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.     

Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was isolated from human postheparin plasma by column chromatography on heparin-Sepharose and phenyl-Sepharose and immunoaffinity chromatography with monoclonal antibodies. The purified enzyme had an apparent molecular weight of 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an amino-terminal sequence of Leu-Gly-Gln-Ser-Leu-Lys-Pro-Glu. Partial amino acid sequences of seven cyanogen bromide peptides were obtained. A human hepatoma cDNA library was screened with synthetic oligonucleotides derived from the partial protein sequence. The cloned H-TGL cDNA of 1569 nucleotides predicts a mature protein of 477 amino acids plus a leader sequence of 22 amino acids. Blot hybridization analysis of poly(A)+ mRNA with a putative H-TGL cDNA clone gave a single hybridizing band of 1.7 kilobases. The protein contains four consensus N-glycosylation sequences based on the cDNA sequence. Comparison of the enzyme sequence with that of other lipases reveals highly conserved sequences in regions of putative lipid and heparin binding. The carboxyl terminus of H-TGL contains a highly basic sequence which is not reported to be present in rat H-TGL or other members of the lipase gene family.     

陆地棉脂肪酶基因克隆及氨基酸序列分析

根据陆地棉(Gossypium hirsutum L.)EST序列设计一对引物,采用RT-PCR方法从萌发的棉籽中获得了脂肪酶(triacylglycerol acylhydrolases,EC 3.1.1.3)基因,该基因编码483个氨基酸;比对结果显示,棉籽脂肪酶与拟南芥、水稻、蓖麻等脂肪酶相似性较低,具有由Ser-Asp-His组成的三联体催化活性中心,在亲核Ser残基周围有GXSXG保守序列.软件预测显示该脂肪酶为可溶性蛋白,分子量约为55.4 kD,等电点为9.07,不含N端信号肽,亚细胞定位可能是过氧化物酶体或胞质溶胶.     

Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis

Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.     

Molecular cloning and expression of the gene encoding a phospholipase A1 from Aspergillus oryzae.

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction. The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp). The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation. PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues. The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor. PLA1 has two possible N-glycosylation sites (Asn27 and Asn55). PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases. The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii. The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional.     

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.     

Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli

Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.     

Analysis of Bacillus megaterium lipolytic system and cloning of LipA,a novel subfamily I.4 bacterial lipase

The lipolytic system of Bacillus megaterium 370 was investigated, showing the existence of at least two secreted lipases and a cell-bound esterase. A gene coding for an extracellular lipase was isolated and cloned in Escherichia coli. The cloned enzyme displayed high activity on short to medium chain length (C(4)-C(8)) substrates, and poor activity on C(18) substrates. On the basis of amino acid sequence homology, the cloned lipase was classified into subfamily I.4 of bacterial lipases.     

来源于青霉XMZ-9两个低温脂肪酶的基因克隆、原核表达与性质测定

低温脂肪酶在低温条件下仍具有较高活性,在食品添加剂、洗涤添加剂及有机合成等产业具有非常独特的应用前景。从低温菌株中分离低温脂肪酶基因是开发新的低温脂肪酶的有效手段。首先利用油脂同化平板与三丁酸甘油酯-维多利亚蓝平板从冰川土样中筛选分离获得一株具有较高脂肪酶活性的真菌,18S rDNA鉴定其属于青霉属,命名为Penicillium sp.XMZ-9。根据真菌脂肪酶多序列比对获得的保守区,设计简并引物,利用降落PCR与染色体步移的方法从Penicillium sp.XMZ-9中克隆到2个完整的脂肪酶基因,分别记为LipA与LipB。LipA全长1 014 bp,无内含子,编码337个氨基酸。而LipB全长1 232 bp,cDNA长1 122 bp,含有2个内含子,编码373个氨基酸。将两基因的cDNA序列克隆到pET30a(+)载体上,转化大肠杆菌Escherichiacoli BL21(DE3)。经低温诱导表达后,LipA大部分表达为包涵体,包涵体经复性后具有脂肪酶活性,并表现出低温适应性;LipB则大部分表达为可溶性蛋白,Ni-亲和层析柱纯化后,其亦具有低温脂肪酶活性。青霉菌株XMZ-9的获得与低温脂肪酶的克隆表达研究,为研究低温菌株与低温酶的适冷机制提供了宝贵的资源,也为进一步开发利用低温脂肪酶奠定了基础。     

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.     

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).     

Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases

The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.     

Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi

The gene coding for the lipase of Pseudomonas fragi was cloned into Escherichia coli JM83 by inserting Sau3A-generated DNA fragments into the BamH I site of pUC9. The plasmid isolated, pKKO, was restriction mapped and the position of the lipase gene on the 2.0 kb insert was pinpointed by subcloning. DNA sequencing revealed that the open reading frame comprises 405 nucleotides and gives a preprotein of 135 amino acids with a predicted Mr of 14643. By comparing the putative lipase amino acid sequence with porcine pancreatic, rat lingual and Staphylococcus hyicus lipases the amino acid sequence around the reactive serine was found to be common among the types of lipase which have been reported.     

