1Department of Agronomy, University of WisconsinMadison and 2Cereal Crops Research Unit, Agricultural Research Service, US Department of Agriculture, 1575 Linden Drive, Madison, WI 53706, USA
3 To whom correspondence should be addressed, at the Cereal Crops Research Unit. e-mail: cahenson{at}facstaff.wisc.edu
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Abstract |
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Keywords: Hordeum vulgare/maltase/peptide bond cleavage
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Introduction |
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The hydrolysis of starch to simple sugars is also an important step in the production of beer, distilled beverages and ethanol fuel. The thermostability of -glucosidase is important to fermentation industries because industrial starch hydrolysis occurs at relatively high temperatures (6075°C). At these temperatures,
-glucosidase has been largely inactivated and its contribution to the production of fermentable sugars is limited (Muslin et al., 2000
). The thermolability of
-glucosidase results in reduced efficiency of starch degradation and can result in an economic loss because of wasted raw materials (Muslin et al., 2003
).
Previous studies have examined the effects of N-glycosylation on the thermostabilities of various proteins. Lige et al. (2001) examined the effect of the systematic removal of the three N-glycosylation recognition sites (NGRS) on the thermostability and activity of recombinant peroxidase from peanut. A NGRS is a three amino acid sequence of asparagine, then any amino acid but proline and serine or threonine (NXS or NXT) and the glycan chain is attached to the asparagine residue. Lige et al. (2001
) determined that the removal of one of the three recognition sites significantly reduced thermostability and removal of the other two recognition sites significantly decreased activity levels of the recombinant mutant enzymes. Terashima et al. (1994
) demonstrated that the removal of the single NGRS in rice
-amylase1A significantly decreased thermostability and altered the temperature dependence of the kinetic parameters Vmax and Km. Meldgaard and Svendsen (1994
) compared the half-lives of Bacillus (1,31,4)-ß-glucanases that were produced in either Escherichia coli (no glycosylation possible) or yeast (glycosylation possible). All but one of the enzymes produced by yeast were more thermostable than the same enzymes produced by E.coli. Meldgaard and Svendsen (1994
) proved that the glucanases produced in yeast were glycosylated and that enzymatic deglycosylation reduced the enzymes thermostability. Han and Lei (1999
) showed that recombinant phytase had decreased thermostability when glycan chains were removed.
We previously reported significant variations in the thermostabilities of four plant -glucosidases, with the
-glucosidase from barley being the most thermolabile and the sugar beet (Beta vulgaris)
-glucosidase being the most thermostable (Muslin et al., 2002
). The barley
-glucosidase lost 90% of its activity after 10 min at 55°C. In contrast, the sugar beet
-glucosidase retained >90% of its activity after 10 min at 75°C. The present study examined the effects of adding and removing NGRS on the thermostability of barley recombinant
-glucosidase. An alignment of the deduced amino acid sequences from cDNA isolated from barley and sugar beet seeds revealed considerable differences in the number and position of NGRS. NGRS present in the barley sequence and absent in the sugar beet sequence were removed via site-directed mutagenesis from the barley protein. Recognition sites absent in the barley sequence and present in the sugar beet sequence were added via mutagenesis into the barley
-glucosidase. The thermostabilities of the resulting mutant enzymes were determined and compared with that of the recombinant wild-type enzyme.
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Materials and methods |
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Chemicals were purchased from Sigma (St Louis, MO) unless stated otherwise.
Mutagenesis and sequencing
Mutagenesis was performed according to Muslin et al. (2002). Generation of mutated cDNA was carried out using the primers listed in Table I to add or remove NGRS in the translated protein sequence. The mutants are identified by the original amino acid, the sequence position and the new amino acid (e.g. N298D is the mutant with the asparagine at position 298 mutated to an aspartate). The mutated cDNAs were sequenced using an automatic DNA sequencer by the Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL).
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The mutated cDNAs were subcloned into the EcoRI site of the Pichia pastoris expression vector pPIC9K (Invitrogen, Carlsbad, CA) and transformed into P.pastoris GS115 using the Pichia EasyComp kit (Invitrogen). Histidine autotrophs were induced with methanol following the instructions in the Pichia Expression kit (Invitrogen). The recombinant wild-type and mutant -glucosidases were secreted by P.pastoris into the culture media. Crude preparations were obtained by removing the yeast cells via centrifugation (2000 g, 10 min) and the resulting supernatants were used for thermostability studies.
