用重叠PCR合成植物甜蛋白brazzein基因

目的:为在泡盛曲霉(Aspergillus awamori)中进行表达,采用重叠PCR合成了植物甜蛋白brazzein基因。方法:根据非洲热带植物Pentadiplandra brazzeana产生的天然甜蛋白brazzein的氨基酸序列及泡盛曲霉糖化酶基因glaA的密码子偏爱性,设计并化学合成了2对3’-端互补的寡聚核苷酸,通过PCR延伸获得2条末端有部分重叠的双链核苷酸片段,再通过重叠PCR扩增,合成了用于泡盛曲霉表达的植物甜蛋白brazzein基因。结果:将brazzein基因克隆到pMD18-T载体,随机挑取6个重组质粒测序,结果1个重组质粒有连续4个碱基缺失,3个重组质粒各有1个碱基缺失,2个重组质粒携带的brazzein基因核苷酸序列完全正确。结论:合成的brazzein基因大小162 bp,编码54个氨基酸,推断的氨基酸序列与Pentadiplandra brazzeana产生的天然brazzein完全一致,表明植物甜蛋白brazzein基因成功合成。     

Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus

The glucoamylase from the thermophilic fungus Thermomyces lanuginosus has a molecular weight of 66 kDa and was characterized with isoelectric point, pH and temperature optimum of 3.8-4.0, 5.0 and 70 degrees C, respectively. In addition, the activation energy is 60.4 kJ/mol, Km is 3.5 mM and kcat is 25.3 s(-1). The glucoamylase was partially sequenced on the protein level, and the complete glucoamylase gene including its promoter (but excluding its terminator region) was cloned and sequenced. The glucoamylase protein comprises 617 amino acid residues and shows 60% identity with the glucoamylase from the thermophilic fungus Talaromyces emersonii. cDNA encoding Thermomyces lanuginosus glucoamylase was expression cloned into Pichia pastoris, producing approximately 7.4 U/ml. It was concluded that alternative mRNA splicing as it might occur in Aspergillus niger glucoamylase is not responsible for the occurrence of different glucoamylase isoforms in Thermomyces lanuginosus.     

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

AIMS: Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. METHODS AND RESULTS: First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4.5-5.0 and 65 degrees C. The enzyme was stable at 60 degrees C, the enzyme activity kept 80% after 60 min incubation at 70 degrees C. The half-life was 40 and 10 min under incubation at 80 and 90 degrees C respectively. CONCLUSIONS: A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.     

Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera.

The complete nucleotide sequence of the glucoamylase gene GLU1 from the yeast Saccharomycopsis fibuligera has been determined. The GLU1 DNA hybridized to a polyadenylated RNA of 2.1 kilobases. A single open reading frame codes for a 519-amino-acid protein which contains four potential N-glycosylation sites. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. Glucoamylase was purified from a culture fluid of the yeast Saccharomyces cerevisiae which had been transformed with a plasmid carrying GLU1. The molecular weight of the protein was 57,000 by both gel filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 2,000. The amino-terminal sequence of the protein began from the 28th amino acid residue from the first methionine of the putative precursor. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that GLU1 encodes glucoamylase. A comparison of the amino acid sequence of glucoamylases from several fungi and yeast shows five highly conserved regions. One homology region is absent from the yeast enzyme and so may not be essential to glucoamylase function.     

Construction of a starch-utilizing yeast by cell surface engineering.

We have engineered the cell surface of the yeast Saccharomyces cerevisiae by anchoring active glucoamylase protein on the cell wall, and we have endowed the yeast cells with the ability to utilize starch directly as the sole carbon source. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The constructed plasmid containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The glucoamylase activity as not detected in the culture medium, but it was detected in the cell pellet fraction. The glucoamylase protein transferred to the soluble fraction from the cell wall fraction after glucanase treatment but not after sodium dodecyl sulfate treatment, indicating the covalent binding of the fusion protein to the cell wall. Display of the fused protein was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. The transformant cells could surely grow on starch as the sole carbon source. These results showed that the glucoamylase was anchored on the cell wall and displayed as its active form. This is the first example of an application of cell surface engineering to utilize and improve the metabolic ability of cells.     

