The final purification scheme of AMY. AS stands for ammonium sulfate.
Chromatography, at both preparative and analytical levels, has been a key experimental technique in the study of proteins, primarily liquid chromatography in the separation, purification, and analysis. Recent developments in the study of proteins lean towards simplification and miniaturization, thus chromatography becomes less involved and explored. For example, development of protein tags and their associated affinity matrices enables purification of a protein in one step. This chapter describes the identification and structure-function relation study of amylolytic enzymes from
S. fibuligera R64 secretes amylolytic enzyme
Degradation of starch into sugars is performed by amylolytic enzymes, such as α-amylase, glucoamylase, β-amylase, isoamylase, pullulanase, exo(1-4)-α-D-glucanase, α-D-glycosidase, and cyclomaltodextrin-D-glucotransferase (Fig. 1) .
The use of amylolytic enzymes for the ethanol production in the course of renewable energy requires an ability to act on raw starch, allowing the use of biomass as the starting material. The ability of
α-Amylase is commonly used in food, beverage, paper industries , in textile industry and as additive in detergents [9, 10], for renewable energy [11, 12] and medical purpose . Glucoamylase has been the most important enzyme in food industry, mainly in the production of sugar or ethanol . Glucoamylase is normally employed in combination with amylolytic enzymes that are able to act on more complex polysaccharides, such as α-amylase and pullulanase . Recently, amylolytic enzymes are employed in the production of lactic acid and ethanol by-product by lactic acid bacteria (LAB) , demonstrating that the application of amylolytic enzymes continues to expand.
Interestingly, numbers and characteristics of amylolytic enzymes secreted by
Of 136 isolates screened from various places in Indonesia,
Extra-cellular amylolytic enzyme production by strain R64 is relatively simple, using a medium that contains of 1% sago starch and 1% yeast extract. The enzyme was harvested after 4-5 days of cultivation in a one-litre bioreactor, with constant aeration rate 1 vvm, volumetric oxygen transfer coefficient (
3. Isolation of the amylolytic enzyme complex
The amylolytic enzyme complex from strain R64, consisting of α-amylase (AMY) and glucoamylase (GLL1), is secreted into the production medium. Thus, the enzyme complex was harvested by simply cold-centrifugation (~4oC) at 6000
4. Purification of AMY and GLL1 in chromatography columns
Since the final step in the isolation procedure in the pilot experiment was precipitation with high concentration of ammonium sulfate, subsequent purification on a size-exclusion chromatography (SEC) column appears to be the most appropriate approach because it also functions as a desalting procedure. SEC column requires minimum size of sample upon application (recommended less than 3-5% of the column volume), which can easily be overcome by dissolving the protein precipitate from ammonium sulfate precipitation in a small volume. Another option is purification using an anion or cation exchanger chromatography (abbreviated as AEX and CEX, respectively) columns. However, this either type of column requires the removal of salts prior to sample application. This requirement can be countered by either diluting the sample until the conductivity of the sample solution is low or by dialysis against a buffer that contains low salt concentration.
The ammonium sulfate precipitate containing AMY and GLL1 was immediately dissolved in a small portion (3-5 ml) of 25 mM Tris-HCl buffer, pH 8.0. Unfortunately, AMY and GLL1 were not separated in an SEC column as suggested from the enzyme activity assays. In a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis, AMY and GLL1 was not separated and appeared as one protein band with a molecular mass of 55±2 kDa.Further, purification in a DEAE-cellulose AEX column has also failed to separate the two enzymes. This finding indicated a similarity in the distribution of charge on the surface of AMY and GLL1. In addition, there was a recommendation to avoid the use of carbohydrate- based column material for the purification of proteins with an affinity towards carbohydrate, because of the possible interaction between the protein and the supporting column matrix . Thus, both SEC and AEX columns were obliterated from the separation of AMY from GLL1.
