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PubMed1998

Cloning of Novel Maltooligosaccharide-Producing Amylases as Antistaling Agents for Bread.

For better understanding of the antistaling effect of starch-hydrolyzing enzymes, maltose-, maltotriose-, or maltotetraose-producing enzymes were applied to bread mix and the retrogradation rate of the bread was determined using differential scanning calorimetry. A new amylase isolated from Bacillus subtilis SUH 4-2, which selectively produces maltose and maltotriose from starch solution (amylase II), and another amylase from Streptomyces albus KSM-35, mainly producing maltotetraose and maltotriose (amylase IV), were cloned, characterized, and evaluated as antistaling agents for bread. Addition of amylase II or amylase IV significantly reduced the bread staling rate during 7 days of storage (p < 0.05), and especially amylase IV was as effective as a commercial enzyme, Novamyl. Analyses of the maltooligosaccharide composition of bread suggest that maltotriose and maltotetraose produced by the enzyme reaction are responsible for retarding bread retrogradation.

Journal Article↗
PubMed1999

Crystal structure of a maltogenic amylase provides insights into a catalytic versatility.

Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. Compared with many different amylases that are able to hydrolyze only alpha-D-(1,4)-glycosidic bonds, maltogenic amylases exhibit catalytic versatility: hydrolysis of alpha-D-(1,4)- and alpha-D-(1,6)-glycosidic bonds and transglycosylation of oligosaccharides to C3-, C4-, or C6-hydroxyl groups of various acceptor mono- or disaccharides. It has been speculated that the catalytic property of the enzymes is linked to the additional approximately 130 residues at the N terminus that are absent in other typical alpha-amylases. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2.8 A. The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (alpha/beta)(8) barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical alpha-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transglycosylation and to allow binding of a branched oligosaccharide for hydrolysis of alpha-D-(1,4)-glycosidic or alpha-D-(1,6)-glycosidic bond. The (alpha/beta)(8) barrel of the enzyme is the preserved scaffold in all the known amylases. The structure represents a novel example of how an enzyme acquires a different substrate profile and a catalytic versatility from a common active site and represents a framework for explaining the catalytic activities of transglycosylation and hydrolysis of alpha-D-(1,6)-glycosidic bond.

Amino Acid Sequence↗
PubMed1985

Molecular cloning of alpha-amylase genes from Drosophila melanogaster. II. Clone organization and verification.

Restriction maps of an alpha-amylase structural gene clone, lambda Dm65, and of four putative alpha-amylase pseudogene clones are presented. Two alpha-amylase structural genes, inverted with respect to each other, are contained in lambda Dm65. Subregions of internal DNA sequence homology within lambda Dm65 and of cross-homology between the presumptive pseudogene clones and lambda Dm65 were determined. Subregions of cross-homology between the Drosophila clones and the mouse alpha-amylase cDNA clone, pMSa104, were also determined. The presence of functional alpha-amylase structural genes in lambda Dm65 was verified by injection of appropriate subclones into the germinal vesicle of Xenopus oocytes, followed by incubation of the oocytes under conditions that allowed coupled transcription and translation of injected genes to occur. Subclones of the 3.8- and 5.6-kb EcoRI fragments of lambda Dm65 were shown to code for alpha-amylase isozymes 1 and 3, respectively, of Drosophila melanogaster Canton-S. Both subclones are homologous to RNA of a size sufficient to accommodate the alpha-amylase-coding information. No RNA species homologous to other subcloned EcoRI fragments of lambda Dm65 was detected.

Animals↗
PubMed1997

Grain Development Mutants of Barley ([alpha]-Amylase Production during Grain Maturation and Its Relation to Endogenous Gibberellic Acid Content).

Barley (Hordeum vulgare L. Himalaya) mutants with altered grain morphology were isolated to investigate whether defects in grain development, possibly involving gibberellins (GAs) and abscisic acid, would lead to altered patterns of [alpha]-amylase gene expression. Following treatment with sodium azide, 75 mutants, typically showing grain shriveling, were identified. At grain maturity 15 of the 75 mutants had higher [alpha]-amylase activities in shriveled grains compared with either phenotypically normal grains that developed on the same heterozygous plant or with grains of cv Himalaya. Studies of four of these mutants demonstrated increased levels of both high- and low-isoelectric point [alpha]-amylase isozymes midway through grain development. This category of mutant has been designated pga, for premature grain [alpha]-amylase. One such mutant (M326) showed an endosperm-determined inheritance pattern. When crossed into a (GA-deficient) dwarfing background there was a 10- to 20-fold reduction in [alpha]-amylase activity, suggesting a requirement for GA biosynthesis. Endogenous GAs and abscisic acid were quantified by combined gas chromatography-specific ion monitoring in normal and mutant grains of heterozygous M326 plants during the period of [alpha]-amylase accumulation. Mutant grains had significantly higher (5.8-fold) levels of the bioactive GA1 compared with normal grains but much lower (approximately 10-fold) levels of the 2[beta]-hydroxylated ("inactive") GAs, typical of developing barley grains (e.g. GA8, GA34, GA48). We propose that a reduced extent of 2[beta]-hydroxylation in the mutant grains results in an increased level of GA1, which is responsible for premature [alpha]-amylase gene expression.

