Mutations to alter Aspergillus awamori glucoamylase selectivity. IV. Combinations of Asn20->Cys/Ala27->Cys, Ser30->Pro, Gly137->Ala, 311–314 Loop, Ser411->Ala and Ser436->Pro

Hsuan-Liang Liu, Clark Ford1 and Peter J. Reilly2

1 Departments of Chemical Engineering and Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Six previously constructed and nine newly constructed Aspergillus awamori glucoamylases with multiple mutations made by combining existing single mutations were tested for their ability to produce glucose from maltodextrins. Multiple mutations have cumulative effects on glucose yield, specific activity and thermostability. No general correlation between glucose yield and thermostability was observed, although mutations that presumably impede unfolding at high temperatures uniformly increase thermostability and generally increase glucose yield. Peak glucose yields decrease with increasing temperature. The best combination of high glucose yield, high specific activity and high thermostability occurs in Asn20->Cys/Ala27->Cys/Ser30->Pro/Gly137->Ala glucoamylase.

Keywords: additivity/glucoamylase/glucose yield/isomaltose/selectivity/site-directed mutagenesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Glucoamylase [{alpha}-(1,4)-D-glucan glucohydrolase; EC 3.2.1.3, GA], one of the most widely used industrial enzymes, not only hydrolyzes {alpha}-1,4-glucosidic linkages from the non-reducing ends of starch and related oligo- and polysaccharide chains, but also much more slowly hydrolyzes {alpha},ß-1,1-, {alpha}-1,2-, {alpha}-1,3- and {alpha}-1,6-glucosidic linkages (Hiromi et al., 1966aGo,bGo; Meagher and Reilly, 1989Go). Glucoamylase also synthesizes the same bonds at high glucose concentrations, obeying the law of microscopic reversibility (Pazur and Okada, 1967Go; Hehre et al., 1969Go; Watanabe et al., 1969aGo,bGo; Pazur et al., 1977Go; Nikolov et al., 1989Go). Production of isomaltose [{alpha}-D-glucopyranosyl-(1->6)-D-glucose, iG2] and other di- and oligosaccharides limits glucose yields at high soluble solids concentrations to about 95–96% of theoretical (Nikolov and Reilly, 1991Go; Teague and Brumm, 1992Go).

Roughly 11 million metric tons of glucose is produced annually in the United States with GA from concentrated maltodextrin solutions (Corn Annual, 1998Go). 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. Tyr116->Trp, 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 [{alpha}-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 (311–314 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 Tyr312->Trp 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 Asn20->Cys/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., 1998Go) but also increased glucose yields at 35, 45 and 55°C compared with wild-type GA (Liu et al., 1998Go). The Gly137->Ala mutation, which presumably stiffened an {alpha}-helix to increase GA thermostability (Chen et al., 1996Go), led to higher glucose yields at higher temperatures (Liu et al., 1998Go). Two other previously constructed thermostable mutated GAs, Ser30->Pro (Allen et al., 1998Go) and Ser436->Pro (Li et al., 1997Go), and one thermosensitive mutated GA, Ala27->Pro (Li et al., 1997Go), gave lower glucose yields than did wild-type GA (Liu et al., 1998Go). 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 Ser119->Tyr, 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, 1994Go; Svensson et al., 1995Go). Frandsen et al. (1995) altered GA selectivity by the Arg305->Lys mutation, because Arg305 stabilizes {alpha}-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 {alpha}-1,6- over {alpha}-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., 1997Go).

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/Ser30->Pro, Ser30->Pro/Gly137->Ala, SS/Ser30->Pro/Gly137->Ala (Allen et al., 1998Go), SS/Gly137->Ala, SS/Ser436->Pro and Gly137->Ala/Ser436->Pro (Li et al., 1998Go) mutations. These mutations were made to increase GA thermostability, which was additive in all cases except SS/Ser436->Pro GA (Allen et al., 1998Go; Li et al., 1998Go). The Ser30->Pro/Gly137->Ala and SS/Ser30-> Pro/Gly137->Ala GAs are the most stable GAs yet produced (Allen et al., 1998Go).

