Improvement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)

Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.

Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

Influenza virus hemagglutinin-specific cytotoxic T cell response induced by polypeptide produced in Escherichia coli.

Journal of Experimental Medicine ◽

10.1084/jem.162.2.663 ◽

1985 ◽

Vol 162 (2) ◽

pp. 663-674 ◽

Cited By ~ 29

Author(s):

A Yamada ◽

M R Ziese ◽

J F Young ◽

Y K Yamada ◽

F A Ennis

Keyword(s):

Influenza Virus ◽

Recombinant Dna ◽

Cytotoxic T Lymphocyte ◽

Nonstructural Protein ◽

T Cell Response ◽

Cytotoxic T Cell ◽

Protein Immunization ◽

We have tested the abilities of various polypeptides of A/PR/8/34 (H1N1) virus, constructed by recombinant DNA techniques, to induce influenza virus-specific secondary cytotoxic T lymphocyte (CTL) responses. A hybrid protein (c13 protein), consisting of the first 81 amino acids of viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutinin (HA), induced H-2-restricted, influenza virus subtype-specific secondary CTL in vitro, although other peptides did not. Using a recombinant virus, the viral determinant responsible for recognition was mapped to the HA2 portion of c13 protein. Immunization of mice with c13 protein induced the generation of memory CTL in vivo. The CTL precursor frequencies of A/PR/8/34 virus- and c13 protein-immune mice were estimated as one in 8,047 and 50,312, respectively. These results indicate that c13 protein primed recipient mice, even though the level of precursor frequency was below that observed in virus-immune mice.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2372-2382.1992 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2372-2382

Author(s):

K M Arndt ◽

S L Ricupero ◽

D M Eisenmann ◽

F Winston

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Direct Repeat ◽

Single Amino Acid ◽

Wild Type ◽

Dna Binding Specificity ◽

Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

Role of the reaction-structure coupling in temperature compensation of the KaiABC circadian rhythm

10.1101/2021.10.11.464015 ◽

2021 ◽

Author(s):

Masaki Sasai

Keyword(s):

Temperature Compensation ◽

Oscillation Amplitude ◽

Thermal Fluctuations ◽

Reaction Rates ◽

System Level ◽

Single Amino Acid ◽

Structural Transitions ◽

Residue Substitution

When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. We analyzed this temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model suggests that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.

Mutations in the Vaccinia Virus A33R and B5R Envelope Proteins That Enhance Release of Extracellular Virions and Eliminate Formation of Actin-Containing Microvilli without Preventing Tyrosine Phosphorylation of the A36R Protein

Journal of Virology ◽

10.1128/jvi.77.22.12266-12275.2003 ◽

2003 ◽

Vol 77 (22) ◽

pp. 12266-12275 ◽

Cited By ~ 36

Author(s):

Ehud Katz ◽

Brian M. Ward ◽

Andrea S. Weisberg ◽

Bernard Moss

Keyword(s):

Vaccinia Virus ◽

Envelope Protein ◽

Actin Polymerization ◽

Single Amino Acid ◽

Wild Type Virus ◽

Virus Release ◽

Type Virus ◽

Wild Type ◽

Extracellular Virus

ABSTRACT The spread of vaccinia virus in cell cultures is mediated by virions that adhere to the tips of specialized actin-containing microvilli and also by virions that are released into the medium. The use of a small plaque-forming A36R gene deletion mutant to select spontaneous second-site mutants exhibiting enhanced virus release was described previously. Two types of mutations were found: C-terminal truncations of the A33R envelope protein and a single amino acid substitution of the B5R envelope protein. In the present study, we transferred each type of mutation into a wild-type virus background in order to study their effects in vitro and in vivo. The two new mutants conserved the enhanced virus release properties of the original isolates; the A33R mutant produced considerably more extracellular virus than the B5R mutant. The extracellular virus particles contained the truncated A33R protein in one case and the mutated B5R protein in the other. Remarkably, both mutants failed to form actin tails and specialized microvilli, despite the presence of an intact A36R gene. The synthesis of the A36R protein as well as its physical association with the mutated or wild-type A33R protein was demonstrated. Moreover, the A36R protein was tyrosine phosphorylated, a step mediated by a membrane-associated Src kinase that regulates the nucleation of actin polymerization. The presence of large numbers of adherent virions on the cell surface argued against rapid dissociation as having a key role in preventing actin tail formation. Thus, the A33R and B5R proteins may be more directly involved in the formation or stabilization of actin tails than had been previously thought. When mice were inoculated intranasally, the A33R mutant was highly attenuated and the B5R mutant was mildly attenuated compared to wild-type virus. Enhanced virus release, therefore, did not compensate for the loss of actin tails and specialized microvilli.

Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function.

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2231 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2231-2242 ◽

Cited By ~ 72

Author(s):

J E Rudolph ◽

M Kimble ◽

H D Hoyle ◽

M A Subler ◽

E C Raff

Keyword(s):

Amino Acid ◽

Sequence Divergence ◽

Amino Acid Sequences ◽

Single Amino Acid ◽

Structural Constraints ◽

Wild Type ◽

Beta Tubulin ◽

Developmentally Regulated ◽

Beta 2

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.

A Novel Deletion Mutation in pmrB Contributes to Concurrent Colistin Resistance in Carbapenem-Resistant Escherichia coli Sequence Type 405 of Clinical Origin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00220-20 ◽

2020 ◽

Vol 64 (6) ◽

Author(s):

Ching-Hsun Wang ◽

L. Kristopher Siu ◽

Feng-Yee Chang ◽

Yu-Kuo Tsai ◽

Yi-Tsung Lin ◽

...

Keyword(s):

Escherichia Coli ◽

Recombinant Dna ◽

Selective Pressure ◽

Carbapenem Resistance ◽

Sequence Type ◽

Wild Type ◽

Colistin Resistance ◽

Content Type ◽

Carbapenem Resistant ◽

ABSTRACT We report the first clinical Escherichia coli strain EC3000 with concomitant chromosomal colistin and carbapenem resistance. A novel in-frame deletion, Δ6-11 (RPISLR), in pmrB that contributes to colistin resistance was verified using recombinant DNA techniques. Although being less fit than the wild-type (WT) strain or EC3000 revertant (chromosomal replacement of WT pmrB in EC3000), a portion of serially passaged EC3000 strains preserving colistin resistance without selective pressure raises the concern for further spread.

The characterization of ligand-specific maize (Zea mays) profilin mutants

Biochemical Journal ◽

10.1042/bj3580049 ◽

2001 ◽

Vol 358 (1) ◽

pp. 49-57 ◽

Cited By ~ 10

Author(s):

David R. KOVAR ◽

Bj⊘rn K. DRØBAK ◽

David A. COLLINGS ◽

Christopher J. STAIGER

Keyword(s):

Zea Mays ◽

Hair Cells ◽

Plant Cells ◽

Single Amino Acid ◽

Actin Assembly ◽

Wild Type ◽

Ligand Interaction ◽

Binding Partners

Profilins are low-molecular-mass (12–15kDa) cytosolic proteins that are major regulators of actin assembly in all eukaryotic cells. In general, profilins from evolutionarily diverse organisms share the ability to bind to G-actin, poly-(l-proline) (PLP) and proline-rich proteins, and polyphosphoinositides. However, the functional importance of each of these interactions remains unclear and might differ between organisms. We investigated the importance of profilin's interaction with its various ligands in plant cells by characterizing four maize (Zea mays) profilin 5 (ZmPRO5) mutants that had single amino acid substitutions in the presumed sites of ligand interaction. Comparisons in vitro with wild-type ZmPRO5 showed that these mutations altered ligand association specifically. ZmPRO5-Y6F had a 3-fold increased affinity for PLP, ZmPRO5-Y6Q had a 5-fold decreased affinity for PLP, ZmPRO5-D8A had a 2-fold increased affinity for PtdIns(4,5)P2 and ZmPRO5-K86A had a 35-fold decreased affinity for G-actin. When the profilins were microinjected into Tradescantia stamen hair cells, ZmPRO5-Y6F increased the rate of nuclear displacement in stamen hairs, whereas ZmPRO5-K86A decreased the rate. Mutants with a decreased affinity for PLP (ZmPRO5-Y6Q) or an enhanced affinity for PtdIns(4,5)P2 (ZmPRO5-D8A) were not significantly different from wild-type ZmPRO5 in affecting nuclear position. These results indicate that plant profilin's association with G-actin is extremely important and further substantiate the simple model that profilin acts primarily as a G-actin-sequestering protein in plant cells. Furthermore, interaction with proline-rich binding partners might also contribute to regulating profilin's effect on actin assembly in plant cells.

