1 Departments of Chemical Engineering and Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA
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Abstract |
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Keywords: additivity/glucoamylase/glucose yield/isomaltose/selectivity/site-directed mutagenesis
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Introduction |
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Roughly 11 million metric tons of glucose is produced annually in the United States with GA from concentrated maltodextrin solutions (Corn Annual, 1998). A 1% increase in yield through GA protein engineering, eliminating approximately one-quarter of the remaining reaction by-products, would increase glucose yield by about 110 000 tons. This, if it were to take place, almost surely would be the largest yield increase of any product attributed to protein engineering.
This is the fourth of a series of articles reporting the ability of mutated Aspergillus awamori/Aspergillus niger GAs to increase glucose yields from hydrolysis of highly concentrated maltodextrin solutions. This article reports production of GAs with multiple mutations, the single mutations having been produced previously. In addition to achieving higher glucose yields, we wished to determine if the principle of additive mutational effects in proteins could be applied to GA selectivity.
In the first two articles, Fang et al. (1998a,b) tested 11 mutations at positions not totally conserved around the GA active site. Tyr116Trp, Ser119
Gly, Ser119
Trp, Gly121
Ala, Arg241
Lys, Ser411
Ala, Ser411
Gly and Gly121
Ala/Ser411
Gly GAs all gave significantly higher glucose yields at 55°C than wild-type GA. They found an inverse correlation between peak glucose yield and the ratio of the initial rate of glucose condensation to form iG2 to the initial rate of maltodextrin hydrolysis to form glucose (initial rate ratio), but no direct correlation between glucose yields and ratios of catalytic efficiencies, kcat/KM, for hydrolysis of maltose [
-D-glucopyranosyl-(1
4)-D-glucose] to that of iG2.
In the third article, Liu et al. (1998) increased GA glucose yield by inserting a seven-residue loop between Tyr311 and Gly314 (311314 Loop) to alter the active site so that glucose was released from subsite 1 before subsite +1 was occupied by another glucose residue to form more disaccharides. They also increased glucose yield at higher temperatures by the Tyr312Trp mutation. On the other hand, Lys108
Arg GA gave significantly lower glucose yields than did wild-type GA, while Lys108
Met GA could not bind substrate, suggesting that Lys108 is important for both GA catalysis and substrate binding.
Liu et al. (1998) also determined glucose yields of GAs originally mutated to increase their thermostabilities. The Asn20Cys/Ala27
Cys (SS) mutation, which creates a disulfide bond on the catalytic domain surface and stabilizes GA against unfolding, and which will be considered throughout this article as a single mutation, not only increased GA thermostability (Li et al., 1998
) but also increased glucose yields at 35, 45 and 55°C compared with wild-type GA (Liu et al., 1998
). The Gly137
Ala mutation, which presumably stiffened an
-helix to increase GA thermostability (Chen et al., 1996
), led to higher glucose yields at higher temperatures (Liu et al., 1998
). Two other previously constructed thermostable mutated GAs, Ser30
Pro (Allen et al., 1998
) and Ser436
Pro (Li et al., 1997
), and one thermosensitive mutated GA, Ala27
Pro (Li et al., 1997
), gave lower glucose yields than did wild-type GA (Liu et al., 1998
). Based on the above results, Liu et al. (1998) proposed that there is no direct correlation between GA thermostability and selectivity, although they noted that there was a positive correlation between the two factors when only those mutations in the more hydrophobic folding unit were considered.
Earlier attempts to improve A.awamori/A.niger GA selectivity by the Ser119Tyr, Gly183
Lys and Ser184
His mutations, based on sequence homologies around the active site, led to 2.3- to 3.5-fold increased ratios of catalytic efficiencies for maltose hydrolysis over iG2 hydrolysis (Sierks and Svensson, 1994
; Svensson et al., 1995
). Frandsen et al. (1995) altered GA selectivity by the Arg305
Lys mutation, because Arg305 stabilizes
-1,4-linked substrates from the hydrophilic side of the substrate-binding pocket at subsites -1 and +1. Fierobe et al. (1996) made two individual loop replacements and one double loop replacement combining them in A.awamori GA to mimic part of the Hormoconis resinae GA sequence. The double mutation had roughly double the ratio of catalytic efficiencies for hydrolysis of
-1,6- over
-1,4-linked substrates than did wild-type GA. Frandsen et al. (1996) identified OH-4', OH-6' and OH-4 as critical for iG2 hydrolysis by studying the energetics of the transition-state complex for Glu180
Gln and Asp309
Glu mutant GAs. Later work led to the Trp170
Phe, Asn171
Ser, Gln172
Asn, Thr173
Gly, Gly174
Cys, Tyr175
Phe and Asp176
Asn mutations, none of which materially improved GA selectivity (Stoffer et al., 1997
).
