Secular trends in acute myocardial infarction in relation to physical activity in the general Danish population.

Secular trends in AMI rates were analyzed in relation to physical activity levels. The population attributable risk of physical inactivity was calculated. Participants were randomly selected subjects from a suburb of Copenhagen, Denmark, screened during the years 1964-1991. Occupational physical activity and in leisure time were assessed 1964, 1974, 1976, 1982, 1987 and 1991 by self-administered questionnaire along with smoking habits and alcohol consumption. Blood pressure, weight, height and serum lipids were measured according to WHO-standards. Mortality data were obtained from death certificates, from hospital records or autopsies. Acute myocardial infarctions (AMI) 1964-1994 were included. 13.925 men and women aged 30, 40, 50 and 60 years, were drawn as random samples from a background population of 300.000 inhabitants. A cohort born in 1914 was examined in 1964 and 1974, a cohort born in 1936, was examined in 1976 and 1987; Monica (Monitoring trends and determinants in cardiovascular diseases) I cohort were examined in 1982 and 1987; MONICA II in 1986, and MONICA III in 1991. Mean physical activity level at leisure adjusted for age and sex increased over time (P < 0001). 25% of the men were sedentary, and more women reported a sedentary lifestyle than men. The overall trend was from 1964 to 1992 a decline in physical activity at work (P < 0001) in both gender and all age groups. The difference in AMI incidence rates between leisure time physical activity (LTPA) levels increased over time. No change was found in AMI rates comparing sedentary in different time periods. A remarkable decrease over time in the AMI incidence rate was found in physically active during leisure time. Population attributable risk (PAR) exceeded 40% in both genders in the late 1980s. In conclusion the difference in AMI rates between LTPA subgroups has increased over time. The low AMI rates observed among the most physically active reveal a substantial potential for the prevention of AMI through physical activity. A population attributable risk of more than 40% for physical inactivity suggests a potential for primary prevention through increased physical activity.

Isolation, enzymatic properties, and mode of action of an exo-1,3-beta-glucanase from Trichoderma viride.

An exo-1,3-beta-glucanase has been isolated from cultural filtrate of T. viride AZ36. The N-terminal sequence of the purified enzyme (m = 61 +/- 1 kDa) showed no significant homology to other known glucanases. The 1,3-beta-glucanase displayed high activity against laminarins, curdlan, and 1,3-beta-oligoglucosides, but acted slowly on 1,3-1,4-beta-oligoglucosides. No significant activity was detected against high molecular mass 1,3-1,4-beta-glucans. The enzyme carried out hydrolysis with inversion of the anomeric configuration. Whereas only glucose was released from the nonreducing terminus during hydrolysis of 1,3-beta-oligoglucosides, transient accumulation of gentiobiose was observed during hydrolysis of laminarins. The gentiobiose was subsequently degraded to glucose. The Michaelis constants Km and Vmax have been determined for the hydrolysis of 1,3-beta-oligoglucosides with degrees of polymerization ranging from 2 to 6. Based on these data, binding affinities for subsites were calculated. Substrate binding site contained at least five binding sites for sugar residues.

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.

A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley analysis of substrate preference and specificity by nano-probe NMR.

An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The enzyme preparation, Ara 1, contained two polypeptides with apparent molecular masses of approximately 60 and approximately 66 kDa, a pI of 4.55 and almost identical N-terminal amino-acid sequences. With p-nitrophenyl alpha-L-arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a Km of 0.5 mM and a Vmax of 6.7 micromol. min-1.(mg of protein)-1. Maximum activity was displayed at pH 4.2 and 60 degrees C, and, under these conditions, the half-life of the enzyme was 8 min. The Ara 1 preparation showed no activity against p-nitrophenyl alpha-L-arabinopyranoside or p-nitrophenyl beta-D-xylopyranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano-probe NMR. Ara 1 released arabinose from high-molecular-mass arabinoxylan and arabinoxylan-derived oligosaccharides but was inactive against linear or branched-chain arabinan. Arabinose was readily released from both singly and doubly substituted xylo-oligosaccharides. Whereas single 2-O-linked and 3-O-linked arabinose substituents on non-reducing terminal xylose were released at similar rates, there was a clear preference for 2-O-linked arabinose on internal xylose residues. When Ara 1 acted on oligosaccharides with doubly substituted, non-reducing terminal xylose, the 3-O-linked arabinose group was preferred as the initial point of attack. Oligosaccharides with doubly substituted internal xylose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase which is able to release arabinose from both singly and doubly substituted xylose, and it hydrolyses p-nitrophenyl alpha-L-arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.

