Karl Kristian Thomsen

Hybrid Bacillus (1-3,1-4)-beta-glucanases: engineering thermostable enzymes by construction of hybrid genes.

SummaryHybrid (1-3,1-4)-β-glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)-β-glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)-β-glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)-β-glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65° C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70° C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)-β-glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.

A highly thermostable endo-(1,4)-β-mannanase from the marine bacterium Rhodothermus marinus

Rhodothermus marinus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity. When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 °C and pH 5.4, respectively. Purified, E. coli-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 °C and 90 °C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 °C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan.

Mouse α-amylase synthesized by Saccharomyces cerevisiae is released into the culture medium

A cDNA segment complementary to the mouse salivary amylase messenger RNA has been inserted into the yeast expression vector pMA 56 behind the promoter of the gene encoding the alcohol dehydrogenase I of yeast. Yeast transformants containing plasmids with the normal orientation of the promoter and the mouse α-amylase cDNA gene produce amylase and release the enzyme in free form into the medium. In one litre cultures with a final cell density of 2·107 cells ml−1 75 μ of amylase is found corresponding to 0.1% of the cell protein. It has been estimated that almost 90% of the amylase synthesized by the cells is excreted. A handy plate assay for colonies excreting α-amylase is described.

Improvement of bacterial β-glucanase thermostability by glycosylation

The relationship between enzyme stability and glycosylation was examined for two different Bacillus (1,3-1,4)-β-glucanases following expression of the corresponding genes in Escherichia coli and in Saccharomyces cerevisiae. Both of the (1,3-1,4)-β-glucanases secreted from yeast cells were glycosylated and a pronounced difference in the type and extent of glycosylation was observed. Thermostability analysis of the glycosylated enzymes and their unglycosylated counterparts synthesized by E. coli disclosed a substantially higher thermotolerance of the glycosylated enzymes. At 70 °C the half-life of the glycosylated form of B. macerans (1,3-1,4)-β-glucanase was 26 min, as compared to 10 min for the unglycosylated form of the enzyme. Using the same conditions, the half-life of the B. amyloliquefaciens-B. macerans hybrid (1,3-1,4)-β-glucanase was 5 min for the unglycosylated enzyme and about 100 min when the enzyme was glycosylated.

Hybrid bacillus endo-(1-3,1-4)-β-glucanases: construction of recombinant genes and molecular properties of the gene products

Hybrid β-glucanase genes were constructed by the reciprocal exchange of the two halves of the isolated β-glucanase genes from Bacillus amyloliquefaciens and B. macerans. The β-glucanase hybrid enzyme 1 (H1) contains the 107 amino-terminal residues of mature B. amyloliquefaciens β-glucanase and the 107 carboxyl-terminal amino acid residues of B. macerans β-glucanase. The reciprocal β-glucanase hybrid enzyme 2 (H2) consists of the 105 amino-terminal residues from the B. macerans enzyme and the carboxyl-terminal 107 amino acids from B. amyloliquefaciens. The biochemical properties of the two hybrid enzymes differ significantly from each other as well as from both parental β-glucanases.Hybrid β-glucanase H1 exhibits increased thermostability in comparison to other β-glucanases, especially in an acidic environment. This hybrid enzyme has maximum activity between pH 5.6 and 6.6, whereas the pH-optimum for enzymatic activity of B. amyloliquefaciens β-glucanase was found to be at pH 6 to 7 and for B. macerans at pH 6.0 to 7.5 Hybrid enzyme 1 being more heat stable than both parental enzymes represents a case of intragenic heterosis.Hybrid β-glucanase 2 (H2) was found to be more thermolabile than the naturally occurring β-glucanases it was derived from and the pH-optimum for enzymatic activity was determined to be between pH 7 and pH 8.

Purification and characterization of hordolisin, a subtilisin-like serine endoprotease from barley

Summary A protease was purified from green barley (Hordeum vulgare L.) malt with an apparent molecular mass of 74 kD and a pI of 6.9. Activity was assayed using an internally quenched fluorogenic peptide substrate, and the inhibitor profile indicated that the catalytic site contained a serine residue. This was confirmed by labelling with [14C]-diisopropyl fluorophosphate. The N-terminal amino acid sequence of the purified protease was homologous to plant subtilisin-like serine endoproteases, such as cucumisin. The barley protease (trivial name hordolisin) had a pH optimum of 6 and was stable up to 60 °C. The substrate specificity of hordolisin was determined from P4 to P3′ using a series of internally quenched fluorogenic peptide substrates. Hordolisin was similar to Savinase and subtilisin BPN′, except that Ala was better accepted at P1, and Arg was preferred at P1′. Hordolisin does not appear to be important in the degradation of the hordein storage proteins during barley grain germination.