In this article we report glucose yields and other kinetic and thermostability data from maltodextrin hydrolysis catalyzed by six previously made GAs with the SS/Ser30Pro, Ser30
Pro/Gly137
Ala, SS/Ser30
Pro/Gly137
Ala (Allen et al., 1998
), SS/Gly137
Ala, SS/Ser436
Pro and Gly137
Ala/Ser436
Pro (Li et al., 1998
) mutations. These mutations were made to increase GA thermostability, which was additive in all cases except SS/Ser436
Pro GA (Allen et al., 1998
; Li et al., 1998
). The Ser30
Pro/Gly137
Ala and SS/Ser30
Pro/Gly137
Ala GAs are the most stable GAs yet produced (Allen et al., 1998
).
We also constructed GAs with the following nine multiple mutations, SS/311314 Loop, SS/Ser411Ala, Ser30
Pro/311314 Loop, Ser30
Pro/Ser411
Ala, Gly137
Ala/311314 Loop, Gly137
Ala/Ser411
Ala, 311314 Loop/ Ser411
Ala, Ser30
Pro/Gly137
Ala/311314 Loop and Ser30
Pro/Gly137
Ala/Ser411
Ala, and tested them for selectivity, thermostability and specific activity. All were composed of previously made single mutations.
Of these mutated amino acid residues, Asn20 is the C-terminal residue of the first -helix in A.awamori var. X100 GA (Aleshin et al., 1992
), while Ala27 and Ser30 are both located in a type II ß-turn on the extended loop between the first and second
-helices (Figure 1
). Gly137 is located in the middle of the fourth
-helix, and residues Tyr311 to Gly314 are between
-helices 9 and 10. Ser411 is located in a ß-strand between
-helices 12 and 13, while Ser436 is in a random coil in a packing void of unknown function (Aleshin et al., 1994
). Of the above residues, only Ala27 and Ser30 are located in conserved regions, and neither of them is totally conserved (Coutinho and Reilly, 1994a
,b
, 1997
).
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Materials and methods |
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Materials
Glucose and maltose were obtained from Sigma. Maltrin® M100, M180 and M250 maltodextrins, of DE 10, 18 and 25, respectively, and with average degrees of polymerization of 10, 6 and 4, were donated by Grain Processing Corporation. Other materials were as in Fang et al. (1998a).
Site-directed mutagenesis
Gly137Ala (Chen et al., 1996
), Ser436
Pro (Li et al., 1997
), SS, SS/Gly137
Ala, SS/Ser436
Pro, Gly137
Ala/Ser436
Pro (Li et al., 1998
), Ser411
Ala (Fang and Ford, 1998
), 311314 Loop (Liu et al., 1998
), Ser30
Pro, SS/Ser30
Pro, Ser30
Pro/Gly137
Ala and SS/Ser30
Pro/Gly137
Ala (Allen et al., 1998
) GAs were constructed as described earlier.
Site-directed mutagenesis to produce nine multiple mutations was performed by Promega Altered Sites II in vitro mutagenesis system.
For the 311314 Loop/Ser411Ala mutation, an XbaIHindIII fragment of pGEM-GA containing the wild-type GA cDNA (Fang and Ford, 1998
) was inserted into the Promega pALTER-1 vector to make a GA cDNA-containing vector to be used as the double-stranded DNA template (Fang et al., 1998b
). The previously made 311314 Loop mutation containing GA cDNA in the pALTER-1 vector was used as a template, using the oligonucleotide primer 5'-GGC GAG CAG CTT GCA GCA CGC GAC CTG AC-3' (Fang et al., 1998a
) synthesized in the Iowa State University Nucleic Acid Facility. Nucleotides for the desired GA mutation are in bold, while the one silent mutation designed to decrease the primer hairpin melting temperature is underlined.
