黑曲霉脂肪酶：基因克隆、表达及体外复性

根据黑曲霉F044脂肪酶N-端氨基酸序列,运用生物信息学方法,找到与黑曲霉脂肪酶基因同源的候选基因A84689。根据该基因序列,设计引物直接PCR扩增得到黑曲霉脂肪酶全长基因anl。anl全长1044bp,含3个内含子,编码297个氨基酸(含信号肽27个氨基酸),与其它脂肪酶基因没有明显同源性。将编码成熟脂肪酶的anl连接到pET28a载体上得到重组表达质粒,转化大肠杆菌BL21(De3),诱导表达并纯化出目的蛋白。通过大量稀释和DEAESepharoseFastFlow层析相结合的方法,变性后的纯化蛋白在体外实现再折叠复性。     

疏棉状嗜热丝孢菌脂肪酶基因的克隆及其在毕赤酵母中的高效表达

疏棉状嗜热丝孢菌Thermomyces lanuginosus可产生具有重要工业生产价值的脂肪酶。根据已报道的相应序列设计特异引物,综合运用PCR、RT-PCR技术克隆到脂肪酶基因的全长DNA和cDNA序列。其中DNA序列长1071bp,包含876bp的开放阅读框以及3段内含子;cDNA序列长885bp。结构基因编码蛋白包含292个氨基酸,前17个氨基酸构成信号肽。序列提交GenBank,登录号分别为EU022703和EU370914。将脂肪酶基因cDNA序列的开放阅读框克隆到酵母分泌型表达载体pPIC9K中,转化毕赤酵母GS115得到重组子且实现了分泌表达。将重组子诱导产酶,在培养温度30℃、甲醇添加量1%的情况下,小规模发酵量达0.93mg/mL,其分泌表达的最高酶活为7.2U/mL。重组酶最适反应温度和pH分别是60℃和8.0。表达蛋白在60℃保温1h后仍有完全酶活,具有较高的热稳定性。     

Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae

Abstract Aspergillus oryzae IFO4202 produces at least two extracellular lipolytic enzymes L1 and L2 (cutinase, and mono- and diacylglycerol lipase, respectively). Southern hybridization of restriction enzyme-digested genomic DNA fragments with 23mer oligonucleotides synthesized according to the amino acid sequence of the L2 as probe suggested the presence of the L2 gene (tentatively designated as mdlB ) and an additional weakly hybridizing region. A fragment containing the genomic mdlB gene was cloned in Escherichia coli . Nucleotide sequencing of the fragment revealed an open reading frame, comprising 1021 nucleotides, which contains two introns (51 and 52 nucleotides). Putative polyadenylation signals were found 182 and 287 bp downstream of the stop codon. The deduced amino acid sequence of the mdlB gene corresponds to 306 amino acid residues including a leader sequence of 28 amino acids and is highly similar to that of the mdlA gene of Penicillium camembertii . Three residues presumed to form the catalytic triad (serine, aspartic acid and histidine) of lipases were also conserved.     

来源于Aspergillus candidus的乳糖酶基因的克隆及序列分析

从一株产乳糖酶的亮白曲霉（Aspergillus candidus)中克隆到了乳糖酶基因组DNA及cDNA序列（EMBL AC－CESSION No.AJ431643),序列分析表明,乳糖酶基因组DNA序列长3458bp,其中含有8个内含子,cDNA编码区长3015bp,共编码1005个氨基酸,前19个氨基酸为信号肽序列,氨基酸序列中共含有11个潜在的糖基化位点。将此基因在不同来源的乳糖酶基因序列进行比较发现,该基因与绝大多数乳糖酶基因同源性较低。虽与米曲霉ATCC20423的乳糖酶序列同源性较高,但其在酶学性质上更优于后者,亮白曲霉的乳糖酶基因可能是一个具有更广阔的生产应用前景的新基因。     

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.     

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.     