Application of nano-probe NMR for structure determination of low nanomole amounts of arabinoxylan oligosaccharides fractionated by analytical HPAEC-PAD.

A methodology for NMR analysis of low nanomole amounts of oligosaccharides fractionated by analytical HPAEC is presented. Arabinoxylan derived oligosaccharides purified by HPAEC-PAD on an analytical column, by single injections, were analyzed with nano-probe NMR and MALDI-TOF MS to provide full structural assignment. The NMR data were obtained with a 500 MHz NMR spectrometer equipped with a 1H-observe nano-probe. Both one- and two-dimensional experiments on arabinoxylan samples in the low nanomole range were performed, including 1H-1H DQF-COSY, 1H-1H TOCSY and 1H-1H ROESY. These experiments allowed, in combination with MALDI-TOF MS and literature NMR data, a complete structural determination of several tetra-, penta-, hexa- and heptasaccharides. Two new structures: alpha-L-Araf-(1 --> 2)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-D-Xylp and alpha-L-Araf-(1 --> 2)[alpha-L-Araf-(1 --> 3)]-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-D-Xylp) were characterized, as well as some previously published structures.

LEUCOCYANIDIN REDUCTASE ACTIVITY AND ACCUMULATION OF PROANTHOCYANIDINS IN DEVELOPING LEGUME TISSUES

Proanthocyanidin (PA) and anthocyanin accumulation and location in developing leaves, flowers, and seeds of the legumes Medicago sativa, Lotus japonicus, Lotus uliginosus, Hedysarum sulfurescens, and Robinia pseudacacia were investigated by quantitative measurements and by histological analysis after staining with 1% vanillin/HCl, butanol/HCl, or 50% HC1. M. sativa leaves and flowers, L. japonicus leaves, and R. pseudacacia flowers do not contain PAs, but seeds of all investigated species contain PAs. Anthocyanins are absent in the seed coats of all five species and in leaves of L. japonicus. PA content generally increases as a function of development in leaves, but declines in flowers. With the exception of H. sulfurescens, flower PAs are synthesized in the parenchyma cells of the standard petal, while anthocyanins are located in the neighboring epidermal cells. Leucocyanidin reductase (LCR) catalyzes the conversion of 2,3-trans-3,4-cis-leucocyanidin to (+)-catechin and is the first enzyme in the PA-specific pathway. LCR activity was only detected in PA-containing tissues and generally declined during tissue development.

Construction of a yeast vector directing the synthesis and release of barley (1→3, 1→4)-β-glucanase

Two cloned cDNA sequences from barley aleurone, representing complementary parts of a barley (1→3, 1→4)-β-glucanase gene were joined together and fused in frame to a DNA fragment encoding the mouse α-amylase signal peptide. The triplet determining the amino terminal amino acid residue of native β-glucanase was changed from ATC (isoleucine) to CTA (leucine) in order to accommodate the α-amylase signal sequence in the correct reading frame. This chimaeric cDNA was inserted in the yeast expression vector pMA56 behind the promoter from the yeast alcohol dehydrogenase I gene. Yeast cells harbouring such plasmids synthesize β-glucanase and secrete the enzyme to the culture medium.

THE GLYOXYSOMAL 3-KETOACYL-COA THIOLASE PRECURSOR FROM BRASSICA NAPUS HAS ENZYMATIC ACTIVITY WHEN SYNTHESIZED IN ESCHERICHIA COLI

Glyoxysomal 3‐ketoacyl‐CoA thiolase is the last enzyme in the β‐oxidation of fatty acids in plant glyoxysomes. A full‐length cDNA of the glyoxysomal 3‐ketoacyl‐CoA thiolase from Brassica napus and a truncated version, lacking the N‐terminal targeting signal were cloned in a T7 promoter‐based vector. Both recombinant proteins were expressed in Escherichia coli and activity was measured. Full‐length and truncated 3‐ketoacyl‐CoA thiolase have comparable activity in E. coli. Moreover, full‐length 3‐ketoacyl‐CoA thiolase was purified from E. coli and N‐terminal sequencing of the protein confirmed that the precursor form indeed is enzymatically active.