We also constructed GAs with the following nine multiple mutations, SS/311–314 Loop, SS/Ser411->Ala, Ser30->Pro/311–314 Loop, Ser30->Pro/Ser411->Ala, Gly137->Ala/311–314 Loop, Gly137->Ala/Ser411->Ala, 311–314 Loop/ Ser411->Ala, Ser30->Pro/Gly137->Ala/311–314 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 {alpha}-helix in A.awamori var. X100 GA (Aleshin et al., 1992Go), while Ala27 and Ser30 are both located in a type II ß-turn on the extended loop between the first and second {alpha}-helices (Figure 1Go). Gly137 is located in the middle of the fourth {alpha}-helix, and residues Tyr311 to Gly314 are between {alpha}-helices 9 and 10. Ser411 is located in a ß-strand between {alpha}-helices 12 and 13, while Ser436 is in a random coil in a packing void of unknown function (Aleshin et al., 1994Go). Of the above residues, only Ala27 and Ser30 are located in conserved regions, and neither of them is totally conserved (Coutinho and Reilly, 1994aGo,bGo, 1997Go).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1. Three-dimensional view of A.awamori/A.niger GA, showing mutated residues.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Methods for enzyme production and purification, protein concentration determination, 30% (w/v) malto-oligosaccharide hydrolysis and 30% (w/v) glucose condensation reactions were described by Fang et al. (1998a). Specific activities were measured as by Fang et al. (1998b).

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

Gly137->Ala (Chen et al., 1996Go), Ser436->Pro (Li et al., 1997Go), SS, SS/Gly137->Ala, SS/Ser436->Pro, Gly137->Ala/Ser436->Pro (Li et al., 1998Go), Ser411->Ala (Fang and Ford, 1998Go), 311–314 Loop (Liu et al., 1998Go), Ser30->Pro, SS/Ser30->Pro, Ser30->Pro/Gly137->Ala and SS/Ser30->Pro/Gly137->Ala (Allen et al., 1998Go) 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 311–314 Loop/Ser411->Ala mutation, an XbaI–HindIII fragment of pGEM-GA containing the wild-type GA cDNA (Fang and Ford, 1998Go) 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., 1998bGo). The previously made 311–314 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., 1998aGo) 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 BamHI–PstI fragments containing the SS, Ser30->Pro, Gly137->Ala or Ser30->Pro/Gly137->Ala mutations, which were restriction-digested from the phagemid vector pGEM7Z(+), were separately ligated to the long BamHI–PstI fragments containing the 311–314 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., 1994Go).

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 ({Delta}G{ddagger}) for thermoinactivation of mutant and wild-type GAs were obtained from a plot of ln (kd/T) versus 1/T. The function {Delta}{Delta}G{ddagger} is the difference between {Delta}G{ddagger} values for mutated and wild-type GAs, being positive when the mutated GA is more stable.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specific activities

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 IGo. 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 311–314 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.


View this table:
[in this window]
[in a new window]
 
Table I Specific activities, glucose yields and relative thermostabilities of wild-type and mutant GAs
 
Maltodextrin hydrolysis

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 2Go. 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.




View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2. Glucose formation during the incubation of 30% (w/v) DE 10 maltodextrin with 1.98 µM GA in 0.05 M NaOAc buffer, pH 4.4, at 55°C. (a) Wild-type ({circ}, —), SS/Ser30->Pro ({triangleup}, — —), SS/Gly137->Ala ({square}, — — —), SS/Ser411->Ala ({triangledown}, — — — —), SS/Ser436->Pro ({bullet}, ......), Ser30->Pro/Gly137->Ala ({blacktriangleup}, — ··), Gly137->Ala/Ser436->Pro ({blacksquare}, — ····), SS/Ser30->Pro/Gly137->Ala ({blacktriangledown}, —); (b) wild-type ({circ}, —), SS/311–314 Loop ({blacktriangledown}, — —), SS/Ser411->Ala ({triangledown}, — — —), Ser30->Pro/311–314 Loop ({blacktriangleup}, — — — —); Ser30->Pro/Ser411->Ala ({triangleup}, ......), Gly137->Ala/311–314 Loop ({blacksquare}, — ··), Gly137->Ala/Ser411->Ala ({square}, — ····), 311–314 Loop/Ser411->Ala ({bullet}, —), Ser30->Pro/Gly137->Ala/311–314 Loop ({lozenge}, — —) and Ser30->Pro/Gly137->Ala/Ser411->Ala ({blacklozenge}, — — —).

