Protein engineering in the α-amylase family: catalytic mechanism,substrate specificity,and stability

Most starch hydrolases and related enzymes belong to the -amylase family which contains a characteristic catalytic (/)₈-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the -amylases, the cyclodextrin glucanotransferases, and the oligo-1,6-glucosidases. Throughout the -amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of -amylases, changed the distribution of -, - and -cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative -1,4:-1,6 dual-bond specificity of neopullulanase. Barley -amylase isozyme hybrids and Bacillus -amylases demonstrate the impact of a small domain B protruding from the (/)₈-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as -amylase, starch branching and debranching enzymes, and amylomaltase.Abbreviations CGTase cyclodextrin glucanotransferase - SBD starch binding domain - TAA taka-amylase A - TIM triose-phosphate isomerase. The mutations are described with the one-letter code, i.e. D164A is a mutant in which A in the mutant is substituted for D in the wild-type.     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.     

Starch hydrolysing enzymes in the developing barley grain

Summary The enzymes -amylase (-1, 4-glucan 4-glucanohydrolase, 3.2.1.1), -amylase (-1,4-glucan maltohydrolase, 3.2.1.2) and phosphorylase (-1,4-glucan: orthophosphate glucosyltransferase, 2.4.1.1) were assayed in whole grains of barley throughout the maturation period. -amylase and phosphorylase had peaks of activity between 25 and 30 days after anthesis. On the other hand the activity of -amylase in both the available and latent forms reached a maximum value at 35 days after anthesis which did not decrease thereafter. -amylase activity was also assayed throughout development in the endosperm, aleurone, testa pericarp and embryo. Latent -amylase reached a constant maximum value in endosperm at 35 days but available -amylase reached a peak of activity at 25 days and then declined to zero at 45 days. Only latent -amylase was associated with the aleurone layer and activity rose to a maximum value at 35 days. The testa pericarp had mainly latent -amylase whose activity fell from an early maximum at 21 days to zero at 35 days. No hydrolytic activity was associated with the embryo. The phosphorylase activity was low and mainly associated with the endosperm fraction.     

Sterol conjugates of two phenotypically different calli of Beta vulgaris

Steryl glycosides are the predominant form of sterol at 88% of the total sterol in non-betalain producing calli of Beta vulgaris. The total sterol decreases and sterol form shifts from steryl glycosides to 97% free sterol upon the transition of non-betalain to betalain producing calli. A substantial decrease in stigmasterol (24--ethylcholesta-5,22E-dien-3-ol) and sitosterol (24-ethylcholest-5-en-3-ol) levels is observed during this transition, and alters the ratio of ⁷:⁵ sterols. Spinasterol (24- ethyl-5-cholesta-7,22E-dien-3-ol) is the dominant sterol at 43% and 95% of the total sterol in non-betalain producing and betalain producing calli. The level of 22-dihydrospinasterol (24-ethyl-5-cholest-7-en-3-ol) is reduced in both calli to 3% from 25% in leaves. Lanosterol (4,4,14-trimethyl-cholesta-8(9),24-dien-3-ol) and cycloartenol (9,19-cyclopropyl-4,4,14-trimethyl-cholest-24-en-3-ol) were identified in betalain and nonbetalain producing callus respectively.     

Interaction sites on phosphorylase kinase for calmodulin

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the subunit and a peptide from the subunit present in a region deleted in the isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca²⁺ binding to calmodulin in presence of peptides was measured. By this way, the peptides 542–566, 547–571, 660–677 and 597–614 have been found to bind specifically to calmodulin.Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the and subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in as well as to two regions in and . Exogenous calmodulin can bind to two regions in and in . A binding stoichiometry of 0.8mol of calmodulin/ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from to calmodulin. Therefore, binding of exogenous calmodulin to activates the enzyme. A model for switching endogenous calmodulin between , and and modulation of ATP binding to as well as Mg²⁺/ADP binding to by calmodulin is presented.     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

Some observations on cyclodextrin-mediated boosting of secreted amylolytic enzymes byLactobacillus amylovorus

The amounts of a 160-kDa amylase and a 140-kDa -amylase (A. Burgess-Cassler and S. H. Imam, Curr. Microbiol. 23:207–213, 1991) secreted into culture medium by the starchutilizingLactobacillus amylovorus were enhanced by the use of cyclodextrin (CD) as the carbon source. The levels of total extracellular -amylase obtained with glucose as the carbon source could be boosted severalfold by use of CD. The best enhancer was -CD, and the rank order of best to least effective was -CD>-CD=-CD>glucose.Another amylase, a 65-kDa -amylase, which degraded para-nitrophenyl-(1,4)-d-glucopyranoside, was also detected in this study. The most effective enhancer in this case was -CD, and the rank order was -CD>-CD>-CD glucose. Despite its ability to degradep-nitrophenylated glucose, this enzyme did not convert maltose to glucose. It showed a cleared zone on starch zymograms and did degrade short maltodextrins to maltose. Neither this new -amylase nor the 140-kDa -amylase exhibited any detectable ring-decyclizing (cyclodextrinase) activity against -or -CD.Other extracellular amylases (not characterized here) appeared to be similarly enhanced by CDs. Although the precise mechanism by which this effect is accomplished remains undefined, CDs can be useful inducing agents, boosting the expression and/or secretion of otherwise low-level enzymes, either as additives to growth media or as sole carbon source.     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins

Summary The functional R6K origin is composed of two DNA elements, one of 580 bp carrying the origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previosly identified as also required for and origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for origin activity. The function of the origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the origin depends on the R6K replication initiation protein .Within the 580 bp of the origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the replicon. Deletion of the 96 bp or 98 bp results in inactivation of the and the origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the and the origins of R6K. DNA homology analysis performed on , and origin sequences, also reveals 10–23 bp sequences in the and the origins that are related to the family of 22 bp direct repeats in the origin which were shown previously to be binding sites for the protein.     

