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N W Ho

Publications and source records attributed to N W Ho.

18 recordsLinked to original sources
PubMed1991

Genetic transformation of xylose-fermenting yeast Pichia stipitis. Scientific note.

A plasmid-mediated transformation system has been developed for the xylose-fermenting yeast Pichia stipitis. We found that plasmid vectors containing the Saccharomyces cerevisiae 2 mu replicon and the kanamycin resistance gene (KmR) could be introduced into the Pichia cells and maintained as extrachromosomal elements. Pichia transformants containing such vectors will be resistant to the antibiotic geneticin that can be inactivated by the protein product of KmR. Plasmids identical to those used for transformation can be recovered from the Pichia transformants. Protocols for transformation of P. stipitis by the CaCl2-polyethylene glycol-protoplast process or by direct electroporation of intact Pichia cells have both been developed.

Escherichia coli↗
PubMed1990

Purification, characterization, and amino terminal sequence of xylose reductase from Candida shehatae.

D-Xylose is a major component of the carbohydrates derived from agricultural residues and forest products. Among more than two hundred known xylose-utilizing yeasts, only a few species are known to be able to ferment xylose anaerobically. Candida shehatae is one of such xylose-fermenting yeasts. Xylose reductase (E.C. 1.1.1.21) is a key enzyme responsible for xylose metabolism in xylose-utilizing as well as xylose-fermenting yeasts. In this paper, we report the development of a convenient and reliable procedure for the purification of xylose reductase from C. shehatae to near homogeneity. The amino acid composition and N-terminal sequence of the enzyme have also been analyzed. C. shehatae seems to contain only a single xylose reductase, but the enzyme has a dual coenzyme specificity for both NADPH and NADH. The enzyme is remarkably stable at room temperature and 4 degrees C.

Aldehyde Reductase↗
PubMed1988

A novel xylB-based positive selection vector.

Expression of a plasmid-borne Escherichia coli xylulokinase gene (xylB) under the control of the lac promoter yields constitutively high levels of xylulokinase activity. When a plasmid containing this lac-xylB fusion (pLEK100) is transformed into a xylB- mutant the Xyl+ phenotype is restored on xylose-containing media. When the same transformants are plated on xylitol medium, growth inhibition is observed. Positive selection is achieved by cloning DNA into the unique restriction sites of pLEK100, to disrupt xylB expression, transforming E. coli, and then plating transformants on xylitol medium. With this protocol only transformants with insert containing plasmids will be obtained. This results in a considerable reduction in the time and effort needed to construct genomic libraries or perform routine DNA cloning experiments. Three unique sites are available which are suitable for positive selection of DNA fragments, via the disruption of translation (BglII) or transcription (HindIII, SalI, and BglII) of the xylB gene.

Escherichia coli↗
PubMed1987

Positive selection vectors based on xylose utilization suppression.

High levels of xylose isomerase activity in wild-type Escherichia coli strains results in a Xyl- phenotype. This phenomenon was exploited for the development of a versatile positive selection system. The xylA promoter was deleted with the exonuclease BAL 31 and the resulting structural gene was inserted into the SmaI site of pUC9, yielding the prototype vector, pLX100. In this construct xylA expression is placed under the transcriptional control of the lac promoter. Transformation of any wild-type E. coli strain with pLX100 results in high levels of xylose isomerase and a Xyl- phenotype. Decreasing the activity below a critical level (approx. 100 u) restores the Xyl+ phenotype. pLX100 contains contiguous restriction sites for HindIII, PstI, BamHI and XhoI, suitable for positive selection cloning experiments. E. coli transformants containing pLX100 cannot grow in minimal medium with xylose unless a DNA fragment is inserted into any one of the unique restriction sites. This makes the plasmid an ideal positive-selection cloning vector.

Aldose-Ketose Isomerases↗
PubMed1985

Cloning and expression of the Escherichia coli D-xylose isomerase gene in Bacillus subtilis.

A DNA fragment containing the Escherichia coli D-xylose isomerase gene and D-xylulokinase gene had been isolated from an E. coli genomic bank constructed by Clarke and Carbon. The D-xylose isomerase gene coding for the synthesis of an important industrial enzyme, xylose isomerase, was subcloned into a Bacillus-E. coli bifunctional plasmid. It was found that the intact E. coli gene was not expressed in B. subtilis, a host traditionally used to produce industrial enzymes. An attempt was then made to express the E. coli gene in B. subtilis by fusion of the E. coli xylose isomerase structural gene downstream to the promoter of the penicillinase gene isolated from Bacillus licheniformis. Two such fused genes were constructed and they were found able to be expressed in both B. subtilis and E. coli.

Aldose-Ketose Isomerases↗
PubMed1984

Cloning and characterization of the xyl genes from Escherichia coli.

Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase ( xylA ), xylulokinase ( xylB ), and regulatory ( xylR ) or transport ( xylT ) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10 -15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp Bg/II fragment and a 2.6 kbp HindIII-Sa/I fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.

Aldose-Ketose Isomerases↗
PubMed1981

Esterification of terminal phosphate groups in nucleic acids with sorbitol and its application to the isolation of terminal polynucleotide fragments.

The exposure of mono- and polynucleotides to 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and high concentrations of sorbitol results in the esterification of their monosubstituted phosphate groups. The presence of the sorbitol moiety permits these derivatives to bind strongly at pH 8.7 to columns of chromatographic supports containing the dihydroxyboryl group and to be subsequently released by elution with buffers at pH 5.5. The procedure constitutes a method for the isolation of polynucleotide fragments arising from the terminals of nucleic acids. A new method for the preparation of the chromatographic supports involves the synthesis of the 1,3-propanediol cyclic ester of m-[[3-(N-succinimidoxycarbonyl)propanoyl]amino]benzeneboronic acid and its condensation with aminoethylcellulose or amino-ethylpolyacrylamide. The reagent is readily prepared by reaction of N-[m-(dihydroxyboryl)phenyl)]succinamic acid with 1,3-propanediol to protect the boronate moiety followed by esterification with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide.

Animals↗
PubMed1979

Methods for limiting the action of SP3 DNAase and for the determination of the direction of hydrolysis of processive exonucleases.

The action of the exonuclease SP3 DNAase is inhibited by chemical modification of DNA with the cation N-cyclohexyl-N'-beta-(4-methylmorpholinium)-ethylcarbodiimide (CME). The limited activity of the enzyme on CMA-modified DNA makes it possible to demonstrate that the enzyme also initiates its attack on polydeoxyribonucleotides at the 5'-termini. This was determined by the analysis of the products from the digestion of CME-modified DNA containing labeled 5'-terminal phosphate groups. Such procedure can be adopted as a general approach for the determination of the direction of hydrolysis of other processive exonucleases. SP3 DNAase has been shown able to degrade oligo- and polydeoxyribonucleotides with or without 5'-terminal phosphate groups with equal efficiency (Aposhian, H.V., Friedman, N., Nichihara M., Heimer, E.P., and Nussbaum, A.L. (1970) J. Mol. Biol. 49, 367-379). The present work also shows that the enzyme can even hydrolyze oligo- and polynucleotides containing derivatized phosphate groups.

Bacillus subtilis↗
PubMedDate not supplied

Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene. Scientific note.

D-Xylose is a major constituent of hemicellulose, which makes up 20-30% of renewable biomass in nature. D-Xylose can be fermented by most yeasts, including Saccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase, and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source of xylose isomerase produced by recombinant E. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level of xylulokinase activity in S. cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene with an effective promoter. The resulting genetically-engineered yeast can ferment xylose and xylulose more than twice as fast as the parent yeast.

Cloning, Molecular↗