 
Peak glucose yields averaged over the three substrates are shown in Table IGo for all three temperatures. Most GAs with multiple mutations give higher peak glucose yields than does wild-type GA, but those containing the Ser436->Pro mutation have lower yields.

Initial glucose formation rates appear in Table IIGo. 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.


View this table:
[in this window]
[in a new window]
 
Table II Initial rates of glucose and iG2 formation in the hydrolysis of 30% (w/v) maltodextrins and condensation of 30% (w/v) glucose, respectively, and their relative ratios for wild-type and mutant GAs at pH 4.4 and 35, 45 and 55°C
 
Activation energies, obtained by plotting ln (initial rate) versus 1/T, average 81 kJ/mol for 30% (w/v) maltodextrin hydrolyses from 35 to 55°C over different substrates and different mutated and wild-type GAs. Again there are no significant differences between among substrates. Activation energies for wild-type and all multiply-mutated GAs are within the 95% confidence range, meaning that there is a 95% probability that the values are not significantly different, and suggesting that all mutated GAs catalyze the same hydrolysis mechanism as does wild-type GA.

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 3Go. 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 IIGo. Of the GAs with multiple mutations, SS/Ser411->Ala, SS/311–314 Loop and 311–314 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.




View larger version (43K):
[in this window]
[in a new window]
 
Fig. 3. Formation of iG2 for 30% (w/v) glucose condensation with wild-type and mutated GAs. Conditions as in Figure 2Go. (a) Symbols as in Figure 2aGo; (b) symbols as in Figure 2bGo.

 
Activation energies, obtained as above, for isomaltose formation of all multiply-mutated GAs, averaging 66 kJ/mol and all within the 95% confidence range, are much lower than that of wild-type GA (90 kJ/mol), meaning that the GAs with multiple mutations may undergo different active-site conformational changes than does wild-type GA while catalyzing the condensation reaction.

Selectivity for {alpha}-1,6-linked product synthesis versus {alpha}-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 IIGo). Of the multiply-mutated GAs, SS/311–314 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 4Go. 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.





View larger version (79K):
[in this window]
[in a new window]
 
Fig. 4. Peak glucose yields versus initial rate ratios (x103) with 1.98 µM GA in 0.05 M NaOAc buffer, pH 4.4. DE 10 ({circ}, —, bold letters); DE 18 ({triangleup}, — —, normal letters) and DE 25 ({square}, ......, italicized letters) maltodextrins. A, wild-type; B, SS/Ser30->Pro; C, SS/Gly137->Ala; D, SS/311–314 Loop; E, SS/Ser411->Ala; F, SS/Ser436->Pro; G, Ser30->Pro/Gly137->Ala; H, Ser30->Pro/311–314 Loop; I, Ser30->Pro/Ser411->Ala; J, Gly137->Ala/311–314 Loop; K, Gly137->Ala/Ser411->Ala; L, Gly137->Ala/Ser436->Pro; M, 311–314 Loop/Ser411->Ala; N, SS/Ser30->Pro/Gly137->Ala; O, Ser30->Pro/Gly137->Ala/311–314 Loop GAs. (a) 35°C; (b) 45°C; (c) 55°C.

 
Irreversible thermoinactivation

Table IGo shows the changes of {Delta}G{ddagger} from that of wild-type GA ({Delta}{Delta}G{ddagger}) at 65 and 75°C, with data taken here and elsewhere. A rough approximation is that {Delta}{Delta}G{ddagger} 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 311–314 Loop/Ser411->Ala mutation decreases thermostability the most.