Cloning and characterization of a new functional human aldehyde dehydrogenase gene

We cloned a new functional ALDH gene (ALDHx) from a human genomic library in cosmid pWE-15 by screening with a 29-nucleotide probe partially matched to a conserved region of the ALDH1 and ALDH2 genes. The new ALDHx gene does not contain introns in the coding sequence for 517 amino acid residues. The degree of resemblance between the deduced amino acid sequences of the new ALDHx gene and the ALDH2 gene is 72.5% (alignment of 517 amino acid residues), while that between the ALDHx and the ALDH1 gene is 64.6% (alignment of 500 amino acid residues). The amino acid residues (Cys-162, Cys-302, Glu-268, Glu-487, Gly-223, Gly-225, Gly-229, Gly-245 and Gly-250), which exist in both ALDH1 and ALDH2 isozymes and have been implicated in functional and structural importance, are also preserved in the deduced sequence of the new ALDHx gene. Northern blot hybridization with ALDHx probe revealed the existence of a unique mRNA band (3.0 kilobases) in the human liver and testis tissues. Using the new ALDHx probe, we cloned the cDNA of the gene from a human testis cDNA library in lambda gt11 vector. The nucleotide sequence of the cDNA differs from that of the genomic sequence at three nucleotide positions resulting in the exchange of 2 deduced amino acid residues. These positions are polymorphic as further demonstrated by the PCR amplification of the targeted region followed by nucleotide sequence analysis of the genomic DNA from eight unrelated individuals. Alignment of the genomic and cDNA sequence indicates that although the ALDHx gene appears to have no intron in its coding sequence, an intron of 2.6 kilobases is found to interrupt the 5'-untranslated (5'-UT) sequence. Primary extension and S1 mapping analysis indicate the existence of at least two 5'-UT exons. The new ALDHx gene was assigned to chromosome 9 by Southern blot hybridization of DNA samples from a panel of rodent-human hybrid cell lines.     

扩展青霉脂肪酶基因克隆、密码子优化及表达

【目的】克隆扩展青霉脂肪酶基因,实现具强催化活性的脂肪酶的异源高效表达。【方法】利用RT-PCR扩增扩展青霉CICC 40356脂肪酶(PEL)cDNA序列,利用重叠延伸PCR(Over-lap extension PCR)技术对PEL的10个稀有氨基酸密码子和表达载体pPIC9Kα信号肽的9个氨基酸密码子进行了优化,获得了改造过的脂肪酶基因PELM和表达载体pPIC9KM。并构建了带有脂肪酶自身信号肽的pPIC9K-PEL1、pPIC9KM-PELM1、pPIC3.5K-PEL1、pPIC3.5K-PELM1和不带有脂肪酶自身信号肽的pPIC9K-PEL2、pPIC9KM-PELM2六个重组质粒。利用对硝基苯酚棕榈酸酯(pNPP)为底物检测工程菌脂肪酶的酶活。在此基础上,对工程菌的酶学性质进行了研究。【结果】扩展青霉脂肪酶基因cDNA序列分析结果表明该序列与已报道PEL cDNA序列仅相差3个碱基,同源性高达99%。6个重组工程菌在甲醇诱导下,均表现出pNPP水解活性,28℃诱导100h时酶活达到最高,发酵上清的酶活分别为3.65 U/mL、30.49 U/mL、90.85 U/mL、212.05 U/mL、15.29 U/mL、76.32 U/mL。SDS-PAGE结果表明重组脂肪酶分子量均约28 kDa。酶学性质研究表明,重组脂肪酶PELM最适温度为35℃,最适pH为9.5,在pH7.0-10.0范围内该脂肪酶均较稳定,Ca2+和Mg2+对其有激活作用,Fe2+、Zn2+、Cu2+则有抑制作用,EDTA能使之快速失活。以不同碳链长度的对硝基苯酚酯为底物检测其底物特异性,结果显示其对中链酯(C8-C12)有较强的水解能力,最适底物为为C8的pNP酯。【结论】密码子优化后的扩展青霉脂肪酶基因在毕赤酵母中获得理想的表达,其酶活力比未优化的野生脂肪酶的提高了2.3-2.5倍,表明定点突变对其基因本身更改特有稀有密码子是实现PEL功能蛋白的异源高效表达的有效策略之一。     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> lipase: gene cloning,over-expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and in vitro refolding

