共查询到20条相似文献,搜索用时 15 毫秒
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J. M. Crous I. S. Pretorius W. H. van Zyl 《Applied microbiology and biotechnology》1996,46(3):256-260
First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1
P
) and terminator (PGK1
T
) sequences on a multicopy episomal plasmid. The resulting construct PGK1
P
-abf B-PGK1
T
was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.
Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996 相似文献
3.
The α-glucosidase (AGL) from Aspergillus niger has been applied to produce isomaltooligosaccharides. In the present study, various factors which affect the yield of recombinant AGL, produced by engineered Pichia pastoris, were investigated. The expression level reached 5.5 U ml?1 in bioreactor after optimization of parameters of initial induction cell density, induction temperature and methanol concentration. In addition, it was found that coexpression of protein disulfide isomerase (PDI) inhibited the growth of the engineered P. pastoris strains and had an adverse effect on the production of AGL, while codon optimization of native A. niger α-glucosidase encoding gene (aglu) resulted in a significant enhancement of enzyme production, which reached 10.1 U ml?1. We believe that yield of AGL is increased by codon optimization as a result of enhanced translation efficiency as well as more stable mRNA secondary structure. In contrast, PDI coexpression under the control of alcohol oxidase promoter (PAOX1) seems to be less efficient in helping disulfide bond formation in AGL while probably induce unfolded protein response, which further leads to cell apoptosis and increased protein degradation. 相似文献
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Mrázek H Benada O Man P Vaněk O Křen V Bezouška K Weignerová L 《Protein expression and purification》2012,81(1):106-114
α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase. 相似文献
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S. G. Zhao M. C. Wu C. D. Tang S. J. Gao H. M. Zhang J. F. Li 《Applied Biochemistry and Microbiology》2012,48(5):473-481
Using 3′ and 5′ rapid amplification of cDNA ends (RACE) techniques, the full-length cDNA sequence of the Anman5A, a gene that encodes an acidophilic β-mannanase of Aspergillus niger LW-1 (abbreviated to AnMan5A), was identified from the total RNA. The cDNA sequence was 1417 bp in length, harboring 5′- and 3′-untranslated regions, as well as an open reading frame (ORF) which encodes a 21-aa signal peptide, a 17-aa propeptide and a 345-aa mature peptide. Based on the topology of the phylogenetic tree of β-mannanases from glycoside hydrolase (GH) family 5, the AnMan5A belongs to the subfamily 7 of the GH family 5. Its 3-D structure was modeled by the bitemplate-based method using both MODELLER 9.9 and SALIGN programs, based on the known β-mannanase crystal structures of Trichoderma reesei (1QNO) and Lycopersicon esculentum (1RH9) from the GH family 5. In addition, the complete DNA sequence of the Anman5A was amplified from the genomic DNA using the pUCm-T vector-mediated PCR and conventional PCR methods. The DNA sequence was 1825 bp in length, containing a 5′-flanking regulatory region, 2 introns and 3 exons when compared with the full-length cDNA. 相似文献
6.
Yang Hong Hongxiao Han Jinbiao Peng Yaojun Shi Jinming Liu Xiangrui Li 《Experimental parasitology》2010,126(4):517-525
The development of an effective vaccine against the schistosome is thought to be the most desirable means to control schistosomiasis, even though there is an effective means of chemotherapy with praziquantel. A full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 5 protein (SjPSMA5) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 747 bp and encoded 248 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA5 is up-regulated in 18-day and 32-day schistosomes, and the level of expression in male is around fourfold higher than that in female worms at 42 days. The SjPSMA5 was subcloned into pET28a(+) and expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA5 (rSjPSMA5) was immunogenic. After immunization of BALB/c mice with rSjPSMA5, reductions of 23.29% and 35.24% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies and cells were significantly higher (P < 0.01) in the group vaccinated with rSjPSMA5 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA) and flow cytometry. The study suggested that rSjPSMA5 induced partial immunoprotection against S. japonicum in BALB/c mice, and it could be a potential vaccine candidate against schistosomiasis. 相似文献
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M. Ludwig Ursula Bolkenius Lucia Wickert Peter Marynen Frank Bidlingmaier 《Human genetics》1998,102(5):576-581
The human amiloride-sensitive epithelial sodium channel (ENaC) is a member of the degenerin/ENaC family of ion channels and
regulates fluid and electrolyte absorption across a number of epithelia, including kidney, colon and lung. Native ENaC has
been shown to be a multimer made up of at least three homologous subunits (α, β, γ) and mutations affecting the channel complex
have been identified in various human diseases. “Gain of function” mutations in one of the three ENaC subunits have been found
to cause pseudoaldosteronism (Liddle’s syndrome) and ENaC “reduction of function” mutations are found in patients affected
with the recessive form of pseudohypoaldosteronism (PHA) type 1. In this report, we describe the genomic organisation of the
humanαENaC gene. Human αENaC consists of 13 exons spanning 17 kb on chromosome 12p13 and contains at least eight Alu sequences. In addition to the intron/exon
boundaries, we have deciphered almost all the intron sequences and 475 bp of the CCAAT-less and TATA-less 5′ flanking region.