Values of {Delta}{Delta}G{ddagger} for GAs with multiple mutations can be approximately determined by adding the {Delta}{Delta}G{ddagger} values of GAs containing their single mutations, as found earlier (Allen et al., 1998Go; Li et al., 1998Go). In addition, specific activities at 50°C and thermostabilities at 65°C are positively correlated (R = 0.85).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Glucoamylase selectivity has been improved by single mutations (Sierks and Svensson, 1994Go; Svensson et al., 1995Go; Fang et al., 1998aGo,bGo; Liu et al., 1998Go), loop replacements (Fierobe et al., 1996Go) and loop insertions (Liu et al., 1998Go), but there had been no research until this study on the effect of multiple mutations on GA selectivity, except for that of Gly121->Ala/Ser411->Gly GA (Fang et al., 1998bGo). In this study, we tested GAs with multiple mutations for their selectivities and compared them with all the GAs containing their single mutations to check if the principle of additive mutational effects in proteins can also be applied to GA selectivity.

The strategy of additive mutagenesis has been one of the most powerful and successful tools in stabilizing proteins against irreversible thermoinactivation, such as with {lambda} repressor (Hecht et al., 1986Go), subtilisin (Cunningham and Wells, 1987Go; Pantoliano et al., 1989Go), kanamycin nucleotidyltransferase (Liao et al., 1986Go), T4 lysozyme (Matsumura et al., 1989Go) and GA (Allen et al., 1998Go; Li et al., 1998Go); in engineering subtilisin selectivity (Russell and Fersht, 1987Go; Wells et al., 1987aGo,bGo); in improving glutathione reductase coenzyme selectivity (Scrutton et al., 1990Go); and in enhancing the catalytic efficiency of a weakly active subtilisin variant (Carter et al., 1989Go). This principle can be expressed as {Delta}{Delta}GX,Y = {Delta}{Delta}GX + {Delta}{Delta}GY + {Delta}GI, where {Delta}{Delta}GX, {Delta}{Delta}GY and {Delta}{Delta}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, 1985Go). The coupling energy {Delta}GI (Carter et al., 1984Go) 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, {Delta}GI is negligible and the above equation can be simplified to {Delta}{Delta}GX,Y = {Delta}{Delta}GX + {Delta}{Delta}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, Ser30->Pro, Gly137->Ala, 311–314 Loop, Ser411->Ala and Ser436->Pro mutations are demonstrated in Figures 5–9GoGoGoGoGo, 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 [{alpha}-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 {alpha}-1,6 bonds found in maltodextrins, resulting in less complete substrate conversion at peak glucose yields.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Cumulative effects of mutations on GA selectivity for combinations of SS, Ser30->Pro and Gly137->Ala mutations. Variations of peak glucose yields from those of wild-type GA and initial rate ratios (x103) of singly-mutated GAs are at the corners, those of doubly-mutated GAs are at the midpoints of the sides, and those of the triply-mutated GA are in the center. Values have been averaged for DE 10, 18 and 25 maltodextrins. (a) 35°C; (b) 45°C; (c) 55°C.

 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. Cumulative effects of mutations on GA selectivity for combinations of SS, Gly137->Ala and Ser436->Pro mutations. Notations as in Figure 5Go.

 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7. Cumulative effects of mutations on GA selectivity for combinations of SS, 311–314 Loop and Ser411->Ala mutations. Notations as in Figure 5Go.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 8. Cumulative effects of mutations on GA selectivity for combinations of Ser30->Pro, Gly137->Ala and 311–314 Loop mutations. Notations as in Figure 5Go.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 9. Cumulative effects of mutations on GA selectivity for combinations of Ser30->Pro, Gly137->Ala and Ser411->Ala mutations. Notations as in Figure 5Go.

 
Before this series of articles, GA selectivities were determined as the ratios of catalytic efficiencies for maltose hydrolysis over iG2 hydrolysis, and these were measured at low substrate concentrations. However, Fang et al. (1998a,b) and Liu et al. (1998) found that this ratio is not directly related to glucose yield, and instead correlated the latter with the ratio of initial iG2 formation rate to initial glucose formation rate at high substrate concentrations. We have done the same here.