Journal Article↗
PubMed1971

Amylase synthesis and stability in crested wheatgrass seeds at low water potentials.

Drying of seeds of Agropyron desertorum (Fisch. ex Link) Schult. did not result in breakdown of alpha-amylase nor impair the ability of seeds to resume its synthesis when moistened again. beta-Amylase activity did not change during 5 days of germination at a water potential of 0 atmosphere nor during 40 days of incubation at -40 atmospheres. Seeds synthesized alpha-amylase at 0, -20, and -40 atmospheres, but not at -60 atmospheres. At 0 and -20 atmospheres, the log of alpha-amylase activity was linearly related to hastening of germination. But at -40 atmospheres, seeds synthesized alpha-amylase during a time when there was little hastening of germination. Thus, it appears that other biochemical reactions are less drought-tolerant than synthesis of alpha-amylase. It is concluded that inhibition of alpha-amylase synthesis is not a controlling factor in the germination of these seeds at low water potentials.

Journal Article↗
PubMed1972

A Correlation between a Ribonucleic Acid Fraction Selectively Labeled in the Presence of Gibberellic Acid and Amylase Synthesis in Barley Aleurone Layers.

The effects of gibberellic acid on the incorporation of radio-active uridine and adenosine into RNA of barley aleurone layers were investigated using a double labeling method combined with acrylamide gel electrophoresis. After 16 hours of incubation, gibberellic acid stimulated the incorporation of label into all species of RNA, but the effects were very small (0-10%) for ribosomal and transfer RNA and comparatively large (up to 300%) for RNA sedimenting between 5S and 14S. This result was obtained for both isolated aleurone layers and for layers still attached to the endosperm. A similar but less marked pattern occurred in layers incubated for 8 hours, but the effect was not observed after 4 hours. The gibberellic acid-enhanced RNA labeling was not due to micro-organisms. The following evidence was obtained for an association between the gibberellic acid-enhanced RNA synthesis and alpha-amylase synthesis: (a) synthesis of alpha-amylase took place in parallel with incorporation of label into gibberellic acid-RNA; (b) actinomycin D inhibited amylase synthesis and gibberellic acid-RNA by similar percentages; (c) 5-fluorouracil halved incorporation of label into ribosomal RNA but had no effect on amylase synthesis and gibberellic acid-RNA; and (d) abscisic acid had little effect on synthesis of RNA in the absence of gibberellic acid, but when it was included with gibberellic acid the synthesis of both enzyme and gibberellic acid-RNA was eliminated. We conclude that large changes in the synthesis of the major RNA species are not necessary for alpha-amylase synthesis to occur but that alpha-amylase synthesis does not occur without the production of gibberrellic acid-RNA. Gibberellic acid-RNA is probably less than 1% of the total tissue RNA, is polydisperse on acrylamide gels, and could be messenger species for alpha-amylase and other hydrolytic enzymes whose synthesis is under gibberellic acid control.

Journal Article↗
PubMed1977

Occurrence of alpha-amylase in the axis of germinating peas.

alpha-Amylase was found in the axis portion of ungerminated pea seeds (Pisum sativum var. Alaska). The occurrence of this enzyme was demonstrated with crude homogenates (also containing beta-amylase) using three different methods: the hydrolysis of beta-limit dextrin, the change in absorption spectra for the iodine-starch complex, and the increase in reducing materials relative to the decrease in starch. The first method was used to quantitate the changes in alpha-amylase activity during germination. The increase in total amylase activity (primarily beta-amylase) paralleled germination; the accumulation of alpha-amylase activity was not initiated for an additional day. The increased alpha-amylase activity was related to epicotyl growth. Approximately half of this activity was found in the etiolated stem, the distribution being higher in growing than in nongrowing portions.