Cloning and expression of a lipase gene from rice (Oryza sativa cv. Dongjin)

Lipases are useful enzymes that are primarily responsible for the hydrolysis of acylglycerides during lipid processing. We have cloned a lipase gene from a rice seed coat cDNA library (Oryza sativa cv. Dongjin). The cDNA was 1,445 bp in length and encoded 361 amino acid residues (GenBank accession No. AY580163). The deduced amino acid sequence had 82 and 56% identity to Oryza sativa (cv. Chuchung) and Arabidopsis thaliana lipase genes, respectively, and there was a GxSxG consensus motif near the catalytic triad at the active serine site. The deduced sequence had little homology to mammalian and microbial lipases. When the Oryza sativa lipase gene was expressed in Escherichia coli with the pET expression system, activity was found mainly in the pellet fraction. The purified product had lipolytic activity towards tributyrin and was about 40 kDa in size.     

Cloning and molecular modelling of turkey pancreatic lipase: structural explanation of the increased interaction power with lipidic interface

Starting from total pancreatic mRNAs, turkey pancreatic lipase (TPL) cDNA was synthesized by RT-PCR and cloned into the PGEM-T vector. Amino acid sequence of the TPL is compared to that of human pancreatic lipase (HPL). A 3-D structure model of TPL was built using the 3-D structure of HPL as template, given the high amino acid sequence homology between the two lipases. Based on this model, the enhanced interaction power of TPL, as compared to that of HPL, into a phosphatidylcholine monolayer film, could be explained. We concluded that an increase in the exposed hydrophobic residues on the surface of TPL would be responsible for an enhanced interaction with a lipidic interface.     

The lipA gene of Serratia marcescens which encodes an extracellular lipase having no N-terminal signal peptide.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonas fluorescens B52 and P. fluorescens SIK W1, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.     

Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.     

Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.     

Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species

An extracellular lipase catalyzing the synthesis of macrocyclic lactones in anhydrous organic solvents was purified to homogeneity from Pseudomonas nov. sp. 109, and characterized. The lipase showed a pI of 5.3 on isoelectric focusing and a Mr of 29,000 +/- 1,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With respect to substrate specificity, optimum chain length for acyl moiety varied depending on the type of reaction catalyzed: C18 in monomer lactone formation, C11 or shorter in dimer lactone formation, and C8 in ester hydrolysis. The amino-terminal 19 amino acid residues of the purified lipase were determined as Ser-Thr-Tyr-Thr-Gln-Thr-Lys-Tyr-Pro-Ile-Val-Leu-Ala-His-Gly-Met-Leu-Gly- Phe, and the gene encoding the lipase was identified by hybridization to a synthetic 20-nucleotide probe, cloned, and sequenced. Nucleotide sequence analysis predicted a 311-amino acid open reading frame, a putative ribosome-binding site, and a 26-amino acid sequence at the amino terminus of the sequence that is not found in the mature protein. This 26-amino acid sequence has many of the characteristics common to known signal peptides. The lipase gene encoded a sequence of Val-Asn-Leu-Ile-Gly-His-Ser-His-Gly-Gly which is very well conserved among lipases, and showed 38-40% overall homology to the amino acid sequences of lipases from Pseudomonas fragie and Pseudomonas cepacia, but showed little homology to those of other lipases, suggesting that some structural features are required for catalyzing macrocyclic lactone synthesis in organic solvents and are restricted to lipases of the Pseudomonas origin.     

斜带石斑鱼2种胰脂肪酶基因全长cDNA序列的克隆与分析

利用RT-PCR和RACE方法克隆得到斜带石斑鱼（Epinephelus coioides）肝胰脏中胆盐活化的胰脂肪酶(bile salt-activated lipase,BSAL)和依赖于辅酶的胰脂肪酶(colipase-dependent pancreatic lipase,PL)基因的全长cDNA序列.BSAL基因全长cDNA序列1 796 bp,编码558个氨基酸,该蛋白序列含有BSAL的全部特征结构区,与其他脊椎动物BSAL的氨基酸序列同源性为49.9%～57.3%.PL基因的全长cDNA序列1 503bp,编码465个氨基酸,该蛋白序列含有PL全部的特征结构区,与其它脊椎动物PL的氨基酸同源性为49.1%～73.9%.系统树分析表明,斜带石斑鱼BSAL和PL与其它物种BSAL、PL和胰脂肪酶相关蛋白(PL-RP)聚于进化树的两个不同分支,属于2种不同的胰脂肪酶.结果证实,在同一鱼类体内也存在BSAL和PL两种胰脂肪酶基因.