Comparison of two glucoamylases from Hormoconis resinae

Two extracellular glucoamylases (EC 3.2.1.3), glucoamylase P and glucoamylase S, were purified to homogeneity from the culture medium of Hormoconis resinae (ATCC 20495; formerly Cladosporium resinae) by a new method. Their apparent molecular masses (71 kDa glucoamylase P; 78 kDa glucoamylase S) and catalytic properties agreed well with those previously reported in the literature. Heat inactivation studies suggested that the high debranching (1,6-glycosidic) activity of glucoamylase P preparations (measured with pullulan) may reside in the same protein molecule as its 1,4-glycosidic activity (measured with soluble starch). Although glucoamylase S had virtually no debranching activity, it cross-reacted with polyclonal antibodies raised against glucoamylase P, and the two enzymes had very similar amino acid compositions. However, peptide mapping and amino-terminal sequencing studies of the peptides showed that the two enzymes have different sequences and must be encoded by different genes.     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

Chemical stabilization of glucoamylase from Aspergillus niger against thermal inactivation

The applicability of crosslinking an enzyme to an oxidized polysaccharide by reductive alkylation to enhance thermostability has been investigated for glucoamylase from Aspergillus niger. Direct covalent coupling of the enzyme to periodate-oxidized dextran in the presence of NaBH(3)CN results in a conjugate which has thermal properties similar to those of the native enzyme. Our working hypothesis postulates that enhancement of thermostability will result from rigidification of the protein's conformation subsequent to the formation of multiple covalent bonds between the protein and the support. On the basis of the known characteristics of glucoamylase from Aspergillus niger, it would seem necessary to introduce additional amino groups in the polypeptide chain of the protein. The incorporation of new amino groups was performed in two phases. First, the glycosidic part of glucoamylase was oxidized by periodate and the resulting aldehyde groups were reductively aminated by a diaminoalkane and NaBH(3)CIM. Secondly, additional amino groups were introduced on carboxyl functions into the previously aminated glucoamylase by a diaminoalkane and a water-soluble carbodiimide in the presence of maltose to protect the active site. The final derivative was then coupled to periodate-oxidized dextran T-70 in the presence of NaBH(3)CN. Starting with native glucoamylase, three successive operations give rise to a conjugate which retained 27% of the initial activity when measured with soluble starch and 39% when measured with maltopentaose. Using substrates of various sizes, it was observed that steric hindrance at the active site may result from covalent coupling to dextran T-70. It was demonstrated in heat inactivation experiments that the thermostability of the conjugate was in all cases superior to that of the native enzymes. Finally, it was observed that the operational stability of the conjugate was at least twice that of native glucoamylase at 70 degrees C on 18% maltodextrin. Additional experiments rule out the possibility that thermosta-bilization of the complex is due to other reasons than the increase in the amino content of the protein prior to crosslinking. Neither chemical modification, reticulation nor change in the net charge of the protein resulted in a derivative of glucoamylase which presented enhanced thermostability after conjugation. We conclude that for enzymes which have a low content of available amino groups, the thermostabilization method proposed previously by the present authors may still be applicable if additional amino groups are introduced into the protein prior to its crosslinking to an oxidized polysaccharide. This new example reinforces the generality of this method of stabilization.     

Cloning,heterologous expression,and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii

The gene encoding a thermostable glucoamylase from Talaromyces emersonii was cloned and, subsequently, heterologously expressed in Aspergillus niger. This glucoamylase gene encodes a 618 amino acid long protein with a calculated molecular weight of 62,827Da. T. emersonii glucoamylase fall into glucoside hydrolase family 15, showing approximately 60% sequence similarity to glucoamylase from A. niger. The expressed enzyme shows high specific activity towards maltose, isomaltose, and maltoheptaose, having 3-6-fold elevated k(cat) compared to A. niger glucoamylase. T. emersonii glucoamylase showed significantly improved thermostability with a half life of 48h at 65 degrees C in 30% (w/v) glucose, compared to 10h for glucoamylase from A. niger. The ability of the glucoamylase to hydrolyse amylopectin at 65 degrees C is improved compared to A. niger glucoamylase, giving a significant higher final glucose yield at elevated temperatures. The increased thermal stability is thus reflected in the industrial performance, allowing T. emersonii glucoamylase to operate at a temperature higher than the A. niger enzyme.     

The effect of point amino acid substitutions in an internal α-helix on thermostability of Aspergillus awamori X100 glucoamylase

Conformational flexibility of α-helices in glucoamylase of the fungus Aspergillus awamori was studied by molecular dynamics methods. Several amino acid substitutions (G127A, P128A, I136L, G137A, and G139A) optimizing intrinsic interactions in one of the α-helices (D) within the hydrophobic core of this protein were constructed and studied. It was found that these point mutations had different effects on the glucoamylase thermal inactivation constant. Unlike amino acid substitution P128A and substitutions G137A and A246C, I136L and G139A displayed a pronounced additive thermostabilizing effect.     