Journal Article↗
PubMed1978

No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds.

alpha-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10(-6)m gibberellic acid (GA(3)) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10(-2)m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified alpha-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of alpha-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10(-4)m 5-FU prior to the addition of GA(3) resulted in decreased thermal stability of GA(3)-induced alpha-amylase and who interpreted this as evidence that the mRNA for alpha-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified alpha-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of alpha-amylase mRNA synthesis.

Journal Article↗
PubMed1980

Starch Degradation in Spinach Leaves: ISOLATION AND CHARACTERIZATION OF THE AMYLASES AND R-ENZYME OF SPINACH LEAVES.

The properties of two amylase activities which differ in their substrate specificity and subcellular location as well as a chloroplast-associated R-enzyme (debranching activity) are reported. An extrachloroplastic amylase is resolved by gel filtration chromatography into two activities of 80,000 and 40,000 daltons. Both extrachloroplastic activities hydrolyze amylopectin and shellfish glycogen and only slowly hydrolyze rabbit liver glycogen, beta-limit amylopectin, and amylose. In contrast, the major chloroplastic amylase attacks all of these glucans at comparable rates. Glucan hydrolysis by both the extrachloroplastic and chloroplastic amylase generates not only maltose but appreciable amounts of other oligosaccharides, whereas maltotetraose hydrolysis produces glucose, maltose, and maltotriose. The action patterns displayed by the amylase activities indicate that both are endoamylases, although they lack the typical Ca(2+) requirement or heat stability of seed endosperm alpha-amylases. Dithiothreitol, glutathione (oxidized or reduced), ascorbate, dehydroascorbate, and dithiothreitol plus thioredoxin have no effect on either the chloroplastic or extrachloroplastic amylase activities.The chloroplastic R-enzyme debranches amylopectin, beta-limit amylopectin, pullulan, and alpha-limit dextrins, but not rabbit liver glycogen. An increase in extinction coefficient and lambda(max) is detected when the debranched amylopectin and beta-limit amylopectin form a complex with I(2)-KI. Based on these properties, the chloroplastic R-enzyme is similar in enzymic activity to the R-enzyme observed in endosperm tissue.

Journal Article↗
PubMed1982

Effect of ethylene on the gibberellic Acid-enhanced synthesis and release of amylase by isolated barley aleurone layers.

Methods were developed and extended to enable the incubation of isolated barley (Hordeum vulgare cv. Himalaya) aleurone layers under carefully controlled conditions for studies on effects of ethylene on amylase synthesis and release. When layers in medium containing gibberellic acid were exposed to ethylene, the synthesis and release of amylase were altered relative to layers maintained in an ethylene-free environment. These ethylene effects were detected at the smallest concentration used, 0.041 nl/ml, indicating a very low threshold value. During the initial 24 h, ethylene accelerated both the appearance of total amylase activity, and the release of this activity from the aleurone layers. On the other hand, ethylene reduced the total amount of amylase activity that was recovered from samples after 48 and 72 h.Ethylene did not stimulate the release of amylase from membrane-bound structures within the aleurone layers, and did not interact with the enzyme directly.The isoelectric patterns of amylase activity and proteins released from control and ethylene-treated aleurone layers in 24 h were identical. Therefore, ethylene promoted only quantitative differences in amylase synthesis rather than qualitative differences.

Journal Article↗
PubMed1983

In Vivo Synthesis and Turnover of alpha-Amylase in Attached and Detached Cotyledons of Vigna mungo Seeds.

alpha-Amylase activity increased in attached cotyledons of germinated Vigna mungo seeds until the 5th day after imbibition and decreased thereafter, whereas in detached and incubated cotyledons the activity continuously increased and, at the 6th day, reached the value more than three times that of the maximum activity of attached cotyledons. Zymograms of the activities and Ouchterlony double immunodiffusion test on the activities of attached and detached cotyledons showed that the increase of activity in detached cotyledons was due to the identical enzyme as in attached tissues. alpha-Amylase contents, determined by single radial immunodiffusion method, changed in parallel with enzyme activity in both attached and detached cotyledons, which also suggested the de novo synthesis of alpha-amylase in V. mungo cotyledons.The rate of incorporation of the label from [(3)H]leucine into alpha-amylase and the ratios of dpm in alpha-amylase/dpm in trichloroacetic acid-insoluble fraction did not show significant difference between attached and detached cotyledons. The results indicated that in attached cotyledons fluctuation of alpha-amylase activity was regulated by both synthesis and degradation of the enzyme, whereas in detached cotyledons alpha-amylase was synthesized and accumulated, because of low degrading activity during incubation.

