scholarly journals Characterization and Heterologous Gene Expression of a Novel Esterase from Lactobacillus casei CL96

2004 ◽  
Vol 70 (6) ◽  
pp. 3213-3221 ◽  
Author(s):  
Young J. Choi ◽  
Carlos B. Miguez ◽  
Byong H. Lee
Keyword(s):  
Lactobacillus Casei ◽  
Heterologous Gene Expression ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Catalytic Triad ◽  
Methylotrophic Bacterium ◽  
Methylobacterium Extorquens ◽  
Reading Frame ◽  
One Step ◽  
Novel Esterase

ABSTRACT A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (PmxaF ), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S � tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37�C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C8), with Km and k cat values of 14 � 1.08 μM and 1,245 � 42.3 S−1, respectively.

scholarly journals Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Allyl Alcohol ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Direct Selection ◽  
Toxic Exposure ◽  
Peroxisome Biogenesis ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

Analysis of alcohol oxidase isozymes in gene-disrupted strains of methylotrophic yeast Pichia methanolica

2001 ◽  
Vol 91 (2) ◽  
pp. 225-227 ◽  
Author(s):  
Tomoyuki Nakagawa ◽  
Yasuyoshi Sakai ◽  
Hiroyuki Mukaiyama ◽  
Tasuku Mizumura ◽  
Tatsuro Miyaji ◽  
...  
Keyword(s):  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Pichia Methanolica

scholarly journals Pyruvate Carboxylase Is an Essential Protein in the Assembly of Yeast Peroxisomal Oligomeric Alcohol Oxidase

2003 ◽  
Vol 14 (2) ◽  
pp. 786-797 ◽  
Author(s):  
Paulina Ozimek ◽  
Ralf van Dijk ◽  
Kantcho Latchev ◽  
Carlos Gancedo ◽  
Dong Yuan Wang ◽  
...  
Keyword(s):  
Pyruvate Carboxylase ◽  
Tricarboxylic Acid Cycle ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Tricarboxylic Acid ◽  
Matrix Proteins ◽  
Growth Media ◽  
Fad Binding

Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.

scholarly journals Peroxisomal assembly: membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii

1990 ◽  
Vol 96 (4) ◽  
pp. 583-590
Author(s):  
M. Veenhuis ◽  
J.M. Goodman
Keyword(s):  
Early Stage ◽  
Morphological Changes ◽  
Polyclonal Antibodies ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Peroxisomal Membrane ◽  
Cell Induction ◽  
The Matrix ◽  
Dihydroxyacetone Synthase

Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance. Monoclonal and polyclonal antibodies are available against peroxisomal matrix and membrane proteins. These were utilized to correlate the induction of specific proteins with the morphological changes occurring during peroxisomal proliferation. Cells cultured in glucose-containing medium contain two to five small microbodies, which are identifiable by catalase staining and immunoreactivity with a monoclonal antibody against PMP47, an integral peroxisomal membrane protein. Three stages of proliferation can be distinguished when cells are switched to methanol as the carbon source. (1) There is an early stage (within 1 h) in which several peroxisomes develop from a preexisting organelle. This is accompanied by an increase in catalase activity and an induction of PMP47, but no detectable induction of alcohol oxidase or dihydroxyacetone synthase is observed. (2) From 1 to 2.5 h there is further division of these microbodies until up to 30 small peroxisomes generally are present in each of one or two clusters per cell. Induction of alcohol oxidase, dihydroxyacetone synthase and PMP20, a protein that is distributed in the matrix and membrane, is detectable during this time. Serial sections reveal that some peroxisomes remain uninduced while others undergo proliferation. Such sections also show no obvious connections between peroxisomes within clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Cloning, Expression, and Characterization of a Novel Thermostable and Alkaline-stable Esterase from Stenotrophomonas maltophilia OUC_Est10 Catalytically Active in Organic Solvents

Catalysts ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 401 ◽  
Author(s):  
Xinwei Gao ◽  
Xiangzhao Mao ◽  
Ping Lu ◽  
Francesco Secundo ◽  
Changhu Xue ◽  
...  
Keyword(s):  
Organic Solvents ◽  
Stenotrophomonas Maltophilia ◽  
Aqueous Media ◽  
Residual Activity ◽  
Catalytic Triad ◽  
Multiple Sequence ◽  
Medium Temperature ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Catalytically Active