The SS mutation introduced an extra disulfide bond on the catalytic domain surface in the extended loop between the first and second {alpha}-helices of GA. This loop, containing a conserved region involved in substrate binding (Coutinho and Reilly, 1997Go), is near another loop containing residue Trp120, which is critical for catalysis (Sierks et al., 1989Go) and important in directing conformational changes controlling the putative rate-limiting product release step (Natarajan and Sierks, 1996Go). The loop appears to be important for both GA thermostability (Li et al., 1998Go) and selectivity (Liu et al., 1998Go). 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., 1998Go; Li et al., 1998Go). Those mutations plus the SS/311–314 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 IGo). It increases glucose yields and initial glucose formation rates both by itself and when it is combined with other mutations (Tables I and IIGoGo, Figures 5–7GoGoGo). 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 IIGo). Although the SS/311–314 Loop and SS/Ser411->Ala GAs are more thermosensitive than SS and wild-type GAs, they are both more thermostable than 311–314 Loop and Ser411->Ala GAs (Table IGo), meaning that adding a mutation that singly confers thermostability to a GA already containing a thermosensitive mutation can make the latter more thermostable.

The Ser30->Pro 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., 1998Go). It was combined previously with the SS and Gly137->Ala mutations (Allen et al., 1998Go) and then here with the 311–314 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 IGo) and a slightly negative to slightly positive effect on glucose yields (Table IGo, Figures 5, 8 and 9GoGoGo). 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 IIGo). It increases GA thermostabilities in singly- and multiply-mutated GAs (Table IGo).

Even though the SS mutation gives a disulfide bond close to position 30, cumulative mutational effects in SS/Ser30->Pro 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., 1998Go) 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 {alpha}-helix 1 and the following extended loop between {alpha}-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 {alpha}-helix 4, which is part of the inner ring of the {alpha},{alpha}-barrel around the active site, was mutated to Ala to increase GA thermostability, presumably by stiffening the helix (Chen et al., 1996Go). The Gly137->Ala mutation increases specific activity in GAs in which it is a part (Table IGo), has a weakly positive to negligible effect on peak glucose yields (Table IGo, Figures 5, 6, 8 and 9GoGoGoGo) and initial glucose formation rates (Table IIGo), sometimes increases and sometimes decreases initial rate ratios (Table IIGo), and increases thermostabilities (Table IGo).

The 311–314 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., 1998Go). The mutation gives higher glucose yields (Table IGo, Figures 7 and 8GoGo) and lower initial rate ratios (Table IIGo), but it also gives GAs with much lower specific activities, initial glucose formation rates and thermostabilities (Tables I and IIGoGo).

The Ser411->Ala mutation is one of a series constructed to increase the optimal pH of GA (Fang and Ford, 1998Go). 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 IIGoGo) due to the destruction of the hydrogen bond. It increases glucose yields (Table IGo, Figures 7 and 9GoGo) 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 IIGo). GAs with this mutation have strongly decreased thermostabilities (Table IGo).

The Ser436->Pro 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 IGo, Figure 6Go) and increased initial rate ratios (Table IIGo). The low specific activity found in Ser436->Pro GA is not carried over to other GAs containing the mutation (Table IGo), and glucose formation rates are in general increased by it (Table IIGo).

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 4Go 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., 1998Go). However, as noted earlier (Liu et al., 1998Go) and here, peak glucose yields are higher and occur 18–24 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 {alpha}-(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 {alpha}-(1,4) and {alpha}-(1,6) glucosidic bonds; i.e. maltotriose, with a nonreducing-end terminal {alpha}-(1,4) bond, and panose [{alpha}-D-glucopyranosyl-(1,6)-{alpha}-D-glucopyranosyl-(1,4)-glucose], with a nonreducing-end terminal {alpha}-(1,6) bond, are closer in hydrolysis rate than maltose, with its {alpha}-(1,4) bond, and iG2, with its {alpha}-(1,6) bond (Meagher and Reilly, 1989Go). Furthermore, the relative absence of {alpha}-(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 IGo and Figure 4Go 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 IIGo). 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 IGo 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.