For the other eight multiple mutations, the short BamHIPstI fragments containing the SS, Ser30Pro, Gly137
Ala or Ser30
Pro/Gly137
Ala mutations, which were restriction-digested from the phagemid vector pGEM7Z(+), were separately ligated to the long BamHIPstI fragments containing the 311314 Loop or Ser411
Ala mutations, which were restriction-digested from the phagemid vectors pGEM7Z(+) and pALTER-I, respectively.
The resulting multiple mutations were verified by DNA sequencing. cDNAs containing the multiple mutations were subcloned into the yeast expression vector YEpPM18 and then transformed into Saccharomyces cerevisiae C468 as previously described (Chen et al., 1994).
Irreversible thermoinactivation
Wild-type and mutant GAs (0.475 µM) were incubated for 12 min in 0.05 M NaOAc buffer, pH 4.4, at six or seven temperatures between 65 and 80°C. Samples were taken at 2 min intervals, quickly chilled on ice, and then stored at 4°C for 24 h before being subjected to residual activity assay at 35°C as in Liu et al. (1998). The first-order inactivation rate coefficients (kd) for mutant and wild-type GAs were obtained from a plot of ln (residual activity) versus inactivation time. The transition-state free energies (G
) for thermoinactivation of mutant and wild-type GAs were obtained from a plot of ln (kd/T) versus 1/T. The function
G
is the difference between
G
values for mutated and wild-type GAs, being positive when the mutated GA is more stable.
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Results |
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The specific activities of mutated and wild-type GAs at 50°C and pH 4.5 using 4% maltose as substrate are shown in Table I. Data were gathered here and taken from earlier work. Two observations may be made: (i) If single mutations yield GAs with specific activities near that of wild-type GA, then multiply-mutated GAs containing those mutations have specific activities higher than those of any of the GAs with the single mutations; (ii) If single mutations, specifically 311314 Loop and Ser411
Ala, yield GAs with very low specific activities compared to wild-type GA, then multiply-mutated GAs containing those mutations usually have slightly higher specific activities than does the GA with the single mutation giving the lowest specific activity.
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Glucose formation from 30% (w/v) DE 10 maltodextrin hydrolyses at 55°C with 1.98 µM wild-type and mutated GAs are shown in Figure 2. Data (not shown) for DE 18 and DE 25 maltodextrin hydrolyses are similar, while hydrolyses at 35 and 45°C are slower but are also essentially similar. Glucose concentrations decrease after reaching their maximal values because of reverse reactions to form di- and oligosaccharides, especially at higher temperatures.
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Initial glucose formation rates appear in Table II. Most multiply-mutated GAs except for those containing the Ser411
Ala mutation have rates similar to or higher than those of wild-type GA. There are relatively few significant differences in initial glucose formation rates among DE 10, 18 and 25 maltodextrins. Specific activities and initial rates on DE 25 maltodextrin are positively correlated, with R values ranging from 0.70 to 0.75 depending on the temperature.
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Glucose condensation reactions
Formation of iG2 from condensation of 30% (w/v) glucose at 55°C with 1.98 µM GA is shown in Figure 3. Reaction profiles at 35 and 45°C are similar but slower, except that iG2 formation rates with wild-type GA decrease more sharply with decreasing temperature than with other GAs. Initial rates appear in Table II
. Of the GAs with multiple mutations, SS/Ser411
Ala, SS/311314 Loop and 311314 Loop/Ser411
Ala GAs have the lowest initial rates at all reaction temperatures, while Gly137
Ala/Ser436
Pro and SS/Ser436
Pro GAs have the highest initial rates.
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Selectivity for -1,6-linked product synthesis versus
-1,4-linked substrate hydrolysis
Ratios of the initial rate of iG2 formation from 30% (w/v) glucose condensation reactions to that of glucose formation from 30% (w/v) DE 10, 18 and 25 maltodextrin hydrolyses were determined for mutated and wild-type GAs at 35, 45 and 55°C (Table II). Of the multiply-mutated GAs, SS/311314 Loop, SS/Ser411
Ala and SS/Gly137
Ala GAs have the lowest ratios, while Gly137
Ala/Ser436
Pro and SS/Ser436
Pro GAs have the highest ratios at all reaction temperatures. These ratios are plotted against peak glucose yields in Figure 4
. Yields are slightly higher at 35°C than at 45 and 55°C, and are slightly higher for DE 25 than for DE 10 and DE 18 maltodextrins. Linear correlations were obtained. Seven of the nine curves had standard errors by linear regression less than 10% of the values of their corresponding slopes, while the other two (DE 25 maltodextrin at 45 and 55°C) gave standard errors that were less than 20% of their slopes. This is compelling evidence that variation of glucose yields with different conditions is significant.
