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β-Mannanase (EC 3.2.1.78) is a key enzyme to hydrolyze the β-mannosidic linkages in mannan and heteromannan. The expression of a wild type β-mannanase (manWT) of Aspergillus sulphureus in Pichia pastoris is not high enough for its application in feed supplement. To earn a high expression level, the manWT gene was firstly optimized
to manM according to the code bias of P. pastoris, which was then inserted into pPICzαA and transformed into P. pastoris strain X-33. In the induction by methanol, β-mannanase was expressed in high level with 32% increase in comparison with the manWT gene expressed in P. pastoris in shaken flask. In a 10-L fermenter, the manM was expressed in 9-fold higher level than that in shaken flask, which yielded
the enzyme activity of 1100 U/mL. This is the first study on codon bias effect on the β-mannanase gene expression level, which helps to achieve high β-mannanase yield and enzymatic activity in P. pastoris. 相似文献
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Teng D Fan Y Yang YL Tian ZG Luo J Wang JH 《Applied microbiology and biotechnology》2007,74(5):1074-1083
β-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive
industry. To improve expression efficiency of recombinant β-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding β-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously
increased from 43.6 to 45.5%. At shaking flask level, β-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml−1 with barley β-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration
is approximately 250 mg l−1. The β-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml−1 with barley β-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed.
Compared to the nonoptimized control, expression level of the optimized β-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal
acidity and temperature of this recombinant enzyme were pH 6.0 and 45°C, respectively. 相似文献
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Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower Kn higher kcat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional. 相似文献
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Abdul Ghaffar Sher Afzal Khan Zahid Mukhtar Muhammad Ibrahim Rajoka Farooq Latif 《Molecular biology reports》2011,38(5):3227-3233
We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml−1 while that of recombinant without intron (xyn669) was 1.26 U ml−1 after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant
xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite
lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis
than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was
quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications. 相似文献
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The endo-β-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase
chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an α-mating
factor in a constitutive expression vector pGAPzαA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast–peptone–dextrose
liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed
with a yield of 120 units/mL under the flask culture at 28°C for 3 days. The enzyme showed optimal activity at 50°C and pH 2.4–3.4.
Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80°C
for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60°C for
30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na2HPO4-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na+, Mg2+, Ca2+, K+, Ba2+, Zn2+, Fe2+, and Mn2+) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed
industry as an additive. 相似文献
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β-1,3-1,4-Glucanase has been broadly used in feed and brewing industries. According to the codon bias of Pichia pastoris, the Bacillus subtilis MA139 β-1,3-1,4-glucanase gene was de novo synthesized and expressed in P. pastoris X-33 strain under the control of the alcohol oxidase 1 promoter. In a 10-L fermentor, the β-1,3-1,4-glucanase was overexpressed with a yield of 15,000 U/mL by methanol induction for 96 h. The recombinant β-1,3-1,4-glucanase exhibited optimal activity at 40°C and pH 6.4. The activity of the recombinant β-1,3-1,4-glucanase was not significantly affected by various metal ions and chemical reagents. To our knowledge, the expression
of this β-1,3-1,4-glucanase from Bacillus sp. in P. pastoris is in relatively high level compared to previous reports. These biochemical characteristics suggest that the recombinant
β-1,3-1,4-glucanase has a prospective application in feed and brewing industries. 相似文献
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Jae-Cheol Lee Periasamy Anbu Won-Ho Kim Myung-Ju Noh Su-Jin Lee Jeong-Woo Seo Byung-Ki Hur 《Biotechnology and Bioprocess Engineering》2008,13(5):524-532
The Δ9-elongase isolated from Thraustochytrium aureum, which contains a high level of polyunsaturated fatty acids (PUFAs), was demonstrated to be associated with the synthesis
of C20 PUFAs. The TaELO gene contains a 825 bp ORF that encodes a protein of 274 amino acids that shares a high similarity with other PUFA elongases.
The expression of the TaELO gene in Pichia pastoris resulted in the elongation of linoleic acid (LA, C18:2; n-6) and α-linolenic acid (ALA, C18:3; n-3) to eicosadienoic acid
(EDA, C20:2; n-6) and eicosatrienoic acid (ETrA, C20:3; n-3), respectively. The endogenous conversion rate of LA and ALA to
EDA and ETrA was 32.68 and 38.57%, respectively. In addition, TaELO was also able to synthesize eicosenoic acid (C20:1; n-9) from oleic acid (OA, C18:1; n-9), even though the conversion level
was low (2.81%). Furthermore, TaELO was able to carry out the 6Δ-elongation of γ-linolenic acid (GLA, C18:3; n-6) to dihomo-γ-linolenic acid (DGLA, C20:3; n-6)
and Δ5-elongation of eicosapentaenoic acid (EPA, C20:5; n-3) to docosapentaenoic acid (DPA, C22:5; n-3). The conversion rate
of GLA to DGLA and EPA to DPA were 93 and 28.36%, respectively. The TaELO protein was confirmed to have multifunctional activities, such as Δ9, Δ6, and Δ5-elongations as well as the elongation of
monounsaturated fatty acid. 相似文献
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Background
Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. 相似文献10.
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An expression plasmid containing the agdA gene encoding Aspergillus oryzae ZL-1 α-glucosidase was constructed and expressed in Pichia pastoris X-33. The molar mass of the purified protein was estimated by SDS-PAGE. HPLC analysis showed that the purified enzyme has
a transglucosylating activity with maltose as substrate. The main component of the enzyme products was panose, while amounts
of isomaltose and isomaltotriose were very low or absent. pH 5.2 and temperature of 37 °C were optimum for enzyme activity. 相似文献
12.
C. violaceum appeared as important bacterium in different applications and mainly these aspects are related to the production of violacein.
This review discusses the last reports on biosynthetic pathways, production, genetic aspects, biological activities, pathological
effects, antipathogenic screening through quorum sensing, environmental effects and the products of C. violaceum with industrial interest. An important discussion is on biological applications in medicine and as industrial products such
as textile and in cosmetics. 相似文献
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The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed
in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under
control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially
in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of
the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically
active lipase from a basidiomycete fungus. 相似文献
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Wun-Rong Hong Mei Ge Zhi-Hong Zeng Li Zhu Min-Yu Luo Lei Shao Dai-Jie Chen 《Biotechnology letters》2009,31(3):449-455
Micromonospora inyoensis produces sisomicin (Sm), an aminoglycoside antibiotic. The gene cluster of sisomicin biosynthesis spanning ca. 47 kb consists
of 37 ORFs encoding various proteins for sisomicin biosynthesis, regulation, resistance and transport. The comparative genetic
studies on the biosynthetic genes of sisomicin and gentamicin (Gm) reveal a similar biosynthetic route and provide a framework
for the future biosynthetic studies.
An erratum to this article can be found at 相似文献
16.
Bimal Kumar Ghimire Eun Soo Seong Jung Dae Lim Kweon Heo Myong Jo Kim Ill-Min Chung John A. Juvik Chang Yeon Yu 《Plant Cell, Tissue and Organ Culture》2008,95(3):265-274
Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l
α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted
on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions,
several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and
light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene.
Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work. 相似文献
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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. 相似文献
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Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH4NO3 and containing 3.0 mg l−1 IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious
root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l−1 cefotaxime and 50 mg l−1 kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium
with 300 mg l−1 cefotaxime and 50 mg l−1 kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l−1) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production
of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of
adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can
be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng. 相似文献
20.
TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD. 相似文献