    Acknowledgments
 
This project was supported by the US Department of Energy through the Consortium for Plant Biotechnology Research, Inc.; by the US Department of Agriculture through the Midwest Advanced Food Manufacturing Alliance, and by Genencor International, Inc. The authors thank Martin Allen, Tsuei-Yun Fang and Yuxing Li for the gifts of many of their mutated GAs, John Robyt's group for their generous help on iG2 quantification by high performance thin layer chromatography, James Meade for the gift of wild-type GA gene and plasmid, Richard Antrim of Grain Processing Corporation for helpful discussions and Pedro Coutinho for modifying Figure 1Go.


    Notes
 
2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ackers,G.K. and Smith,F.R. (1985) Annu. Rev. Biochem., 54, 597–629.[ISI][Medline]

Aleshin,A., Golubev,A., Firsov,L.M. and Honzatko,R.B. (1992) J. Biol. Chem., 267, 19291–19298.[Abstract/Free Full Text]

Aleshin,A.E., Firsov,L.M. and Honzatko,R.B. (1994) J. Biol. Chem., 269, 15631–15639.[Abstract/Free Full Text]

Allen,M., Coutinho,P.M. and Ford,C. (1998) Protein Engng, 11, 783–788.[Abstract]

Balaji,V.N., Mobasser,A. and Rao,S.N. (1989) Biochem. Biophys. Res. Commun., 160, 109–114.[ISI][Medline]

Carter,P.J., Winter,G., Wilkinson,A.J. and Fersht,A.R. (1984) Cell, 38, 835–840.[ISI][Medline]

Carter,P., Nilsson,B., Burnier,J.P., Burdick,D. and Wells,J.A. (1989) Proteins Struct. Funct. Genet., 6, 240–248.[ISI][Medline]

Chen,H.-M., Ford,C. and Reilly,P.J. (1994) Biochem. J., 301, 275–281.[ISI][Medline]

Chen,H.-M., Li,Y., Panda,T., Buehler,F.U., Ford,C. and Reilly,P.J. (1996) Protein Engng, 9, 499–505.[Abstract]

Corn Annual (1998) Corn Refiners Association, Inc., Washington, DC.

Coutinho,P.M. (1996) Ph.D. Dissertation, Iowa State University, Ames, IA.

Coutinho,P.M. and Reilly,P.J. (1994a) Protein Engng, 7, 393–400.[Abstract]

Coutinho,P.M. and Reilly,P.J. (1994b) Protein Engng, 7, 749–760.[Abstract]

Coutinho,P.M. and Reilly,P.J. (1997) Proteins Struct. Funct. Genet., 29, 334–347.[ISI][Medline]

Cunningham,B.C. and Wells,J.A. (1987) Protein Engng, 1, 319–325.[Abstract]

Fang,T.-Y. and Ford,C. (1998) Protein Engng, 11, 383–388.[Abstract]

Fang,T.-Y., Coutinho,P.M., Reilly,P.J. and Ford,C. (1998a) Protein Engng, 11, 119–126.[Abstract]

Fang,T.-Y., Honzatko,R.B., Reilly,P.J. and Ford,C. (1998b) Protein Engng, 11, 127–133.[Abstract]

Fierobe,H.-P., Stoffer,B.B., Frandsen,T.P. and Svensson,B. (1996) Biochemistry, 35, 8696–8704.[ISI][Medline]

Frandsen,T.P., Christensen,T., Stoffer,B. Lehmbeck,J., Dupont,C., Honzatko, R.B. and Svensson,B. (1995) Biochemistry, 34, 10162–10169.[ISI][Medline]

Frandsen,T.P., Stoffer,B., Palcic,M.M., Hof,S. and Svensson,B. (1996) J. Mol. Biol., 263, 79–89.[ISI][Medline]

Hecht,M.H., Sturtevant,J.M. and Sauer,R.T. (1986) Proteins Struct. Funct. Genet., 1, 43–46.[Medline]

Hehre,E.J., Okada,G. and Genghof,D.S. (1969) Arch. Biochem. Biophys., 135, 74–89.[Medline]