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

Rhodothermus marimus 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 degrees C and pH 5.4, respectively. Purified, E. coil-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 degrees C and 90 degrees C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 degrees C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan.

Purification and characterization of barley dipeptidyl peptidase IV.

Barley (Hordeum vulgare L.) storage proteins, which have a high content of proline (Pro) and glutamine, are cleaved by cysteine endoproteases to yield peptides with a Pro next to the N-terminal and/or C-terminal amino acid residues. A peptidase cleaving after Xaa-Pro- at the N terminus of peptides was purified from green barley malt. It was identified as a serine-type dipeptidyl peptidase (DPP), based on inhibitor studies, and the nature of the cleavage product. It is a monomeric glycoprotein with an apparent molecular mass of 105 kD (85 kD after deglycosylation), with a pI of 3.55 and a pH optimum at 7.2. Substrate specificity was determined with a series of fluorogenic peptide substrates with the general formula Xaa-Pro-AMC, where Xaa is an unspecified amino acid and AMC is 7-amino-4-methylcoumarin. The best substrates were Xaa = lysine and arginine, while the poorest were Xaa = aspartic acid, phenylalanine, and glutamic acid. The K(m) values ranged from 0.071 to 8.9 microM, compared with values of 9 to 130 microM reported for mammalian DPP IVs. We discuss the possible role of DPP IV in the degradation of small Pro-containing peptides transported from the endosperm to the embryo of the germinating barley grain.

A transglycosylating 1,3(4)-beta-glucanase from rhodothermus marinus NMR analysis of enzyme reactions.

The enzymatic hydrolysis of polysaccharides by the 1, 3(4)-beta-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such as laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1, 4-beta-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley beta-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1, 3-beta-glycosidic and 1,4-beta glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides. When barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1, 4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-beta-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme-substrate complex and subsequent hydrolysis/transglycosylation.

The laminarinase from thermophilic eubacterium Rhodothermus marinus--conformation, stability, and identification of active site carboxylic residues by site-directed mutagenesis.

A gene (lamR) encoding laminarinase (LamR) was cloned from the marine thermophilic eubacterium Rhodothermus marinus ITI278. The enzyme purified from recombinant Escherichia coli cells hydrolyses mixed 1,3-1,4-beta-glucans (lichenan, barley and oat beta-glucan) and 1,3-beta-homoglucans (laminarin, curdlan) by an endo type action pattern. The CD spectrum of laminarinase is characteristic for a protein with prevailing beta secondary-structural elements, and the fluorescence spectrum points to a surface localisation of the tryptophan residues. A half-transition concentration of 5.4 M guanidinium chloride was measured for the denaturant-induced unfolding of laminarinase monitoring changes of the ellipticity at 222 nm and the fluorescence. Substitution of acidic residues Glu129, Asp131 and Gln134, which are invariant in family 16 glycosyl hydrolases, caused a severe reduction of beta-glucan-hydrolysing activity suggesting their central role in enzymatic hydrolysis. Deletion of Met133 drastically reduced catalytic activity. Met133 is invariant in family 16 laminarinases but not present in the active-site region of bacterial 1,3-1,4-beta-glucanases which also belong to glycosyl hydrolase family 16. Replacement of Met133 by Ala, Cys or Trp did not affect activity against 1,3-1,4-beta-polyglucans and 1,3-beta-polyglucans, but in mutant Met133A the rate of hydrolysis of cellobiosyltriose (G1-4G1-3Gr) was increased about 10 times. Hydrolysis of 1,3-beta-oligosaccharides and 1,4-beta-oligosaccharides (DP 2-7) demonstrated the ability of the enzyme to cleave 1,3-beta-linkages and 1,4-beta-linkages in low-molecular-mass carbohydrates independent of the structure of neighbouring linkages. The laminarinase contains five or six subsites for substrate binding according to the action pattern deduced from hydrolysis of labelled and unlabelled curdlan oligosaccharides of different chain length.

