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At least 1,009 records · Page 56Linked to original sources
PubMed2006

Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch.

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying alpha-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.

Enzymes, Immobilized↗
PubMed2001

Role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines: application of genetically engineered Salmonella typhimurium YG7108 expressing each form of CYP together with human NADPH-cytochrome P450 reductase.

The role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines was examined by Ames test using genetically engineered Salmonella typhimurium (S. typhimurium)YG7108 cells expressing each form of human CYP together with human NADPH-cytochrome P450 reductase (OR). The relationship between the structure of N-alkylnitrosamines and CYP form(s) involved in the activation was evaluated. Eleven strains of S. typhimurium YG7108 cells expressing each form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) were employed. Eight N-alkylnitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylethylamine (NMEA), N-nitrosomethylpropylamine (NMPA), N-nitrosomethylbutylamine (NMBA) and N-nitrosoethylbutylamine (NEBA) were examined. Minimal concentration (MC) value of a promutagen was defined as the concentration of a chemical giving a positive result. Mutagen-producing capacity of CYP, as indicated by induced revertants/nmol promutagen/pmol CYP, for an N-alkylnitrosamine was determined for all forms of CYP. These N-alkylnitrosamines were mainly activated by CYP2E1, CYP2A6 and CYP1A1. N-alkylnitrosamines with relatively short alkyl chains such as NDMA and NMEA were primarily activated by CYP2E1 as judged by mutagen-producing capacity. With the increase of the number of the carbon atoms of the alkyl chains, the contribution of CYP2A6 increased. CYP2A6 played major roles in the activation of NDEA, NDPA, NMPA, NMBA and NEBA. Interestingly, CYP1A1 became a molecular form of CYP playing a major role in the metabolic activation of NDBA.

Aryl Hydrocarbon Hydroxylases↗
PubMed2026

Multistrategy metabolic engineering of Talaromyces pinophilus for α-amylase production from lignocellulosic biomass.

Filamentous fungi are important hosts for industrial enzyme production. Growing demand for α-amylase has increased reliance on food-derived carbon substrates, necessitating fungal strains that efficiently utilize nongrain biomass. In this study, Talaromyces pinophilus Y117 was metabolically engineered to produce α-amylase from lignocellulosic biomass. A strong cellobiohydrolase I gene (cbh1) promoter (Pcbh1Tru) was identified to drive expression. Multiple rounds of multilocus integration of the α-amylase gene were performed using homologous multicopy genomic sequences as recombination arms with a Cre/loxP-based recyclable selection system, yielding the multicopy strain Tp4, which achieved 4124.5 U/mL α-amylase activity in shake-flask fermentation with corncob powder as the sole carbon source. To minimize enzyme degradation, the protease gene 8538 was deleted using the Cre/lox2272 system, generating Tp4Δp. This strain showed a 50% increase in shake-flask α-amylase activity (6208.4 U/mL). In 3-L bioreactor cultivation, Tp4Δp exhibited excellent production performance, achieving 26 712.2 U/mL α-amylase activity. When corncob powder was used as the sole substrate, the cellulose and hemicellulose degradation rates reached 90.00% and 70.01%, respectively, and the enzyme yield reached 213 697.5 U per gram of corncob powder. This engineered strain demonstrates strong potential for industrial applications. The synthesis-degradation synergistic optimization strategy provides a practical approach for engineering filamentous fungal cell factories to produce enzymes directly from lignocellulosic biomass. One sentence summary Metabolic engineering of Talaromyces pinophilus through promoter optimization, multicopy integration, and protease deletion enables efficient α-amylase production from lignocellulosic biomass, achieving 26 712 U/mL in bioreactor fermentation.

Talaromyces↗
PubMed2004

Engineering of chimeric class II polyhydroxyalkanoate synthases.

PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.

Acyltransferases↗
PubMed2005

The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274-->Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R-N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 degrees C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 A resolution.

Adenosine↗
PubMed2001

Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region.

Modification of substrate specificity of an autoprocessing enzyme is accompanied by a risk of significant failure of self-cleavage of the pro-region essential for activation. Therefore, to enhance processing, we engineered the pro-region of mutant subtilisins E of Bacillus subtilis with altered substrate specificity. A high-activity mutant subtilisin E with Ile31Leu replacement (I31L) as well as the wild-type enzyme show poor recognition of acid residues as the P1 substrate. To increase the P1 substrate preference for acid residues, Glu156Gln and Gly166Lys/Arg substitutions were introduced into the I31L gene based upon a report on subtilisin BPN' [Wells et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223]. The apparent P1 specificity of four mutants (E156Q/G166K, E156Q/G166R, G166K, and G166R) was extended to acid residues, but the halo-forming activity of Escherichia coli expressing the mutant genes on skim milk-containing plates was significantly decreased due to the lower autoprocessing efficiency. A marked increase in active enzyme production occurred when Tyr(-1) in the pro-region of these mutants was then replaced by Asp or Glu. Five mutants with Glu(-2)Ala/Val/Gly or Tyr(-1)Cys/Ser substitution showing enhanced halo-forming activity were further isolated by PCR random mutagenesis in the pro-region of the E156Q/G166K mutant. These results indicated that introduction of an optimum arrangement at the cleavage site in the pro-region is an effective method for obtaining a higher yield of active enzymes.