Hiromi,K., Takahashi,K., Hamauzu,Z. and Ono,S. (1966a) J. Biochem., 59, 469–475.[ISI][Medline]

Hiromi,K., Kawai,M. and Ono,S. (1966b) J. Biochem., 59, 476–480.[ISI][Medline]

Li,Y., Reilly,P.J. and Ford,C. (1997) Protein Engng, 10, 1199–1204.[Abstract]

Li,Y., Coutinho,P.M. and Ford,C. (1998) Protein Engng, 11, 661–667.[Abstract]

Liao,H., McKenzie,T. and Hageman,R. (1986) Proc. Natl Acad. Sci. USA, 83, 576–580.[Abstract]

Liu,H.-L., Coutinho,P.M., Ford,C. and Reilly,P.J. (1998) Protein Engng, 11, 389–398.[Abstract]

Matsumura,M., Signor,G. and Matthews,B.W. (1989) Nature, 342, 291–294.[ISI][Medline]

Meagher,M.M. and Reilly,P.J. (1989) Biotechnol. Bioengng, 34, 689–693.[ISI]

Natarajan,S. and Sierks,M.R. (1996) Biochemistry, 35, 15269–15279.[ISI][Medline]

Nikolov,Z.L. and Reilly,P.J. (1991) In Dordick.,J.S. (ed.) Biocatalysts for Industry. Plenum, New York, pp. 37–62.

Nikolov,Z.L., Meagher,M.M. and Reilly,P.J. (1989) Biotechnol. Bioengng, 34, 694–704.[ISI]

Pantoliano,M.W., Whitlow,M., Wood,J.F., Dodd,S.W., Hardman,K.D., Rollence,M.L. and Bryan,P.N. (1989) Biochemistry, 28, 7205–7213.[ISI][Medline]

Pazur,J.H. and Okada,S. (1967) Carbohydr. Res., 4, 371–379.

Pazur,J.H., Cepure,A., Okada,S. and Forsberg,L.S. (1977) Carbohydr. Res., 58, 193–202.[ISI][Medline]

Russell,A.J. and Fersht,A.R. (1987) Nature, 328, 496–500.[ISI][Medline]

Scrutton,N.S., Berry,A. and Perham,R.N. (1990) Nature, 343, 38–43.[ISI][Medline]

Sierks,M.R. and Svensson,B. (1994) Protein Engng, 7, 1479–1484.[Abstract]

Sierks,M.R., Ford,C., Reilly,P.J. and Svensson,B. (1989) Protein Engng, 2, 621–625.[Abstract]

Stoffer,B.B., Dupont,C., Frandsen,T.P., Lehmbeck,J. and Svensson,B. (1997) Protein Engng, 10, 81–87.[Abstract]

Svensson,B., Frandsen,T.P., Matsui,I., Juge,N., Fierobe,H.-P., Stoffer,B. and Rodenburg,K.W. (1995) In Petersen,S.B., Svensson,B. and Pedersen,S. (eds) Carbohydrate Bioengineering. Elsevier, Amsterdam, pp. 125–145.

Teague,W.M. and Brumm,P.J. (1992) In Schenck,F.W. and Hebeda,R.E. (eds) Starch Hydrolysis Products: Worldwide Technology, Production and Applications. VCH, New York, pp. 45–77.

Watanabe,T., Kawamura,S., Sasaki,S. and Matsuda,K. (1969a) Stärke, 21, 18–21.

Watanabe,T., Kawamura,S., Sasaki,S. and Matsuda,K. (1969b) Stärke, 21, 44–47.

Wells,J.A., Cunningham,B.C., Graycar,T.P. and Estell,D.A. (1987a) Proc. Natl Acad. Sci. USA, 84, 5167–5171.[Abstract]

Wells,J.A., Powers,D.B., Bott,R.R., Graycar,T.P. and Estell,D.A. (1987b) Proc. Natl Acad. Sci. USA, 84, 1219–1223.[Abstract]

Received July 22, 1998; revised October 26, 1998; accepted November 11, 1998.