Secular trends in blood pressure levels in Denmark 1964-1991.

BACKGROUND: Hypertension is an essential risk factor for development of cardiovascular diseases. Prospective studies show a reduction in risk of myocardial infarction with reduction of blood pressure. In Denmark there was a decrease in ischaemic heart disease mortality during the period (1968-1992) with around 34% in 30-65 year old men and 30% in women. OBJECTIVE: To assess the changes in casual blood pressure between 1964 and 1991 in seven cross-sectional population studies. SETTING: Centre of Preventive Medicine, University of Copenhagen, DK-2600 Glostrup. POPULATION: 10359 subjects, equal numbers of men and women, age exactly 30, 40, 50 and 60 years drawn as random samples from a background population of 300000 inhabitants and surveyed in 1964-1974 and five cross-sectional studies 1976, 1978, 1982-1984, 1986-1987 and 1991. METHODS: Blood pressure was measured according to WHO criteria by one technician in each survey. Alcohol consumption and physical activity were measured by a self-administered questionnaire. The weight and height were measured by standardized methods. Data on mortality from ischaemic heart disease were obtained from death certificates recorded by the National Board of Health. RESULTS: Blood pressure increased with increasing age in both genders and was significantly higher in men than in women. Median blood pressure in 50 year old men in 1964 was 135/85 mmHg and in 1991 it was 123/79, whereas in women in 1964 it was 140/85, against 119/74 in 1991. The prevalence of hypertensives among 30 and 40 year olds declined throughout the period. The performance of blood pressure measurements, technical variation, examination programme, seasonal variation and inter-observer variation were potential bias sources and influenced blood pressure levels, but cannot be shown to be responsible for the declining trend in blood pressure and hypertension. Women became a little more physical active in leisure time and men less active. Women consumed less alcohol than men, but the amounts slightly increased by the end of the period. Body mass index >25 was seen less frequently in women than in men and this increased in men over the period. Sale of antihypertensive drugs increased in Denmark over the 1964-1991 period. There seems to be good agreement between the changes in blood pressure in the population and the decline in mortality from stroke and coronary heart disease in Denmark, which is influenced by other risk factors as well. CONCLUSION: Blood pressure distributions have shifted towards lower values in 1964-1991. Prevalence of hypertension declined up to 1983. Risk factor changes as well as treatment for hypertension contribute to this.

Crystal structure of barley 1,3-1,4-beta-glucanase at 2.0-A resolution and comparison with Bacillus 1,3-1,4-beta-glucanase.

Both plants and bacteria produce enzymes capable of degrading the mixed-linked beta-glucan of the endosperm cell walls of cereal grains. The enzymes share the specificity for beta-1,4 glycosyl bonds of O-3-substituted glucose units in linear polysaccharides and a similar cleavage mechanism but are unrelated in sequence and tertiary structure. The three-dimensional structure of the 1,3-1, 4-beta-glucanase isoenzyme EII from barley was determined from monoclinic crystals at a resolution of 2.0 A. The protein is folded into a betaalpha8 barrel structure as has been shown previously (Varghese, J. N., Garrett, T. P. J., Colman, P. M., Chen, L., Hoj, P. B., and Fincher, G. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2785-2789) by diffraction analysis at lower resolution of tetragonal crystals. It contains one N-glycosylation site which is described in detail with the sugar moieties attached to residue Asn190. The geometry and hydration of the barley 1,3-1,4-beta-glucanase is analyzed; a model beta-glucan fragment is placed into the binding site by molecular dynamics simulation, and the beta-glucan binding grooves of the plant and bacterial enzymes are compared. Their active sites are shown to have a small number of common features in generally dissimilar geometries that serve to explain both the identical substrate specificity and the observed differences in inhibitor binding.