From the N-terminal amino acid sequence of the lipase from Aspergillus niger F044, a potential homologous gene A84689 to the lipanl (the gene encoding the lipase from Aspergillus niger F044) was identified. A pair of primers were designed according to the nucleotide sequence of A84689, and the lipanl was cloned by PCR. Nucleotide sequencing revealed that the lipanl has an ORF of 1,044 bp, containing three introns. The deduced amino acid sequence corresponds to 297 amino acid residues. The cloned cDNA fragment encoding the mature lipase from Aspergillus niger F044 was over-expressed in Escherichia coli BL21(De3) and the recombinant protein was refolded in vitro by dilution followed by DEAE Sepharose Fast Flow chromatography.     

Human hepatic lipase. Cloned cDNA sequence, restriction fragment length polymorphisms, chromosomal localization, and evolutionary relationships with lipoprotein lipase and pancreatic lipase

Human hepatic lipase is an important enzyme in high density lipoprotein (HDL) metabolism, being implicated in the conversion of HDL2 to HDL3. Three human hepatic lipase cDNA clones were identified in two lambda gt11 libraries from human liver. The cDNA-derived amino acid sequence predicts a protein of 476 amino acid residues, preceded by a 23-residue signal peptide. Four potential N-glycosylation sites are identified, two of which are conserved in rat hepatic lipase. On alignment with human, mouse, and bovine lipoprotein lipase, the same two sites were also conserved in lipoprotein lipase in all three species. Stringent conservation of the cysteine residues was also evident. Comparative analysis of amino acid sequences shows that hepatic lipase evolves at a rapid rate, 2.07 x 10(-9) substitutions/site/year, about four times that in lipoprotein lipase and half that in pancreatic lipase. Further, hepatic lipase and pancreatic lipase appear to be evolutionarily closer to each other than either of them is to lipoprotein lipase. Southern blot analysis revealed high frequency restriction fragment length polymorphisms of the hepatic lipase gene for the enzymes HindIII and MspI. these polymorphisms will be useful for haplotype and linkage analysis of the hepatic lipase gene. Using cloned human hepatic lipase cDNA as a hybridization probe, we performed Southern blot analysis of a panel of 13 human-rodent somatic cell hybrids. Concordance analysis of the various hybrid clones indicates that the hepatic lipase gene is located on the long arm of human chromosome 15. Analysis of hybrids containing different translocations of chromosome 15 localized the gene to the region 15q15----q22.     

水稻H3.2型组蛋白基因RH3.2A的克隆与盐胁迫下的表达分析

组蛋白H3与其他类型的组蛋白分子H2A,H2B,H4共同构成了真核生物核小体的八聚体核心。研究发现组蛋白H3的多种翻译修饰,如甲基化、乙酰化、磷酸化等在调控基因转录过程种发挥了重要的作用。本研究从盐胁迫处理的水稻幼苗组织中分离了一个新的水稻组蛋白H3基因RH3．2A,编码具有136个氨基酸残基的多肽,与多种植物的组蛋白H3蛋白具有高度的氨基酸一致性。多序列比较发现,除了基因结构差异之外,还有3个位置的氨基酸残基（32、88、91）在H3．1与H3．2型组蛋白H3中存在差异。研究了RH3．2A基因在高盐和ABA胁迫下的表达,结果发现在水稻根部RH3．2A基因受高盐的强烈诱导,而在叶片RH3．2A基因的表达则不受高盐诱导,此外RH3．2A基因也受外源ABA的诱导,结合启动子分析的结果,我们认为RH3．2A基因可能参与了依赖于ABA的高盐胁迫应答反应。文章讨论了植物组蛋白H3基因在高盐胁迫应答反应中可能的作用。     