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Table I shows the changes of
G
from that of wild-type GA (
G
) at 65 and 75°C, with data taken here and elsewhere. A rough approximation is that
G
values of 1 and 1 kJ/mol correspond to about 1 and 1°C changes in operating temperature, respectively, to maintain the same enzyme stability. Ser30
Pro/Gly137
Ala and SS/Ser30
Pro/ Gly137
Ala mutations increase GA thermostability the most, while the 311314 Loop/Ser411
Ala mutation decreases thermostability the most.
Values of G
for GAs with multiple mutations can be approximately determined by adding the
G
values of GAs containing their single mutations, as found earlier (Allen et al., 1998
; Li et al., 1998
). In addition, specific activities at 50°C and thermostabilities at 65°C are positively correlated (R = 0.85).
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Discussion |
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The strategy of additive mutagenesis has been one of the most powerful and successful tools in stabilizing proteins against irreversible thermoinactivation, such as with repressor (Hecht et al., 1986
), subtilisin (Cunningham and Wells, 1987
; Pantoliano et al., 1989
), kanamycin nucleotidyltransferase (Liao et al., 1986
), T4 lysozyme (Matsumura et al., 1989
) and GA (Allen et al., 1998
; Li et al., 1998
); in engineering subtilisin selectivity (Russell and Fersht, 1987
; Wells et al., 1987a
,b
); in improving glutathione reductase coenzyme selectivity (Scrutton et al., 1990
); and in enhancing the catalytic efficiency of a weakly active subtilisin variant (Carter et al., 1989
). This principle can be expressed as
GX,Y =
GX +
GY +
GI, where
GX,
GY and
GX,Y are the free energy changes associated with the measured variables for the single mutations X and Y and the multiple mutation X,Y, respectively (Ackers and Smith, 1985
). The coupling energy
GI (Carter et al., 1984
) is the free energy of interaction between sites X and Y. When the side chains at sites X and Y are remote from one another and there are no large structural perturbations or changes in the reaction mechanism or rate-determining step,
GI is negligible and the above equation can be simplified to
GX,Y =
GX +
GY. We have used the increase of glucose yield for a mutated GA over that of wild-type GA instead of the change in transition-state energy to show the cumulative effect on GA selectivity, since the main purpose here was to achieve higher glucose yields by combining mutations.
The cumulative effects on the incremental glucose yield and initial rate ratio of iG2 to glucose formation by various combinations of SS, Ser30Pro, Gly137
Ala, 311314 Loop, Ser411
Ala and Ser436
Pro mutations are demonstrated in Figures 59
, all at 35, 45 and 55°C but with results for DE 10, 18 and 25 maltodextrins averaged. In general the multiply-mutated GAs have initial rate ratios between those of the individual mutations of which they are comprised. Increases of glucose yield by multiple mutations are roughly additive, demonstrating the validity of the additivity principle, but are smaller when the incremental glucose yields of both the original mutated GAs are already very high than when they are lower. This means that some limitations occur in increasing glucose yield by mutating GA. For example, by-products such as maltulose [
-D-glucopyranosyl-(1
4)-D-fructose] produced during maltodextrin production prevent glucose yield from being complete, and decreasing the ability of GA to synthesize iG2 by mutation also limits hydrolysis of
-1,6 bonds found in maltodextrins, resulting in less complete substrate conversion at peak glucose yields.
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The SS mutation introduced an extra disulfide bond on the catalytic domain surface in the extended loop between the first and second -helices of GA. This loop, containing a conserved region involved in substrate binding (Coutinho and Reilly, 1997
), is near another loop containing residue Trp120, which is critical for catalysis (Sierks et al., 1989
) and important in directing conformational changes controlling the putative rate-limiting product release step (Natarajan and Sierks, 1996
). The loop appears to be important for both GA thermostability (Li et al., 1998
) and selectivity (Liu et al., 1998
). The SS mutation may stabilize either or both the loops, thus stabilizing the functional conformation.