A thermostable and alkaline-stable novel esterase (Est7) was identified through the whole genome sequencing of Stenotrophomonas maltophilia OUC_Est10. The open reading frame of this gene encoded 617 amino acid residues. After heterologous expression in Escherichia coli BL21 (DE3), the purified Est7 was separated as a single protein and presented a molecular mass of 70.6 kDa. Multiple sequence alignment indicated that Est7 had a typical catalytic triad (Ser-Asp-His) and the conserved sequence (GDSL) typical of the family II lipid hydrolase proteins. Est7 showed good stability in alkaline buffers, especially in Tris-HCl buffer at pH 9.0 (residual activity 93.8% after 96 h at 4 °C) and in the medium temperature conditions (residual activity 70.2% after 96 h at 45 °C and pH 8.0). The enzyme also retained higher stability toward several hydrophilic and hydrophobic organic solvents (e.g., after incubation in 100% acetonitrile or in n-hexane the enzyme retained about 97% and 84% of the activity in the absence of organic solvent, respectively). Furthermore, Est7 could catalyze the transesterification reaction of vinylacetate with 2-phenylethanol and cis-3-hexen-1-ol to their corresponding acetate esters in petroleum ether or tert-butyl methyl ether. These results indicate Est7 as a promising biocatalyst for applications of Est7 in non-aqueous media.

cDNA cloning, heterologous expression and characterization of a cell wall invertase from copper tolerant population of Elsholtzia haichowensis

Biologia ◽  
2015 ◽  
Vol 70 (8) ◽  
Author(s):  
Chen Liu ◽  
Zhongrui Xu ◽  
Shenwen Cai ◽  
Luan Zhang ◽  
Zhiting Xiong
Keyword(s):  
Cell Wall ◽  
Evolutionary Relationship ◽  
Methylotrophic Yeast ◽  
Cell Wall Invertase ◽  
Reading Frame ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Acid Protein ◽  
Elsholtzia Haichowensis ◽  
A Cell ◽  
Theoretical Molecular Mass

AbstractThe main objective of the present study was to clone, heterologously express and characterize a novel cell wall invertase (FCWI) from a Cu tolerant population of Elsholtzia haichowensis. The full-length FCWI cDNA contained an open reading frame (ORF) of 1671 bp which encoded a 556-amino-acid protein. The theoretical molecular mass and pI of the deduced protein were 62.5 kDa and 9.29, respectively. Phylogenetic analysis showed that FCWI had a closer evolutionary relationship to cell wall invertase of dicot. FCWI was expressed in methylotrophic yeast Pichia pastoris and purified to near homogeneity. Recombinant FCWI enzyme had pH optima of 4.0 and temperature optima of 50◦C. Activity analyses in the presence of various metal cations indicated that FCWI was completely inhibited by Hg

Molecular cloning and life stage expression pattern of a new acetylcholinesterase gene from the plant-parasitic nematode Meloidogyne incognita

Nematology ◽  
2003 ◽  
Vol 5 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Jean-Baptiste Laffaire ◽  
Marie-Noëlle Rosso ◽  
Stéphanie Jaubert ◽  
Pierre Abad
Keyword(s):  
Meloidogyne Incognita ◽  
Life Stage ◽  
Distinct Group ◽  
Catalytic Triad ◽  
Pcr Analysis ◽  
Parasitic Nematodes ◽  
Reading Frame ◽  
C Elegans ◽  
Second Stage ◽  
Before And After

AbstractA new cDNA, named Mi-ace-2, encoding an acetylcholinesterase, was isolated from Meloidogyne incognita. The full-length cDNA, carrying the trans-spliced SL1 leader sequence, was 2122 bp long with an open reading frame of 2058 bp. The predicted protein shared 32.3% identity with the previously identified MI ACHE and 49.1% identity with the C. elegans ACE-2. The conserved motifs involved in the catalytic triad, the choline binding site and 11 aromatic residues lining the catalytic gorge were present in the MI-ACE-2 deduced protein. RT-PCR analysis showed that Mi-ace-2 is transcribed in second stage juveniles before and after hatching, in females and in males. Phylogenetic analysis showed that MI-ACE-2 and ACE-2 were clustered in a distinct group closely related to the acetylcholinesterases secreted by animal-parasitic nematodes.

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