Primary structure of Beijing duck apolipoprotein A-1

The primary structure of Beijing duck apolipoprotein A-1 was determined by sequencing peptide fragments derived from tryptic and endoproteinase Asp-N digestion of the protein, and alignment with homologous chicken apo A-1. All of the peptide fragments were isolated by high-pressure liquid chromatography (HPLC) with a Vydac C18 column using a trifluoroacetic acid (TFA) buffer system. The N-terminus of the protein was determined to be aspartic acid by directly sequencing 52 residues of the intact protein. The C-terminus was alanine. The protein contains 240 amino acid residues. By analysis of the whole protein and its tryptic peptides, a six amino acid (Arg-Tyr-Phe-Trp-Gln-His) prosegment was determined. No cross-reactivity between duck and human apo A-1 with a goat antiserum against human apo A-1 was found. Sequence analysis of apo A-1 of other species indicates that amino acid substitutions in rat are more extensive than in other mammals. Isoleucine residues in apo A-1 are inversely correlated to the homology of human to other species, except dog.     

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein

A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.     

Complete amino acid sequence of yeast thioltransferase (glutaredoxin)

The amino acid sequence of a thioltransferase isolated from Saccharomyces cerevisiae was determined. The protein was cleaved by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The peptides generated were purified by reverse phase HPLC. Sequencing of intact protein and its fragments were achieved by automated Edman degradation. The protein contains 106 amino acid residues with two cysteines. Yeast thioltransferase showed 51% structural similarity to pig liver thioltransferase and 34% to E. coli glutaredoxin.     

Construction of an alpha-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae.

A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.     

Automated Gas Chromatographic Monitoring of Ethylene Evolution out of Rice Seedlings Infected with Blast Fungus

To find amino acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLU1 DNA encoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHX linker DNA. Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of 11 proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. A series of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements were produced by site-directed mutagenesis. Analyses for thermal stability and temperature dependency of these mutant proteins revealed that Ala81, Asp89, Trp94, Arg96, Asp97, and Trp166 are required for wild-type levels of activities, and that at least Ala81 and Asp89 are not essential to catalytic activities, but act in thermal stability.     

Cloning of the alpha-amylase cDNA of Aspergillus shirousamii and its expression in Saccharomyces cerevisiae.

alpha-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined. The alpha-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active alpha-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.     

Amino acid sequence of the L-arabinose-binding protein from Escherichia coli B/r.

The amino acid sequence of the L-arabinose-binding protein of Escherichia coli B/r was determined by sequenator analyses of reduced and S-pyridylethylated L-arabinose-binding protein and fragments derived by chemical and enzymatic cleavage of the native protein. The fragments were the products of cleavage by cyanogen bromide. BNPS-skatole, hydroxylamine, mild acid hydrolysis, limited trypsin digestion, chymotrypsin subdigestion, and subdigestion with Staphylococcus aureus protease V8. The COOH-terminal sequence was determined using bovine carboxypeptidases A and B and amino acid analyses. The L-arabinose-binding protein was determined to contain 306 amino acid residues, the sequence of which is presented below.     

Import of proteins into mitochondria: nucleotide sequence of the gene for a 70-kd protein of the yeast mitochondrial outer membrane.

The nucleotide sequence of the yeast chromosomal gene coding for the 70-kd protein of the mitochondrial outer membrane was determined. The deduced amino acid sequence of the protein agrees with the experimentally determined size and amino acid composition of the purified protein and correctly predicts the fragments obtained by cleaving the protein at its single tryptophan residue. The deduced NH2-terminal sequence features an uninterrupted stretch of 28 uncharged amino acids flanked on both sides by basic amino acids. By sequencing a truncated version of the gene it was found that the corresponding polypeptide product lacks the 203 carboxy-terminal amino acids of the authentic 70-kd protein. As shown in the accompanying paper, this protein fragment still becomes attached to the mitochondrial outer membrane in vivo.     

Structure of the antitumor protein neocarzinostatin. Amino acid sequence

The amino acid sequence of the antitumor protein neocarzinostatin has been proposed, as shown in Fig. 7, by sequence analysis of tryptic peptides and fragments obtained by digestion of the protein with chymotrypsin, pepsin, acid protease, and dilute hydrochloric acid. Neocarzinostatin consists of 109 amino acid residues and its molecular weight, calculated from the proposed structure, is 10,717.     

Isolation and characteristics of thermostable pullulanase from Clostridium thermohydrosulfuricum produced by Escherichia coli cells

The expressed gene (pul) for a thermostable pullulanase from Clostridium thermohydrosulfuricum was cloned into Escherichia coli. The enzyme was purified from cell extracts of E. coli by thermoinactivation, ammonium sulphate precipitation and gel exclusion. The purified enzyme was characterized as monomer with both pullulanase and glucoamylase activities. The general physico-chemical and catalytic properties of this enzyme were obtained. In particular, pullulanase and glucoamylase activities were stable and optimally active at 65 degrees C. The pH optimum for activity was 5.8. The amino acid composition and amino acid sequence of N-terminal end were estimated.