Amino Acid Sequence↗
PubMedDate not supplied

Reconstitution of a metabolic pathway with triple-cistronic IRES-containing retroviral vectors for correction of tetrahydrobiopterin deficiency.

BACKGROUND: Tetrahydrobiopterin (BH4) is an essential cofactor for catecholamine and serotonin neurotransmitter biosynthesis. BH4 biosynthesis is carried out in a three-enzyme pathway involving GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SR). Treatment of genetic defects leading to BH4 deficiency requires neurotransmitter replacement since synthetic cofactor does not efficiently penetrate the blood-brain barrier. Autologous fibroblasts transplanted into the brain as depository cells for drug delivery might offer an alternative. However, normal fibroblasts do not express GTPCH, and fibroblasts from PTPS patients lack two biosynthetic enzymes for BH4 production. METHODS: We engineered primary fibroblasts by the use of triple-cistronic, retroviral vectors for cofactor production. RESULTS: Constitutive SR activity in these cells enabled BH4 biosynthesis by transducing GTPCH and PTPS cDNAs together with a selective marker coupled in a single transcript with two IRES-elements in tandem. Upon reaching a critical concentration (> 400 pmol/mg protein) of intracellular BH4, the fibroblasts efficiently released cofactor even under non-dividing conditions. CONCLUSION: The use of triple-cistronic vectors for single transduction to reconstitute metabolic pathways or to treat multi-genetic diseases may be useful for engineering, for instance, depository cells for various organs, including the nervous system.

Biopterins↗
PubMed2000

Autohydrolysis of plant polysaccharides using transgenic hyperthermophilic enzymes.

Commercial bioprocessing of plant carbohydrates, such as starch or cellulose, necessitates the use of commodity enzyme additives to accelerate polysaccharide hydrolysis. To simplify this procedure, transgenic plant tissues constitutively producing commodity enzymes were examined as a strategy for accelerating carbohydrate bioprocessing. Hyperthermophilic glycosyl hydrolases were selected to circumvent enzyme toxicity, because such enzymes are inactive at plant growth temperatures and are therefore physiologically benign. Transgenic tobacco lines were established that produced either a hyperthermophilic alpha-glucosidase or a beta-glycosidase using genes derived from the archaeon Sulfolobus solfataricus. Western blot and immunoprecipitation analyses were used to demonstrate the presence of recombinant enzymes in plant tissues. Transgenic enzyme levels exhibited an unusual delayed pattern of accumulation while their activities survived plant tissue preservation. Transgenic plant protein extracts released glucose from purified polysaccharide substrates at appreciable rates during incubation in high-temperature reactions. Glucose was also produced following enzymatic treatment of plant extracts enriched for endogenous polysaccharides. Direct conversion of plant tissue into free sugar was evident using whole plant extracts of either transgenic line, and could be significantly accelerated in a synergistic manner by combining transgenic line extracts.

Blotting, Western↗
PubMed2004

Chemical strategies for activity-based proteomics.

The assignment of molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes presents a major challenge for the field of proteomics. To address this problem, chemical approaches have been introduced that utilize small-molecule probes to profile dynamics in enzyme activity in complex proteomes. These strategies for activity-based protein profiling enable both the discovery and functional analysis of enzymes associated with human disease.

Animals↗
PubMed1997

Overexpression of insulin degrading enzyme: cellular localization and effects on insulin signaling.

To investigate the role of insulin degrading enzyme (insulysin, EC 3.4.24.56) in insulin signaling, Chinese hamster ovary cells overexpressing the human insulin receptor were genetically engineered to also stably overexpress the rat insulin degrading enzyme. In comparison to the parental cells, these cells expressed 2.7-fold elevated levels of enzyme and insulin degradation was also increased 2-fold. These cells also exhibited a more rapid decrease in receptor tyrosine phosphorylation after removal of insulin. Moreover, low concentrations of insulin were less effective at stimulating proliferation of the cells overexpressing the enzyme. Finally, a fraction of the overexpressed enzyme as well a fraction of the endogenous enzyme could be detected on the plasma membrane surface of these cells. These results support the hypothesis that this enzyme may function in insulin signaling by degrading the insulin molecule.

Animals↗
PubMed1997

Identification of a novel allele at the human NAT1 acetyltransferase locus.