Enzyme assay
-Glucosidase activities were measured by the release of glucose from maltose. Unless stated otherwise, the enzyme was incubated for 18 h at 30°C with 20 mM maltose in 50 mM sodium succinate (pH 4.5). The glucose released was quantified by determining the reduction of NAD+ with the coupled reactions of hexokinase and glucose-6-phosphate dehydrogenase (Im and Henson, 1995
).
Thermostability testing of wild-type and mutated -glucosidases
Crude enzyme extracts of recombinant wild-type and mutated -glucosidases were incubated for 10 min at temperatures from 0 to 60°C at pH 5. After cooling to room temperature, the remaining enzyme activity was assayed. We have previously demonstrated that the crude wild-type and purified wild-type recombinant
-glucosidase (rAGL) preparations had essentially the same T50 values [47.8 and 47.6°C, respectively (Muslin et al., 2002
)], so crude enzyme preparations were used to obtain the data presented here. For statistical analysis of thermostability, a two-tailed Students t-test was performed using each colony as a separate replication and assuming unequal variance. The mean T50 values and standard deviations were calculated using replications of 416 separate colonies.
Testing of pH dependence of thermostability
Three separate extracts each of recombinant wild-type and mutated -glucosidases were dialyzed (18 h, 4°C) against 4 l of 25 mM sodium succinate, pH 4, 5 or 6. The dialyzed enzyme extracts were incubated for 10 min at temperatures from 0 to 60°C. After cooling to room temperature, the enzyme activity was assayed. We determined that 200 mM assay buffer was required to ensure that the assay was conducted at pH 4.5 regardless of the pH of the thermostability test.
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Results and discussion |
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The effect on thermostability of the removal of the NGRS present at position 340 was explored by replacing the threonine (T) with an alanine (A). The T50 of the T340A rAGL was 56.1°C, which is 7.3°C higher than that of the wild-type (Table II). The thermostability profile of this mutant shows that it retains full activity at temperatures as high as 50°C and has lost only 1015% of its activity at 53°C (Figure 2). In contrast, the wild-type enzyme has lost 90% of its activity at 53°C. Because the sugar beet
-glucosidase has a proline (P) in position 340, we previously removed this NGRS from the barley
-glucosidase by replacing the T at position 340 with a P (Muslin et al., 2002
). That mutation resulted in a 10°C increase in the T50 and an enzyme that was fully stable at 55°C. Because the T340A and T340P mutations produced similar increases in enzyme thermostability, we conclude that the removal of the NGRS site was the dominant stabilizing factor and insertion of the P slightly enhanced the effect of the NGRS removal.
The effects of the additions of NGRS at positions 463 and 694 on thermostability are shown in Figure 3. The replacement of the aspartate (D) with a serine (S; D463S) had no significant impact on thermostability (Figure 3 and Table II). Either the added recognition site at position 463 was not glycosylated or the site was glycosylated and the glycan chain imparted no protection against thermal denaturation. The insertion of the NGRS at position 694 had a significant (P = 5.3 x 1013) positive effect on the mutant enzymes thermostability (Figure 3). The D694N (aspartate to asparagine) rAGL had a T50 of 55.5°C whereas the wild-type rAGL had a T50 of 48.8°C (Table II). The D694N enzyme lost only 20% of its activity at 53°C compared with the >90% activity loss of the wild-type enzyme at the same temperature (Figure 3).
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In conclusion, the effects of adding and removing NGRS were dependent on their position in the proteins primary structure. Olsen and Thomsen (1991) showed that the type and position of glycosylation on the (1,31,4)-ß-glucanase had more of an effect on thermostability than the amount of glycosylation the protein had attached. The same may be said about the barley recombinant wild-type
-glucosidase as in several cases the removal of the potential glycosylation site increased the thermostability of the enzyme and not all potential NGRS sites may be glycosylated. In one case, the removal of the D and not the addition of the NGRS resulted in a very large increase in the thermostability of the enzyme. One possible application of this information is in enhancing yield of fermentable sugars during the malting and brewing processes, as we previously showed that the addition of a thermostable rAGL to the industrial starch hydrolysis system increased the yield of fermentable sugars (Muslin et al., 2003
). Although transgenic barley containing a thermostable
-glucosidase gene could be generated, the use of genetically modified barley to produce malt beverages is currently not accepted. The fact that a change in only one nucleotide at either position 340 or 694 resulted in significantly increased thermostability combined with the fact that an N at 694 is found in at least two other plant species suggests that it may be worthwhile to screen H.vulgare germplasm for an
-glucosidase with either a P or A at position 340 and either an N or A at position 694.
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Acknowledgements |
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References |
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Received December 16, 2003; revised February 25, 2004; accepted March 1, 2004 Edited by Valerie Daggett
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