Ole Olsen

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.

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.

Transformation of barley by microinjection into isolated zygote protoplasts

Barley zygote protoplasts were mechanically isolated, embedded in agarose droplets, and microinjected with a rice actin promoter Act1–gusA-nos gene construct. On average 62% of the cells survived the injection and of these 55% continued development into embryo-like structures and eventually to plants. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained this fragment. However, among the hundreds of injected zygotes, derived structures and regenerants we only found significant GUS expression in two cases (embryo-like structures nine days after injection). Two lines of green plants, derived from zygotes microinjected with linearized plasmid (line A147-1) or an isolated Act1–gusA-nos gene cassette (line A166-h) proved to be transgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to contain a single fragment of the gusA gene that was transferred to the progeny as a single Mendelian trait. One additional fragment of the gusA gene was identified in this line. The present data show that transformation of barley by microinjection of DNA into isolated zygotes is feasible but also that gene expression rarely is achieved, possibly due to degradation of the introduced DNA.

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.

Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin.

Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7.5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli-pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31% esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.

Expression of an Erwinia pectate lyase in three species of Aspergillus

Abstractu2002Transgenic filamentous fungi of the species Aspergillus niger, A. nidulans and A. awamori expressing and secreting Erwinia carotovora subsp. atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using the A. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide of A. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro-sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0u2005mg/l for expression in A. niger, A. awamori and A. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes.

Processing and secretion of barley (1–3, 1–4)-β-glucanase in yeast

DNA segments encoding signal peptides from mouse α-amylase, yeast acid phosphatase, and yeast invertase were fused in frame to a barley (1–3, 1–4)-β-glucanase cDNA gene and expressed in yeast cells under the control of the phosphoglycerate kinase gene promoter. Pure β-glucanase is obtained by gel filtration of concentrated yeast cell supernatant. It was shown that the glucanase pre-protein was specifically processed and the mature protein efficiently secreted when the yeast invertase signal sequence directed secretion.

Monitoring pathways of β-glucan degradation by enzyme mixtures in situ.

The degradation of β-glucans from cereal cell walls is related to health benefits of whole grain foodstuffs and is a prominent cost in the production of bioethanol and in improving the filterability of malt-based beverages. Detailed assays of β-glucan degradation pathways by enzyme mixtures therefore promise to support the analysis of physiological and the optimization of technological processes. Physiological and biotechnological processes tend to occur in complex mixtures of catalysts and substrates and the development of advanced methodologies for mixture analysis has been attracting a great deal of attention. In situ detection of processes that involve carbohydrate synthesis and degradation encompasses the challenge of detecting monomer identities and linkage patterns, as well as enzymatic stereochemistry and specificity while resolving chemically similar reactants, challenges that are complicated in the additional detection of intermediate degradation steps. In the current study, we show that nuclear magnetic resonance (NMR) spectroscopy near the highest available spectrometer fields permits detailed real-time assays of the degradation of polymeric barley (1→3),(1→4)-β-glucan by commercial enzyme mixtures. Up to six different intermediates can be resolved and structurally assigned within a 0.07 ppm chemical shift range using the anomeric 1H chemical signal in β-(1→3) glycosidic linkages as a structural reporter. More than 16 different glucopyranosyl spin systems are assigned to structural motifs in degradation fragments. The time course of intermediate emergence permits deciphering cleavage pathways and stereochemistry for up to four different enzyme catalyzed steps in situ.

GLYCAN MODIFICATION OF A THERMOSTABLE RECOMBINANT (1-3,1-4)-BETA -GLUCANASE SECRETED FROM SACCHAROMYCES CEREVISIAE IS DETERMINED BY STRAIN AND CULTURE CONDITIONS

High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)-β-glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1p) and theBacillus macerans (1-3,1-4)-β-glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man8-13GlcNAc2, although only traces of Man9GlcNAc2 were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3′-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y.

Analysis of the effect of dG·dC homopolymer tails on expression of a mouse α-amylase cDNA gene in yeast

Yeast expression plasmids with an α-amylase cDNA gene inserted behind theADHI gene promoter direct different levels of amylase secretion to the culture medium. The difference among these plasmid constructions is the length of the 5′ and 3′ nontranslated flanking regions of the cDNA gene. The nucleotide sequences of these regions were determined and it was found that a stretch of 17 dG·dC bp in front of the amylase gene reduces secretion substantially compared to a stretch of 3 or 8 dG·dC bp. The length of the 3′ dC·dG homopolymer tail does not influence the secretion level. Whether the 5′ homopolymer nucleotide region reduces transcription or translation is not known, but a G stretch in the 5′ end of an mRNA may adopt a secondary structure which reduces the translatability. Problems related to nucleotide sequence determination of homopolymer regions are discussed.