Humans possess two N-acetyltransferase isozymes (NAT1 and NAT2). We cloned and sequenced a novel NAT1 allele (Genbank HSU 80835) that contained nucleotide substitutions at -344 (C-->T), -40 (A-->T), 445 [G-->A(Val-->Ile)], 459 [G-->A(silent)], 640 [T-->G(Ser-->Ala)], a 9 base pair deletion between nucleotides 1065 and 1090, and 1095 (C-->A). The novel NAT1 allele which we have designated NAT1*17 is similar to NAT1*11 except for a G445A substitution (Val149-->Ile) in the NAT1 coding region. The G445A (Val149-->Ile) substitution yielded no significant changes in levels of immunoreactivity, as detected by Western blot, nor in intrinsic stability of the recombinant N-acetyltransferase protein. However, the G445A (Val149-->Ile) substitution yielded expression of recombinant NAT1 protein that catalyzed the N-acetylation of aromatic amines and the O- and N,O-acetylation of their N-hydroxylated metabolites at rates up to 2-fold higher than wild-type recombinant human NAT1.

Acetylation↗
PubMed2000

Altering molecular recognition of RNA aptamers by allosteric selection.

In a continuing effort to explore structural and functional dynamics in RNA catalysis, we have created a series of allosteric hammerhead ribozymes that are activated by theophylline. Representative ribozymes exhibit greater than 3000-fold activation upon effector-binding and cleave with maximum rate constants that are equivalent to the unmodified hammerhead ribozyme. In addition, we have evolved a variant allosteric ribozyme that exhibits an effector specificity change from theophylline to 3-methylxanthine. Molecular discrimination between the two effectors appears to be mediated by subtle conformational differences that originate from displacement of the phosphodiester backbone near the effector binding pocket. These findings reveal the importance of abstruse aspects of molecular recognition by nucleic acids that are likely to be unapproachable by current methods of rational design.

Allosteric Regulation↗
PubMed2001

A novel mode of regulation of an RNA-cleaving DNAzyme by effectors that bind to both enzyme and substrate.

We describe a novel and general strategy for controlling the activity of RNA-cleaving nucleic acid enzymes (ribozymes and DNAzymes) via the use of RNA and DNA effectors. Whereas in conventional heteroallosteric enzymes (including ribozymes) control of catalysis is achieved by the binding of effector molecules to the enzyme, in our strategy DNA and RNA regulators bind to both the enzyme and the substrate. The design of this system permits the control of catalysis even in the absence of a detailed knowledge of the secondary and tertiary structure of the relevant ribozyme or DNAzyme. Here, we utilize the ability of RNA and DNA to form branched three-way junctions to regulate the RNA-cleaving activity of the in vitro selected "10-23" DNAzyme by three orders of magnitude. Control is exercised by the ability of a DNA or RNA "regulator" to induce formation of stable and catalytically competent "three-way" enzyme-substrate-regulator complexes, relative to otherwise unstable and catalytically poor enzyme-substrate complexes. Such expansively regulated "three-way" ribozyme/DNAzyme systems might find utility in vivo to bring about the catalyzed destruction of one RNA transcript contingent on the presence in its immediate environment of another gene transcript.

Base Sequence↗
PubMed2005

Trends and challenges in enzyme technology.

Several major developments took place in the field of biocatalysis over the past few years. These include the invention of directed evolution as an extremely useful method for biocatalyst improvement on the molecular level in combination with high-throughput screening systems, methods for accessing "nonculturable" biodiversity using metagenome approaches and progress in sequence-based biocatalyst discovery. In addition, new carriers and tools for immobilization of enzymes have been developed. For the synthesis of optically active compounds impressive examples using new enzymes and major progress in dynamic kinetic resolutions of racemates took place. These achievements are summarized in this review.

Biotechnology↗
PubMed1992

Determination of the rate-limiting step(s) in the biosynthetic pathways leading to penicillin and cephalosporin.

This paper is a review of strategies that have been used, or that could be used, to determine the rate-limiting step(s) in the biosynthetic pathways leading to penicillin or cephalosporin. Information is summarized from published material that involves studies with low-producing strains of Penicillium chrysogenum and Cephalosporium acremonium. We also summarize information derived from some high-producing production strains. Identification of the rate-limiting step(s) was of great interest to us as the first step in a rational program to further improve antibiotic titers of these highly developed strains. A number of approaches that could be used to elucidate the rate-limiting step(s) are described herein.

Acremonium↗
PubMed1999

Homing endonucleases: structure, function and evolution.

'Homing' is the lateral transfer of an intervening genetic sequence, either an intron or an intein, to a cognate allele that lacks that element. The end result of homing is the duplication of the intervening sequence. The process is initiated by site-specific endonucleases that are encoded by open reading frames within the mobile elements. Several features of these proteins make them attractive subjects for structural and functional studies. First, these endonucleases, while unique, may be contrasted with a variety of enzymes involved in nucleic acid strand breakage and rearrangement, particularly restriction endonucleases. Second, because they are encoded within the intervening sequence, there are interesting limitations on the position and length of their open reading frames, and therefore on their structures. Third, these enzymes display a unique strategy of flexible recognition of very long DNA target sites. This strategy allows these sequences to minimize nonspecific cleavage within the host genome, while maximizing the ability of the endonuclease to cleave closely related variants of the homing site. Recent studies explain a great deal about the biochemical and genetic mechanisms of homing, and also about the structure and function of several representative members of the homing endonuclease families.

Animals↗