A single amino acid change converts Aurora-A into Aurora-B-like kinase in terms of partner specificity and cellular function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0900833106 ◽

2009 ◽

Vol 106 (17) ◽

pp. 6939-6944 ◽

Cited By ~ 50

Author(s):

Jingyan Fu ◽

Minglei Bian ◽

Junjun Liu ◽

Qing Jiang ◽

Chuanmao Zhang

Keyword(s):

Amino Acid ◽

Aurora Kinase ◽

Single Amino Acid ◽

Aurora B ◽

Aurora A ◽

Single Amino Acid Change ◽

Equivalent Residue ◽

And Function

Aurora kinase-A and -B are key regulators of the cell cycle and tumorigenesis. It has remained a mystery why these 2 Aurora kinases, although highly similar in protein sequence and structure, are distinct in subcellular localization and function. Here, we report the striking finding that a single amino acid residue is responsible for these differences. We replaced the Gly-198 of Aurora-A with the equivalent residue Asn-142 of Aurora-B and found that in HeLa cells, Aurora-AG198N was recruited to the inner centromere in metaphase and the midzone in anaphase, reminiscent of the Aurora-B localization. Moreover, Aurora-AG198N compensated for the loss of Aurora-B in chromosome misalignment and cell premature exit from mitosis. Furthermore, Aurora-AG198N formed a complex with the Aurora-B partners, INCENP and Survivin, and its localization depended on this interaction. Aurora-AG198N phosphorylated the Aurora-B substrates INCENP and Survivin in vitro. Therefore, we propose that the presence of Gly or Asn at a single site assigns Aurora-A and -B to their respective partners and thus to their distinctive subcellular localizations and functions.

IMMUNIZATION AGAINST AN INHIBIN SUBUNIT PRODUCED BY RECOMBINANT DNA TECHNIQUES RESULTS IN INCREASED OVULATION RATE IN SHEEP

Journal of Endocrinology ◽

10.1677/joe.0.114r001 ◽

1987 ◽

Vol 114 (2) ◽

pp. R1-R4 ◽

Cited By ~ 49

Author(s):

R.G. Forage ◽

R.W. Brown ◽

K.J. Oliver ◽

B.T. Atrache ◽

P.L. Devine ◽

...

Keyword(s):

Fusion Protein ◽

Recombinant Dna ◽

Binding Capacity ◽

Control Groups ◽

Keyhole Limpet Haemocyanin ◽

A Subunit ◽

ABSTRACT Seven Merino–Border Leicester cross–bred ewes were immunized with a purified fusion protein, produced by recombinant DNA methods, of the a subunit of bovine inhibin. Four animals were immunized with the fusion protein alone and three with a conjugate made by coupling the fusion protein to keyhole limpet haemocyanin (KLH) using glutaraldehyde. Each animal received four injections of the fusion protein over 93 days. The animals were synchronized using progestagen sponges and subjected to laparoscopy for the determination of ovulation rates in two consecutive cycles (days 115 and 135). The immunized animals had overall mean ovulation rates for each cycle of 3.4 and 3.4 which was significantly (P < 0.001) above the rates of 1.1 and 1.4 determined for the controls, which had either received no treatment (n=5) or had been immunized with 300 μg KLH (n=4). Analysis of antisera taken on day 115 showed significant fusion protein antibodies and iodinated inhibin–binding capacity in the test but not control groups. Furthermore, antisera to the fusion protein in four out of seven ewes neutralized the inhibin bioactivity of ovine follicular fluid in an in–vitro bioassay. These data demonstrate that neutralization of inhibin can be effected by immunization with bovine inhibin a subunit and that such immunization results in increased ovulation rates as predicted from the biological role of inhibin as a suppressor of FSH.