Journal Article↗
PubMed1984

Possible roles of calcium and calmodulin in the biosynthesis and secretion of alpha-amylase in rice seed scutellar epithelium.

The scutellar epithelial cells of rice (Oryza sativa L. cv Kimmazé) seeds actively secrete alpha-amylase in an early stage of germination. Employing an in vivo system of freshly dissected scutellar tissues, effect of Ca(2+) on the biosynthesis and the secretion of alpha-amylase have been studied. The maximum biosynthetic rate was saturated at about 0.5 mm external Ca(2+) concentrations, whereas the secretion continued to increase to concentrations above 10 mm Ca(2+). In the presence of 1 mm Ca(2+), 0.01 mum A-23187 significantly increased both the biosynthesis and the secretion of alpha-amylase.A cation-specific requirement for Ca(2+) was apparent, since both biosynthesis and extracellular secretion of alpha-amylase were inhibited by 0.1 mm EGTA but were increased above basal rate only with Ca(2+) and Sr(2+); K(+), Mg(2+), and Ba(2+) being ineffective.La(3+) and ruthenium red (selective inhibitors of [Ca(2+) + Mg(2+)]-ATPase) were found to profoundly inhibit the secretion of alpha-amylase. A calmodulin antagonist, W-7, also inhibited the secretion of alpha-amylase at concentrations where the enzyme synthesis was not much affected. Overall data indicate that Ca(2+) movement and secretion of alpha-amylase are tightly linked and it is likely that they are regulated by the cytoplasmic Ca(2+) concentration under possible control by calmodulin.

Journal Article↗
PubMed1984

Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain.

alpha-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20 degrees C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl(2), the alpha-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of alpha-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of alpha-amylase activity in the starchy endosperm in a whole grain.Scutella separated after 1 day of germination released small amounts of alpha-amylase activity into 10 millimolar CaCl(2). This release was linear for at least 1 hour and did not occur at 0 degrees C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of alpha-amylase activity in the starchy endosperm in a whole grain.Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total alpha-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

Journal Article↗
PubMed1986

Hormonal Regulation of alpha-Amylase Gene Transcription in Wild Oat (Avena fatua L.) Aleurone Protoplasts.

The time of appearance and relative amounts of alpha-amylase mRNA in wild oat (Avena fatua L.) aleurone protoplasts incubated with 1 micromolar gibberellin A(4) (GA(4)) were closely correlated with the amounts of alpha-amylase enzyme secreted by the protoplasts. In the absence of GA(4), or when protoplasts were incubated with 25 micromolar abscisic acid (ABA) together with 1 micromolar GA(4) no alpha-amylase mRNA was detected and only very low levels of alpha-amylase were secreted. Nuclei were isolated in high yields (65-71%) from aleurone protoplasts and in an in vitro transcription system displayed characteristics of a faithful DNA-dependent RNA synthesizing system. The time course of incorporation of [(3)H]-UTP suggested that the RNA synthesized was mainly ;run off' transcription and therefore that the transcripts produced in vitro were those being synthesized in the protoplasts at the times when the nuclei were isolated. By hybridizing in vitro synthesized [(32)P]RNA to barley alpha-amylase cDNA and control filters we have estimated that 90 +/- 10 ppm of the transcripts synthesized by nuclei isolated from GA(4) treated protoplasts can be attributed to alpha-amylase sequences and that statistically insignificant amounts of these transcripts are obtained from control and GA(4) plus ABA treatments. The results suggest that GA(4) and ABA influence the transcription of alpha-amylase genes in aleurone protoplasts of wild oat.

Journal Article↗
PubMed1990

Heat shock proteins are not required for the degradation of alpha-amylase mRNA and the delamellation of endoplasmic reticulum in heat-stressed barley aleurone cells.