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 beta-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.

Mitral valve replacement with total preservation of native valve and subvalvular apparatus.

BACKGROUND AND AIMS OF THE STUDY: Preservation of the mitral valve and subvalvular apparatus was introduced clinically in the early 1960s, but for two decades the technique for mitral valve replacement included excision of both leaflets and their attached chordae tendineae. Lately, emphasis has been replaced on retaining the mitral subvalvular apparatus during valve replacement because of its role in left ventricular function. Hence, during the past six years, when performing mitral valve replacement we have, when possible, preserved the valvular and sub-valvular mitral apparatus. METHODS: Between January 1990 and November 1996, complete retention of all mitral tissue in connection with mitral valve replacement was performed in 58 patients (23 women and 35 men). Mean age was 63 years (range: 23 years to 77 years). Coronary bypass was a concomitant procedure in 19 patients; both the mitral and aortic valve was replaced in four cases. Calcified and/or stenotic valves were not a contraindication for the procedure; calcified plaques were removed. Adhesion between anterior and posterior leaflets was treated with sharp dissection. Valve and subvalvular tissue were preserved. The leaflets were reefed within the valve-sutures and compressed between the sewing ring and the native annulus when implanting the valve prosthesis. Chordal tension on the ventricle is thus maintained and the chordae pulled away from the valve effluent. RESULTS: Six patients died in the postoperative period and three had transient neurological symptoms. In no patient was death or transient neurological symptoms a consequence of the retention of mitral leaflets with subvalvular apparatus. CONCLUSIONS: We find the described technique to be useful not only in valve insufficiency but also in valve stenosis when preserving the mitral leaflets with sub-valvular apparatus during valve replacement. The technique is without procedure-related complications and prevents obstruction of left ventricular outflow tract.

Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.

Transplanting two unique beta-glucanase catalytic activities into one multienzyme, which forms glucose.

Endo cellulases of plant pathogenic erwinias degrade cellulose as well as the cellulosic domains of barley (1-3,1-4)-beta-glucan. Depolymerization of the latter substrate is mainly caused by (1-3,1-4)-beta-glucanases, which hydrolyze (1-4)-beta glycosidic linkages adjacent to (1-3)-beta linkages. To construct an enzyme for efficient degradation of barley (1-3,1-4)-beta-glucan, the sequence encoding the catalytic domain and interdomain linker of the cellulase from Erwinia carotovora subspecies atroseptica was fused to that for the heat stable Bacillus hybrid, H(A12-M) delta Y13 (1-3,1-4)-beta glucanase. The chimeric enzyme secreted from Escherichia coli cells did not remain covalently assembled as judged by SDS-PAGE. However, the glycosylated and intact enzyme (denoted CELGLU) is secreted from the yeast Pichia pastoris. CELGLU exhibits both cellulase and (1-3,1-4)-beta-glucanase catalytic activities, and was accordingly classified a true multienzyme. HPLC and NMR analyses revealed that among the products from CELGLU, di- and trimeric oligosaccharides were identical to those produced by the parental cellulase. Tetrameric oligosaccharides, derived from the (1-3,1-4)-beta-glucanase activity of CELGLU, were further degraded by the cellulase moiety to yield glucose and trimers. Compared with the parental enzymes, CELGLU exhibits substantially higher Vmax for degradation of both soluble cellulose and barley (1-3,1-4)-beta-glucan. These findings point to construction of multienzymes as an effective approach for engineering enzymes with novel characteristics.