The SS mutation had been previously combined with other single mutations to investigate their additive effects on GA thermostability (Allen et al., 1998; Li et al., 1998
). Those mutations plus the SS/311314 Loop and SS/Ser411
Ala GA mutations made here show that the SS mutation has a neutral effect on specific activities, except for the SS/Ser436
Pro mutation, when it has a positive effect (Table I
). It increases glucose yields and initial glucose formation rates both by itself and when it is combined with other mutations (Tables I and II
, Figures 57
). Its most notable effect is its very strong ability to depress the ratios of initial iG2 formation rate to glucose formation rate, mainly by its depressing effect on the former (Table II
). Although the SS/311314 Loop and SS/Ser411
Ala GAs are more thermosensitive than SS and wild-type GAs, they are both more thermostable than 311314 Loop and Ser411
Ala GAs (Table I
), meaning that adding a mutation that singly confers thermostability to a GA already containing a thermosensitive mutation can make the latter more thermostable.
The Ser30Pro mutation, having a Pro residue in a Type II ß-turn in a highly conserved region of GA, was constructed to increase GA thermostability (Allen et al., 1998
). It was combined previously with the SS and Gly137
Ala mutations (Allen et al., 1998
) and then here with the 311314 Loop and Ser411
Ala mutations to form various doubly- and triply-mutated GAs. In general it has a neutral to slightly positive effect on GA specific activities (Table I
) and a slightly negative to slightly positive effect on glucose yields (Table I
, Figures 5, 8 and 9
). At 35 and 45°C it has a neutral effect on initial glucose formation rates and a positive effect on the ratios of initial iG2 formation rate to glucose formation rate. At 55°C it increases the former and decreases the latter (Table II
). It increases GA thermostabilities in singly- and multiply-mutated GAs (Table I
).
Even though the SS mutation gives a disulfide bond close to position 30, cumulative mutational effects in SS/Ser30Pro GA still occur. Furthermore, despite the suggestion of Balaji et al. (1989) that a disulfide bond should not be inserted in the protein primary structure within four amino acids of a Pro residue, SS/Ser30
Pro GA is more active, stable (Allen et al., 1998
) and selective than wild-type GA. Li et al. (1997, 1998), Allen et al. (1998) and Liu et al. (1998) have suggested that the region between the C-terminus of
-helix 1 and the following extended loop between
-helices 1 and 2 is important for reversible thermoinactivation and selectivity. Further investigation might focus on single or deletion mutations in this region, as the loop is very thermolabile and is exposed to solvent (P.M.Coutinho, personal communication, 1998).
Gly137, located in the middle of -helix 4, which is part of the inner ring of the
,
-barrel around the active site, was mutated to Ala to increase GA thermostability, presumably by stiffening the helix (Chen et al., 1996
). The Gly137
Ala mutation increases specific activity in GAs in which it is a part (Table I
), has a weakly positive to negligible effect on peak glucose yields (Table I
, Figures 5, 6, 8 and 9
) and initial glucose formation rates (Table II
), sometimes increases and sometimes decreases initial rate ratios (Table II
), and increases thermostabilities (Table I
).
The 311314 Loop mutation was made to mimic the Rhizopus oryzae GA sequence, in the hope of decreasing iG2 formation without affecting maltose hydrolysis (Liu et al., 1998). The mutation gives higher glucose yields (Table I
, Figures 7 and 8
) and lower initial rate ratios (Table II
), but it also gives GAs with much lower specific activities, initial glucose formation rates and thermostabilities (Tables I and II
).
The Ser411Ala mutation is one of a series constructed to increase the optimal pH of GA (Fang and Ford, 1998
). It was meant to remove the hydrogen bond between atom OG of Ser411 and atom OE2 of Glu400, which also hydrogen-bonds to both the catalytic water and to the hydroxyl group of the invariant Tyr48. The mutation strongly decreases specific activities and initial rates of glucose formation in the GAs where it occurs (Tables I and II
) due to the destruction of the hydrogen bond. It increases glucose yields (Table I
, Figures 7 and 9
) and substantially decreases the initial rate ratio only in the GA where it is the only mutation and not in any multiply-mutated GAs in which it is found (Table II
). GAs with this mutation have strongly decreased thermostabilities (Table I
).
