1 Department of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706 and 2 Cereal Crops Research Unit, Agricultural Research Service, U.S. Department of Agriculture, USA
E-mail: cahenson{at}facstaff.wisc.edu
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Abstract |
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Keywords: Hordeum vulgare/maltase/starch degradation
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Introduction |
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The thermal stability of -glucosidase is important because the conversion of starch to fermentable sugars during the industrial production of ethanol (e.g. brewing, fuel ethanol production) typically takes place at temperatures of 6573°C. The thermolability of
-glucosidase results in either reduced efficiency of starch breakdown at the high temperatures used for starch gelatinization or requires that the starch be cooled to a more favorable temperature for enzymatic hydrolysis after the starch is gelatinized.
In order to increase the thermostability of barley -glucosidase, site-directed mutagenesis of the recombinant enzyme was considered. There are no data in the literature on the thermostability of other plant
-glucosidases nor is there a report of the crystal structure of any
-glucosidase. However, there have been studies of other members of the glycosyl hydrolase family 31 in which thermostability was increased by site-directed mutagenesis. The thermostability of fungal glucoamylase (GA) has been well studied. The GA of Aspergillus awamori is maximally active at 50°C and begins to undergo thermal denaturation at 70°C (Chen et al., 1995
). Chen et al. have succeeded in increasing the thermostability of GA by substituting alanines for asparagines (Chen et al., 1994
). Suzuki, working with oligo-1,6-glucosidases from several Bacillus species, demonstrated that an increase in the frequency of proline in ß-turns and in the total number of hydrophobic residues can enhance protein thermostability (Suzuki, 1989
).
In this study we investigate the thermostability of -glucosidases from four plant species, compare their deduced amino acid sequences, and test the effect of substituting a proline for the residue present in the wild-type enzyme on the thermostability of
-glucosidase.
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Materials and methods |
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Chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.
Plant sources
Seeds of a sugarbeet (Beta vulgaris) breeding line (ACS9400461, Wang and Goldman, 1999) were kindly provided by Professor I.Goldman (University of Wisconsin). Arabidopsis (A.thaliana, cv. Columbia) seedlings were grown under a 10 h photoperiod at a temperature of 25°C for 3 weeks before harvesting. Spinach seeds (Spinacia oleracea, cv. Bloomsdale Longstanding, Northrup King) were purchased locally. Barley seeds (Hordeum vulgare, cv. Morex) were imbibed, germinated and kilned as described by Henson and Stone (Henson and Stone, 1989
).
Isolation of crude extracts from plants
Crude extracts from malted barley, seeds of sugarbeet and spinach, and leaves from Arabidopsis were isolated using published protocols (Chiba et al., 1978; Im and Henson, 1995
; Sugimoto et al., 1995
; Monroe et al., 1999
). The extracts were dialyzed (16 h, 4°C) against 50 mM sodium-succinate (pH 4.5).
Enzyme assay
-Glucosidase activities were measured by the release of glucose from maltose. Unless otherwise stated, the enzyme was incubated for 1 h at 30°C with 25 mM maltose in 50 mM sodium-succinate (pH 4.5) during which time substrate hydrolysis rates were linear. The glucose released was quantified by determining the reduction of NAD with the coupled reactions of hexokinase and glucose-6-dehydrogenase (Im and Henson, 1995
).
Thermostability testing of plant extracts
Enzyme extracts were incubated for 10 min at temperatures ranging from 5 to 75°C. The residual rate of maltose hydrolysis was assayed for 1 h at 30°C.
Alignment of -glucosidase sequences from four plant species
Alignment of the published -glucosidase deduced amino acid sequences from barley (GenBank accession number U22450), spinach (D86624), sugarbeet (D89615) and Arabidopsis (AF014806) was done using the program Align Plus-Version 2.0 (Scientific and Educational Software).
Mutagenesis
Mutagenesis was done using the Muta-Gene kit (BIO-RAD, Hercules, CA). Barley -glucosidase cDNA (Tibbot and Skadsen, 1996
) was sub-cloned into the EcoRI site of the phagemid pTZ18U (BIO-RAD). Escherichia coli strain CJ236 was used to generate dU-substituted DNA, and single-stranded DNA was isolated using the helper phage M13K07 (BIO-RAD). Generation of the mutant R336P used the oligonucleotide CGGTGAAGTTGACAGGATCCAAGGTGAAG (5', reverse complement) to replace the codon for arginine (CGT) with a codon for proline (CCT) and to remove a Tth111I site. The mutant T340P was generated using the oligonucleotide GAGCTCGGCGGCGGGGAAGTTTACACGGTC to replace the codon for threonine (ACC) with a codon for proline (CCC) and to remove a Tth111I site. To generate the mutant A742P we used the oligonucleotide CCAGGAGGTGGAACGGGGTCCGGCGC to replace the codon for alanine (GCG) with a codon for proline (CCG) and to remove a RsrII site.
Sequencing
The mutated cDNA was sequenced using the Sanger method (Sanger et al., 1977) with an automatic sequencer by the Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL.
Expression
The mutated cDNA was subcloned into the EcoRI site of the Pichia pastoris vector pPIC9K (Invitrogen, CA) and transformed into P.pastoris GS115 using the Pichia EasyComp kit (Invitrogen). Ten histidine autotrophs (His+) were induced with methanol following the instructions in the Pichia Expression kit (Invitrogen). Pichia colonies that secreted measurable -glucosidase activity were used for thermostability studies.