Received: 23 December 1997 / Accepted: 5 March 1998 相似文献
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《Journal of biotechnology》1987,5(1):87-91
Enterobacter aerogenes genomic library has been constructed using cosmid pJB8 in Escherichia coli. The gene encoding α-acetolactate decarboxylase (ALDC) has been isolated from this library by direct measurement of enzyme activity. The expression of the ALDC gene in E. coli appears to originate from the own promoter. Subsequent subcloning revealed that the ALDC gene locates within 1.7 kb BamHI-PstI fragment. 相似文献
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Xianliang Zheng Bo Fang Dongfei Han Wenxia Yang Feifei Qi Hui Chen Shengying Li 《PloS one》2016,11(8)
α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application. 相似文献
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Scott-Craig JS Borrusch MS Banerjee G Harvey CM Walton JD 《The Journal of biological chemistry》2011,286(50):42848-42854
α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi. 相似文献
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Julan Liao Masayuki Okuyama Keigo Ishihara Yoshinori Yamori Shigeo Iki Takayoshi Tagami 《Bioscience, biotechnology, and biochemistry》2016,80(9):1747-1752
The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater kcat/Km values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides. 相似文献
16.
《Gene》1997,192(2):261-270
We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme. 相似文献
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Michel J. A. Flipphi Henk Panneman Peter van der Veen Jaap Visser Leo H. de Graaff 《Applied microbiology and biotechnology》1993,40(2-3):318-326
Secretion of endo-1,5--l-arabinase A (ABNA) by an Aspergillus niger xylulose kinase mutant upon mycelium transfer to medium containing l-arabitol was immunochemically followed with time to monitor its induction profile. A cDNA expression library was made from polyA + RNA isolated from the induced mycelium. This library was immunochemically screened and one ABN A specific clone emerged. The corresponding abnA gene was isolated from an A. niger genomic library. Upon Southern blot analysis, a 3.1-kb HindIII fragment was identified and subcloned to result in plasmid pIM950. By means of co-tranformation using the A. niger pyrA gene as selection marker, the gene was introduced in both A. niger and A. nidulans uridine auxotrophic mutants. Prototrophic A. niger and A. nidulans transformants overproduced A. niger ABN A upon growth in medium containing sugar beet pulp as the sole carbon source, thereby establishing the identity and functionality of the cloned gene. The DNA sequence of the complete HindIII fragment was determined and the structure of the abnA gene as well as of its deduced gene product were analysed. Gene abnA contains three introns within its structural region and codes for a protein of 321 amino acids. Signal peptide processing results in a mature protein of 302 amino acids with a deduced molecular mass of 32.5 kDa. A. niger abnA is the first gene encoding an ABN to be isolated and characterized.
Correspondence to: L. H. de Graaff 相似文献
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Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained
were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in
fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and
high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified
free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence
of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05
M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following
incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small
amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable
activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan. 相似文献
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Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease
activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect
to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal
peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the
loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley
α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted
and correctly processed by A. niger using its own signal sequence.
Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997 相似文献
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Kenji Aoki Motoo Arai Yasuji Minoda 《Bioscience, biotechnology, and biochemistry》2013,77(13):2479-2486
Monochlorotrifluoro-p-benzoquinone (CFQ) was used for investigating the state of the amino groups of acid-stable α-amylase and acid-unstable α-amylase. About half of the total amino groups in both enzyme molecules were reacted with the reagent. The unreactive amino groups seemed to exist in a different state from the reactive ones. Both enzymes whose amino groups were modified by CFQ still maintained the α-phenylmaltosidase activity in spite of losing or decreasing the amylase activity. These facts suggest that the amino groups of both enzymes were not in the active site but the modification of them caused steric hindrance.The pH-stability of the acid-unstable α-amylase whose one or two amino groups were modified with succinic anhydride or 2,4,6-trinitrobenzene-l-sulfonate (TNBS) increased on the acidic side and decreased on the alkaline side, but further modification of them led to decrease the stability on both sides. 相似文献