Changes in the distribution of integrins and their basement membrane ligands during development of human thyroid follicular epithelium

Interactions between cells and basement membrane components are crucial for the regulation of epithelial cell differentiation and polarization. We have studied by immunohistochemical methods the distribution of integrin adhesion proteins and some of their basement membrane ligands in foetal (16--19 weeks) and adult thyroid follicular epithelia. A diffuse immunoreactivity for only 3, v and 1 integrins was found in foetal follicular epithelium, whereas in adult follicular epithelium these integrins were expressed basally in a polarized manner. Additionally, 3 integrin was seen in a more basolaterally confined manner in adult follicular epithelium. Among basement membrane components, laminin 1, 1, 1 and 2 chains were found in epithelial basement membranes of the foetal thyroid gland, suggestive of the presence of laminins-1 and -3. In contrast, the basement membranes of adult follicular epithelium presented a much weaker immunoreactivity for the laminin 2 chain. Furthermore, immunoreactivity for the laminin 2 chain was occasionally seen in adult thyroid glands, apparently confined to myofibroblasts. Immunoreactivity for type IV collagen 1 and 2 (IV) chains was found in follicular basement membranes of foetal as well as adult thyroid gland. The results suggest that during maturation of foetal thyroid follicular epithelium a distinct polarization of integrins takes place. In mature thyroid follicular epithelium, the presumable adhesion-mediating integrin complexes are 31, v1 and/or v3 mediating adhesion to laminin-1 (1-1- 1) and type IV collagen trimer 12 (IV)     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Oxidation of 17α,20β- and 17α,20α-Dihydroxypregn-4-en-3-ones,Side Products of Progesterone Biotransformation with Recombinant Microorganisms Expressing Cytochrome P-45017α

Progesterone biotransformation with recombinant yeasts Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 expressing bovine adrenocortical cytochrome P-45017 yielded 17-hydroxyprogesterone and two diols, 17,20- and 17,20-dihydroxypregn-4-en-3-ones. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17–20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17-hydroxylation and 20- and 20-reduction. The results widen the possibilities of enzymatic and chemical modifications of steroids.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Contribution to the knowledge of the enzymatic activity of yeasts of clinical interest

The determination of the enzymatic activity of the yeasts has been applied to the identification of species, specially that ofCandida albicans. In order to know its usefulness in species of clinical interest, we have tested the commercial system API ZYM (Bio Mérieux) on 500 isolated strains of different organic samples, belonging to eight genera and twenty species. All the strains showed positivity to Phosphatase alcaline, Esterase (C4), Esterase lipase (C8), Leucine arylamidase and Phosphatase acid, and negativity to Lipase (C14), Trypsin, Chymotrypsin, -galactosidase, -glucoronidase, -manosidase and -fucosidase. Fourteen enzymatic activity patterns were obtained considering the substrates with variable results for the whole of the strains: Valine arylamidase, Cystine arylamidase, Naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase. In the majority of the species, the enzymatic profile did not have very specific results since it is usually shared by more than one species.C. albicans is that which presents the greatest number of enzymatic variations, some of these are similar to those of other common clinical species, such asCandida krusei, Candida parapsilosis andCandida tropicalis. This system is proposed as a rapid method for identification and as an epidemiological marker of medically important yeasts.Abbreviations AGL -glucosidase - BGA -galactosidase - BGL -glucosidase - CAA Cystine arylamidase - NAG N.Acetyl--glucosaminidase - PHO Naphthol-AS-BI-phosphohydrolase - VAA Valine arylamidase     

A novel method for determination of α1,6fucosyltransferase activity using a reducing oligosaccharide from egg yolk as a specific acceptor

A new method for determination of 1,6fucosyltransferase activity has been described. Recently, the disialyl-biantennary undecasaccharide was prepared in high yield from egg yolk [(1996), Carbohydr Lett 2: 137–42]. By treatment of this oligosaccharide with neuraminidase and -galactosidase, we readily obtained an asialo-agalacto-biantennary heptasaccharide (GlcNAc 1,2Man1,6[GlcNAc1,2Man1,3]Man1,4GlcNAc1,4GlcNAc). Using this asialo-agalacto-oligosaccharide as an acceptor, fucosyltransferases from human plasma and extracts of various human hepatoma cell lines were assayed in the presence of GDP-[3H]fucose. The reaction mixture was applied to a column of GlcNAc-binding, Psathyrella velutina lectin coupled gel. All the fucosylated acceptor were bound to the column which was eluted with 50 mM GlcNAc. Structural analyses revealed that only the innermost GlcNAc residue of the acceptor was fucosylated through an 1,6-linkage, and the oligosaccharide prepared could be used as a specific acceptor for 1,6fucosyltransferase. The present method was used to screen plasma 1,6fucosyltransferase in several patient groups, and significantly elevated activities were found in samples from patients with liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.