The Ser436Pro mutation was made by Li et al. (1998) to reduce backbone bond rotation and therefore to decrease entropy during protein unfolding. It also fills a packing void and enhances hydrophobic interactions there. The mutation in fact confers added thermostability upon GAs that contain it but at the cost of decreased glucose yields (Table I
, Figure 6
) and increased initial rate ratios (Table II
). The low specific activity found in Ser436
Pro GA is not carried over to other GAs containing the mutation (Table I
), and glucose formation rates are in general increased by it (Table II
).
The hypothesis that decreasing the ability of GA to synthesize iG2 would increase glucose yield was proved by Fang et al. (1998a,b) and Liu et al. (1998), generally with single mutations, and is confirmed here with multiple mutations. Figure 4 shows the inverse relationship between peak glucose yields and initial rate ratios.
There is usually no significant difference among initial rates of glucose formation with substrates of different chain length at high substrate concentrations, even though catalytic efficiencies are higher for longer substrates than for shorter ones at low substrate concentrations (Liu et al., 1998). However, as noted earlier (Liu et al., 1998
) and here, peak glucose yields are higher and occur 1824 h earlier with DE 25 maltodextrin than with DE 10 and DE 18 maltodextrins. To correct the previous explanation for this, DE 25 maltodextrin appears to have fewer slowly hydrolyzed
-(1,6)-initiated side-chains that do DE 10 and DE 18 maltodextrins (R.L.Antrim, personal communication). This would have a small effect on initial glucose formation rates but would make the average rate of DE 25 maltodextrin hydrolysis with increasing incubation time greater than that of the other two maltodextrins. This is so because decreasing substrate chain length during the course of hydrolysis increases the difference in hydrolysis rate between
-(1,4) and
-(1,6) glucosidic bonds; i.e. maltotriose, with a nonreducing-end terminal
-(1,4) bond, and panose [
-D-glucopyranosyl-(1,6)-
-D-glucopyranosyl-(1,4)-glucose], with a nonreducing-end terminal
-(1,6) bond, are closer in hydrolysis rate than maltose, with its
-(1,4) bond, and iG2, with its
-(1,6) bond (Meagher and Reilly, 1989
). Furthermore, the relative absence of
-(1,6) bonds in DE 25 maltodextrin and its resulting quicker approach to peak glucose yield leads to a higher yield than with the other maltodextrins, since there is less time for GA to form by-products from glucose.
Table I and Figure 4
show that glucose yields tend to be slightly lower at higher reaction temperatures for wild-type GA and most mutated GAs, even though increasing reaction temperature results in progressively lower initial rate ratios (Table II
). The former result but not the latter one agrees with the catalytic efficiency ratios calculated by Coutinho (1996) from literature data on A.awamori/A.niger GA, in which ratios for iG2 hydrolysis (and therefore iG2 synthesis, assuming a stable equilibrium constant) to those of maltose hydrolysis increased with increasing temperature while those for maltooligosaccharide hydrolysis to those of maltose hydrolysis decreased.
Of the 16 GAs in Table I giving glucose yields clearly greater than that of wild-type GA, seven are more thermostable at 65°C than wild-type GA and nine are less stable. Both the GAs with lower glucose yields are more stable than wild-type GA. This confirms the observation of Liu et al. (1998) that there is no general correlation between thermostability and selectivity. However, those GAs containing all mutations that presumably prevent unfolding at higher temperatures (SS, Ser30
Pro, Gly137
Ala, Ser436
Pro) uniformly have increased thermostability and tend to give increased glucose yield.
In summary, changes in selectivity to reduce iG2 formation during glucose condensation and to increase glucose yield have been successfully attained in many of the multiply-mutated GAs studied here. We also have demonstrated that specific activity, glucose yield, initial rate ratios and thermostability in multiply-mutated GAs can be predicted from the corresponding properties of the GAs containing the single mutations, suggesting that the principle of additive mutational effects in proteins is a powerful tool to improve glucose yield and thermostability. Glucoamylase selectivity is affected by the reaction temperature, with glucose yields being higher at lower temperatures. There is no correlation between GA thermostability and selectivity.
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Acknowledgments |
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Notes |
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References |
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Received July 22, 1998; revised October 26, 1998; accepted November 11, 1998.