Thermostability testing of wild-type and mutated -glucosidases
Enzyme extracts from non-mutated, recombinant -glucosidase (rAGL), T340P and A742P were incubated for 10 min at temperatures ranging from 0 to 60°C at a pH of either 6.0 or 4.0. The residual rate of maltose hydrolysis was assayed for 18 h at 30°C at pH 4.5.
Arrhenius plot
Enzyme extracts from rAGL and T340P (pH 6.0) and substrate (pH 4.5) were incubated separately (10 min) at temperatures ranging from 0 to 55°C and assayed for 3.5 h at the same temperatures. The results were plotted as log Vi versus 10-3/T (K).
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Results and discussion |
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The thermostability profiles of barley, sugarbeet, spinach and Arabidopsis -glucosidases are shown in Figure 1
. Sugarbeet
-glucosidase was the most thermostable and still retained greater than 90% of its maximal activity following exposure to 75°C for 10 min. The spinach
-glucosidase was the second most thermostable, followed by those from Arabidopsis and barley. The barley enzyme had only 10% of its maximal activity after exposure to 55°C for 10 min.
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No mutation was made at position 547 because secondary structure predictions made using the program Peptide Structure (Wisconsin GCG Package, Madison, WI), which is based on the theories of Garnier et al. (Garnier et al., 1978), placed this residue in the middle of a ß-sheet (Table I
). According to Watanabe et al. the insertion of a proline residue in the middle of a ß-sheet would not enhance the thermostability of the enzyme (Watanabe et al., 1991
).
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Although no three-dimensional structure has been reported for an -glucosidase, the secondary structure of the barley protein was predicted using the program Peptide Structure. Table I
shows the secondary structure predictions for the residues at positions 336, 340, 547 and 742 (numbers based on barley sequence) in
-glucosidases from barley, sugarbeet, spinach and Arabidopsis. According to Table I
, the threonine at position 340 of barley
-glucosidase is in the first turn of an
-helix. Similarly, the prolines in the same position in the sugarbeet, spinach and Arabidopsis enzymes are also found in the first turn of an
-helix. According to Watanabe, the presence of prolines in the first turn of
-helices and at the second sites of ß-turns often results in protein stabilization (Watanabe et al., 1991
). Therefore, it makes sense that the addition of a proline in the first turn of an
-helix in the barley
-glucosidase made the enzyme more thermostable.
Residue 742 in barley -glucosidase was predicted to be in the second turn of an
-helix (Table I
). According to Watanabe et al., the influence of proline on nearby residues is so strong that the imprudent substitution of proline for another amino acid might result in the destruction of both secondary and tertiary structures as well as the loss of protein function and/or stability (Watanabe et al., 1996
). The substitution of a proline in the second turn of an
-helix (as was done with the mutation A742P) apparently had a destructive effect on the thermostability of the enzyme but not on the enzyme's activity (Figure 3
).
According to the secondary structure program Peptide Structure, residue 336 in barley -glucosidase is in the second site of a ß-turn (Table I
). Thus, we predicted the insertion of a proline at this position would positively affect the enzyme's thermostability. However, the mutation R336P resulted in an inactive enzyme. It should be noted that the secondary structure predictions made using the method of Chou and Fassman (Chou and Fassman, 1978
) indicate that residue 336 is in the last turn of an
-helix (data not shown). Therefore, according to these structural predictions, the addition of a proline at residue 336 could destabilize the enzyme. Such a destabilization occurred as evidenced by the R336P mutation resulting in an inactive
-glucosidase. The secondary structure predictions (Peptide Structure) for sugarbeet and Arabidopsis at position 336 indicate that these prolines are in undefined regions; therefore, the effect these prolines have on enzyme stability cannot be anticipated. According to the program Peptide Structure, the proline at position 336 in spinach is in a ß-sheet. According to Watanabe, this proline should not enhance the protein's thermostability, possibly indicating that it is not this residue that is the cause of this enzyme's thermostability.
The difference in T50 values between the mutant and wild-type enzymes was decreased from 10°C at pH 6 to 7°C at pH 4. These data could indicate that different mechanisms are responsible for thermostability at these two pHs. Such has been documented to be the case for lysozyme. Ahern and Klibanov found that the thermoinactivation of lysozyme can be attributed to one or more of the following mechanisms: deamidation of asparagine residues, hydrolysis of peptide bonds at aspartic acid residues, destruction of disulfide bonds and the formation of non-functional aggregates or other incorrect structures (Ahern and Klibanov, 1985). The relative contributions of these mechanisms to thermoinactivation are dependent upon the individual protein and its environment, e.g. pH. They determined that at pH 4.0 the dominant mechanism of thermoinactivation of lysozyme is deamidation of asparagine residues. At pH 8 destruction of cysteine residues and formation of incorrect structures become the dominant mechanisms of thermoinactivation.
According to an analysis of the deduced amino acid sequences of barley, sugarbeet, spinach and Arabidopsis -glucosidases, the sugarbeet and spinach enzymes are the most related (Monroe et al., 1999
). Perhaps the presence of the conserved prolines in these dicot species, specifically the proline at position 340, represents an evolutionary trend towards a more thermostable enzyme.
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Notes |
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Mention of a proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other suitable products.
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Acknowledgments |
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References |
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Received February 10, 2001; revised August 16, 2001; accepted September 25, 2001.