated by isoelectric focusing using carrier ampholites with pH ranges 3-6 and 4-6. beta-Glucosidases with pI 3.8, 4.2, 4.6, 5.1, 5.6 and 6.2 were found in cellulase preparation from G. candidum 3C. Molecular weight of beta-glucosidases with pI 3.8, 4.2, 4.6 and 6.2, isolated under isoelectric focusing, were estimated by means of gel filtration through Sephadex G-200 to be 35000, 123000, 188000 and 223000 respectively. beta-Glucosidases with pI 3.8, 4.6, 5.6 and 6.2 hydrolyzed cellobiose and did not attack p-nitrophenyl-beta-D-glucopyranoside; those with pI 4.2 and 5.6 hydrolyzed p-nitrophenyl-beta-D-glucopyranoside and plant glucoside, protodioscin, and did not split cellobiose. All the beta-glucosidases studied did not hydrolyze laminaribose, beta-D-methylsylopyranoside, alder O-methylglucuronoxylane, o-nitrophenyl-beta-D-galactopyranoside and p-nitrophenyl-alpha-D-glucopyranoside. beta-Cellobiase with pI 6.2 hydrolzed lactoses, cellobioses with pI 3.8 and pI 5.6 splited gentiobiose. beta-Glucosidase with pI 4.6 did not attack any substrate studied, except cellobiose.     

Separation and characterization of two molecular forms of Geotrichum candidum lipase

Southern blot analysis of the Geotrichum candidum genome with a cloned lipase cDNA as the probe indicated the existence of two genes on the chromosome of the fungus which are homologous to the cDNA. As expected, two forms of lipase (lipases I and II) were actually isolated by hydrophobic interaction chromatography after a multistep procedure including ammonium sulfate fractionation, anion exchange chromatography, and gel filtration of the culture filtrate. Lipase I, the first eluted fraction, was the predominant form, and more than 80% of the total activity was attributed to this form. Amino acid sequence analysis of the amino and carboxyl termini of these two enzyme preparations indicated that lipase I was the product of the lipase gene whose cDNA had previously been cloned and sequenced [Shimada et al. (1989) J. Biochem. 106, 383-388]. Lipase II, on the other hand, had similar amino acid composition, but different terminal sequences which were not found in the primary structure of lipase I deduced from the cDNA sequence. These results gave lines of evidence for the expression of truely different lipase genes and ruled out the possibility that the observed multiple forms are caused by proteolytic digestion. The molecular mass estimated by SDS-PAGE and the isoelectric point of lipase I were 64 kDa and 4.3, while those of lipase II were 66 kDa and 4.3, respectively. The two lipases had essentially the same specific activities, substrate specificities, pH stabilities, and optimal temperatures, but different pH optima and thermal stabilities.     

Evaluation of strains of Geotrichum candidum for lipase production and fatty acid specificity

Summary Three strains of Geotrichum candidum (ATCC 34614, NRRL Y-552 and NRRL Y-553) were examined for lipase production and activity. Variables including medium, pH, temperature, agitation rate and incubation time were examined to define the optimal culture conditions. Growth on oil in complex medium at 30°C, 300 rpm, and pH 7 produced maximal lipase activity. Fatty acid specificity of these strains and of two crude G. candidum enzyme preparations (lipase 26557 RP, Rhône Poulenc and lipase GC-4, Amano) was measured using equimolar mixtures of methyl or butyl esters of palmitic and oleic acids. The lipase from NRRL Y-553 and lipase 26557 RP displayed preferential specificity for hydrolyzing oleic acid esters, while the lipases from ATCC 34614, NRRL Y-552 and lipase GC-4 failed to discriminate between plamitic and oleic acids.     

Multiple crystal forms of lipases from Geotrichum candidum

Multiple stable crystal forms of two lipases from the fungus Geotrichum candidum have been obtained. The diffraction pattern extends to beyond 2.0 A resolution. Similarity of the cell dimensions of various forms suggested similar packing of molecules in different crystals. This was confirmed by rotation function results. Four heavy-atoms derivatives have been identified.     

Identification of Geotrichum candidum at the species and strain level: proposal for a standardized protocol

In this study, the M13 primer was used to distinguish Geotrichum candidum from the anamorphic and teleomorphic forms of other arthrospore-forming species (discriminatory power = 0.99). For intraspecific characterization, the GATA4 primer showed the highest level of discrimination for G. candidum among the 20 microsatellite primers tested. A molecular typing protocol (DNA concentration, hybridization temperature and type of PCR machine) was optimized through a series of intra- and interlaboratory trials. This protocol was validated using 75 strains of G. candidum, one strain of G. capitatum and one strain of G. fragrans, and exhibited a discrimination score of 0.87. This method could therefore be used in the agro-food industries to identify and to evaluate biodiversity and trace strains of G. candidum. The results show that the GATA4 primer might be used to differentiate strains according to their ecological niche.     

A novel Geotrichum candidum lipase with some preference for the 2-position on a triglyceride molecule

Summary A new component of Geotrichum candidum lipase with a unique positional specificity was isolated from culture broth together with a known major component. The purification included DEAE-Sephadex A-50 ion exchange chromatography. Sephadex G-100 gel filtration, and Butyl Toyopearl 650S hydrophobic interaction chromatography. The newly isolated component, though only a minor one, cleaved the 2-positioned ester bond nearly twice as fast as the 1(3)-positioned ester bond of a triglyceride molecule. In contrast, the major component hydrolysed all the ester bonds indiscriminately, which is consistent with the widely accepted positional specificity of the lipase from G. candidum. Offprint requests to: A. Sugihara     

Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

Characterization of Geotrichum candidum lipase III with some preference for the inside ester bond of triglyceride

A new form of Geotrichum candidum lipase with a unique positional specificity was found to exist in the culture broth as a minor component together with the well-documented major form. Unlike the major form, which cleaves both the inside and outside ester bonds of triglyceride indiscriminately, the newly isolated form showed some preference for the inside (2-position) ester bond. The new enzyme was also characterized by its own fatty acid specificity, i.e., an outstandingly high activity towards triolein and methyl oleate among the simple triglycerides and fatty acid methyl esters tested. Moreover, the enzyme possesed a specific activity three times as high as the major form. Notable difference in circular dichroism spectra were observed between the two forms, indicating distinct conformational differences. Edman degradation revealed that the N-terminal sequence of the new form differed from that of the major form, thus demonstrating the existence of a novel lipase gene on the chromosome. Correspondence to: A. Sugihara