Crystallization of barley (1-3,1-4)-beta-glucanase, isoenzyme II.

The (1-3,1-4)-beta-glucanase from barley (Hordeum vulgare, cv Alexis) degrades mixed linked beta-glucan in the cell wall of the starchy endosperm. Isoenzyme II of the (1-3,1-4)-beta-glucanase forms large single crystals when a protein solution is equilibrated against 20% (w/w) polyethylene glycol 8000 at acidic pH using the hanging drop vapour diffusion method. The crystals diffract to better than 2 A resolution. They are monoclinic, space group P2(1), with cell dimensions a = 49.58 A, b = 82.99 A, c = 77.56 A and beta = 104.36 degrees. Two protein molecules are estimated to fill the asymmetric unit.

Crystallization of the hybrid Bacillus (1-3,1-4)-beta-glucanase H(A16-M).

The extremely thermostable hybrid Bacillus (1-3,1-4)-beta-glucanase H(A16-M) has been crystallized with polyethylene glycol by vapour diffusion. The single crystals diffract to a resolution of 2.2 A. The protein crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 70.22(7) A, b = 72.56(1) A, c = 49.97(1) A, and has one molecule per asymmetric unit.

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

Hybrid (1-3,1-4)-beta-glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)-beta-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)-beta-glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)-beta-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 degrees C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70 degrees 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)-beta-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.

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

Hybrid beta-glucanase genes were constructed by the reciprocal exchange of the two halves of the isolated beta-glucanase genes from Bacillus amyloliquefaciens and B. macerans. The beta-glucanase hybrid enzyme 1 (H1) contains the 107 amino-terminal residues of mature B. amyloliquefaciens beta-glucanase and the 107 carboxyl-terminal amino acid residues of B. macerans beta-glucanase. The reciprocal beta-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 beta-glucanases. Hybrid beta-glucanase H1 exhibits increased thermostability in comparison to other beta-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 beta-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 beta-glucanase 2 (H2) was found to be more thermolabile than the naturally occurring beta-glucanases it was derived from and the pH-optimum for enzymatic activity was determined to be between pH 7 and pH 8.

Processing and secretion of barley (1-3,1-4)-beta-glucanase in yeast.

DNA segments encoding signal peptides from mouse alpha-amylase, yeast acid phosphatase, and yeast invertase were fused in frame to a barley (1-3,1-4)-beta-glucanase cDNA gene and expressed in yeast cells under the control of the phosphoglycerate kinase gene promoter. Pure beta-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.

Primary structure of the (1-->3,1-->4)-beta-D-glucan 4-glucohydrolase from barley aleurone.

During germination of barley grains, the cell walls of the starchy endosperm are degraded by (1-->3,1-->4)-beta-glucanases (EC 3.2.1.73) secreted from the aleurone and scutellar tissues. The complete sequence of the aleurone (1-->3,1-->4)-beta-glucanase isoenzyme II comprises 306 amino acids and was determined by sequencing nine tryptic peptides (110 residues) and aligning them with the amino acid sequence deduced from a cDNA clone encoding the 291 NH(2)-terminal residues. Although no amino acid sequence homology with a bacterial (1-->3)(1-->4)-beta-glucanase is apparent, close to 50% homology is found with two large regions of a (1-->3)-beta-glucanase from tobacco pith tissue. The gene for barley (1-->3,1-->4)-beta-glucanase isoenzyme II shares with that for the alpha-amylase isoenzyme 1 a strongly preferred use of codons with G and C in the wobble position (94% and 90%, respectively). Both enzymes are secreted from the aleurone cells during germination. Such one-sided codon usage is not characteristic for the gene encoding the (1-->3)-beta-glucanase of tobacco pith tissue or the hor2-4 gene encoding the B(1) hordein storage protein in the endosperm.