菊芋类金属硫蛋白基因htMT2的克隆及其表达特征分析

从菊芋(Helianthus tuberosus L.)块茎cDNA文库中得到了一个新的植物类金属硫蛋白基因htMT2的cDNA序列,全长509 bp,包括240 bp的开放阅读框、62 bp的5′端非翻译区、207 bp的3′端非翻译区.通过PCR获得了2个htMT2编码区的部分基因组片段htMTG-1及htMTG-2,长度分别为986 bp和982 bp.分析表明两个基因组片段均包含3个外显子及2个内含子,编码一个由79个氨基酸残基组成的多肽,与从htMT2推测的多肽完全一致,该多肽具有植物类金属硫蛋白的典型结构特征,N端及C端结构域富含Cys,分别具有8个和7个Cys残基,上述两个结构域被一个无Cys的中间区分开.Southern杂交结果表明,htMT2在菊芋基因组中以小基因家族的形式存在.Northern杂交结果表明htMT2在叶片、叶柄、茎及块茎中均有表达,在茎中有较高水平的表达,但在根中未检测到杂交信号.经Cu2+处理后,htMT2在茎中的表达量显著降低.与其他2型金属硫蛋白的序列同源性比较及htMT2对金属离子处理的反应均表明,htMT2是一种新的植物类金属硫蛋白基因.     

Gene cloning, overexpression and characterization of a novel organic solvent tolerant and thermostable lipase from Galactomyces geotrichum Y05

Although the lipase of Geotrichum candidum has been extensively reported, little attention has been focused on molecular genetic and biochemical characterizations of Galactomyces geotrichum lipases. A lipase gene from G. geotrichum Y05 was cloned from both genomic DNA and cDNA sources. Nucleotide sequencing revealed that the ggl gene has an ORF of 1692 bp without any introns, encoding a protein of 563 amino acid residues, including a potential signal sequence of 19 amino acid residues. The amino acid sequence of this lipase showed 86% identity to lipase of Trichosporon fermentans WU-C12. The mature lipase gene was subcloned into pPIC9K vector, and overexpressed in methylotrophic Pichia pastoris GS115. Active lipase was accumulated to the level of 100.0 U/ml (0.4 mg/ml) in the shake-flask culture, 10.4-fold higher than the activity of the original strain (9.6 U/ml). This yield dramatically exceeds that previously reported with 23–50 U/ml, 0.06 mg/ml and 0.2 mg/ml. The purified lipase exhibited several properties of significant industrial importance, such as pH and temperature stability, wide organic solvent tolerance and broad hydrolysis on vegetable oils. Such a combination of properties makes it a promising candidate for its application in non-aqueous biocatalysis, such as biodiesel production, selective hydrolysis or esterification for enrichment of PUFAs and oil-contaminated biodegradation, which have been drawn considerable attention currently.     

A novel streptomycete lipase: cloning,sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene

An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects

The nucleotide sequence coding for the fourth component of mouse complement (C4) has been determined from a cloned genomic DNA fragment and a cloned cDNA fragment. The amino acid sequence of the protein was deduced. The single chain precursor protein (pro-C4) consists of 1719 amino acid residues. The mature beta, alpha, and gamma subunits contain 654, 766, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted for the beta chain, three for the alpha chain, and none for the gamma chain. From a comparison with human C4 cDNA sequence an extensive overall sequence homology, 79% in nucleotides and 76% in amino acids, is observed. There is conservation in both the position and number of cysteine residues in human and mouse C4. We compared the mouse C4 amino acid sequences with those of mouse C3 and human alpha 2-macroglobulin and the evolutionary relationship among these three proteins is discussed.     

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.