When barley (Hordeum vulgare) aleurone layers are heat shocked, the synthesis and secretion of alpha-amylase and other secretory proteins is arrested and the synthesis of heat shock proteins (hsps) is induced. alpha-Amylase mRNA, normally a very stable mRNA, is actively degraded during heat shock. In addition, endoplasmic reticulum (ER) is delamellated during heat shock, possibly causing the destabilization of the mRNA for the secreted alpha-amylase. To ascertain whether or not hsps play any role in the destabilization of alpha-amylase mRNA or in the delamellation process of ER, heat shocked cells were treated with the transcription inhibitor cordycepin, which effectively inhibits the synthesis of hsps yet does not affect alpha-amylase synthesis after this enzyme has been fully induced by gibberellic acid (12 hours). In the absence of hsp expression, heat shock still causes the destabilization of alpha-amylase mRNA and the delamellation of ER. Alternatively, the synthesis of hsps may be induced in the absence of temperature increase by incubating cells in the presence of arsenite. Arsenite-induced expression of some hsps in the absence of increased temperature does not result in the destabilization of alpha-amylase mRNA or in the delamellation of ER. If cordycepin or cycloheximide are used to inhibit hsp synthesis during heat shock, the tissue recovers from heat shock with normal recovery kinetics. Although hsps have been implicated in the establishment of thermotolerance, our observations indicate that hsps do not play a role in the other heat shock-induced changes observable in aleurone cells. Furthermore, if the synthesis of hsp mRNA is inhibited during heat shock (by cordycepin) hsp mRNAs are synthesized later, during recovery, indicating that there is a stable inducer of hsp synthesis in aleurone tissues.

Journal Article↗
PubMed1990

Purification of a beta-Amylase that Accumulates in Arabidopsis thaliana Mutants Defective in Starch Metabolism.

Amylase activity is elevated 5- to 10-fold in leaves of several different Arabidopsis thaliana mutants defective in starch metabolism when they are grown under a 12-hour photoperiod. Activity is also increased when plants are grown under higher light intensity. It was previously determined that the elevated activity was an extrachloroplastic beta-(exo)amylase. Due to the location of this enzyme outside the chloroplast, its function is not known. The enzyme was purified to homogeneity from leaves of both a starchless mutant deficient in plastid phosphoglucomutase and from the wild type using polyethylene glycol fractionation and cyclohexaamylose affinity chromatography. The molecular mass of the beta-amylase from both sources was 55,000 daltons as determined by denaturing gel electrophoresis. Gel filtration studies indicated that the enzyme was a monomer. The specific activities of the purified protein from mutant and wild-type sources, their substrate specificities, and K(m) for amylopectin were identical. Based on these results it was concluded that the mutant contained an increased level of beta-amylase protein. Enzyme neutralization studies using a polyclonal antiserum raised to purified beta-amylase showed that in each of two starchless mutants, one starch deficient mutant and one starch overproducing mutant, the elevated amylase activity was due to elevated beta-amylase protein.

Journal Article↗
PubMed1992

Induction of Expression of Genes Coding for Sporamin and beta-Amylase by Polygalacturonic Acid in Leaf-Petiole Cuttings of Sweet Potato.

Sporamin and beta-amylase are two major proteins of tuberous storage root of sweet potato (Ipomoea batatas) and their accumulation can be induced concomitantly with the accumulation of starch in leaves and petioles by sucrose (K Nakamura, M Ohto, N Yoshida, K Nakamura [1991] Plant Physiol 96: 902-909). Although mechanical wounding of leaves of sweet potato only occasionally induced the expression of sporamin and beta-amylase genes, their expression could be reproducibly induced in leaf-petiole cuttings when these explants were dipped in a solution of polygalacturonic acid or chitosan at their cut edges. Polygalacturonic acid seemed to induce expression of the same genes coding for sporamin and beta-amylase that are induced by sucrose. Because polygalacturonic acid and chitosan are known to mediate the induction of wound-inducible defense reactions, these results raise an interesting possibility that beta-amylase, in addition to sporamin, may have some role in the defense reaction. Expression of sporamin and beta-amylase genes could also be induced by abscisic acid, and this induction by abscisic acid, as well as induction by polygalacturonic acid or sucrose, was repressed by gibberellic acid. By contrast, methyl jasmonate did not cause the significant induction of either sporamin or beta-amylase mRNAs. Induction of expression of sporamin and beta-amylase genes by polygalacturonic acid or sucrose was inhibited by cycloheximide, suggesting that de novo synthesis of proteins is required for both of the induction processes.

Journal Article↗
PubMed2006

Role of Trp140 at subsite -6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707.

Maltohexaose-producing amylase (G6-amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short-chain amylose (DP=17). Our previous crystallographic study showed that G6-amylase has nine subsites, from -6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6-amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6-amylase complexes with G6 and maltopentaose (G5). In the active site of the G6-amylase/G5 complex, G5 is bound to subsites -6 to -2, while G1 and G6 are found at subsites +2 and -7 to -2, respectively, in the G6-amylase/G6 complex. In both structures, the glucosyl residue located at subsite -6 is stacked to the indole moiety of Trp140 within a distance of 4A. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short-chain amylose by W140L is lower than that by W140Y or wild-type enzyme. The face-to-face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite -6 and to govern product specificity for G6 production.

Bacillus↗