As purification strategies based on either protein size or charge failed to separate AMY from GLL1, exploiting differences of their protein surface hydrophobicity profiles emerged as an alternative. This strategy was tested on a hydrophobic interaction chromatography (HIC) column, where the purification proteins is based on the hydrophobic character of the surface of the proteins . In an HIC column, proteins in solution are conditioned with high salt concentration, which enforces interaction between proteins and the hydrophobic resin. Separation of AMY from GLL1 in an HIC column is lucrative because it can be carried out immediately after precipitation with high concentration of ammonium sulfate. Thus, the use of the HIC column is complementary to the developed isolation procedure.
4.1. Separation of AMY and GLL1 in an HIC column
The ammonium sulfate precipitated protein was dissolved in 25 mM Tris-HCl buffer, pH 8.0, containing 25% ammonium sulfate (w/v). This protein solution was applied to a butyl-Toyopearl 650M HIC column (Tosoh Bioscience Corp., Tokyo, Japan), which had previously been equilibrated with the same buffer. The column was then eluted with 25 mM Tris-HCl buffer, pH 8.0, containing a decreasing concentration of ammonium sulfate (25-0% of 5% decrement, w/v). The separation profile of AMY and GLL1 in the HIC column is presented in Fig. 2. AMY was eluted at the ammonium sulfate concentration of 15% (w/v) whilst GLL1 was at 5% (w/v). This result demonstrated that AMY and GLL1 were successfully separated based on their hydrophobicity. Moreover, the elution profile suggests that the surface character of GLL1 is more hydrophobic than AMY.
Purification of proteins in HIC column is influenced by the pH of the solution where hydrophobic interaction is stronger at lower pH . This means, less hydrophobic proteins are bound stronger onto the HIC matrix at lower pH value. This phenomenon may explain different separation profile of AMY and GLL1 observed upon purification in an HIC column using phosphate-citrate buffer system at pH 5.8 , where α-amylase activity was detected in the GLL1 fraction. Although this hypothesis is yet to be proven, the separation at neutral to basic pH (above 7.0) is recommended for optimum operation.
The use of HIC column to separate
4.2. Subsequent purification of AMY or GLL1 in chromatography columns
Intended for their characterization, AMY and GLL1 were independently collected and subjected to subsequent purification with DEAE-Toyopearl 650M AEX column. An SDS PAGE analysis showed that these purification steps resulted in pure AMY  and GLL1 . The final purification scheme of AMY is summarized in Table 1. Further, whilst the presence of two and three types of α-amylase and glucoamylase, respectively, were reported upon purification of these amylolytic enzymes from strain KZ by a Mono-Q anion exchanger column , AMY and GLL1 appeared to be the only amylolytic enzyme species from strain R64. Nevertheless, additional analysis was performed to confirm that the isolation of purified AMY and GLL1 was unanimous.
|Total activity (Units)||Total protein|
4.3. Identification of AMY and GLL1 using RP-HPLC for protein
Nowadays, identification of proteins usually employs techniques with sophisticated and delicate instrumentation e.g. peptide-mass finger print mass spectrometry (PMF-MS) . Unfortunately, this technique is rather pricey and furthermore, requires convenient access to the protein database and to the amino acid sequence of the protein. Particularly in the lack of the latter, which is a very common situation in the early stage of protein works, the separation of AMY from GLL1 was confirmed by a more simple and robust technique, exploiting the use of an RP-HPLC system . The analysis using RP-HPLC is based on the fragmentation profile of a protein after proteolytic digestion. Proteolytic digestion of AMY was expected to result in fragmentation profile that differs to that of GLL1, as reflected from their chromatographic profiles.
The analysis of AMY and GLL1 by RP-HPLC was carried out essentially following Soedjanaatmadja and co-workers on the identification of pseudo-hevein from the latex of rubber tree . Firstly, purified AMY was incubated for four hours at 37oC with chymotrypsin (EC. 220.127.116.11), at an AMY to chymotrypsin mass ratio of 100:1, in 200 mM ammonium bicarbonate buffer, pH 8.0. The reaction was stopped by an addition of 10 mM hydrochloric acid, to lower the pH of the solution. Preparation of GLL1 sample was done in the same way, including the substrate to chymotrypsin mass ratio.
The reaction mixture was immediately applied to an RP-HPLC nucleosil 10 C18 column (30 x 0.45 cm) and the separation was performed for 60 minutes, at a flow rate of 1 ml/min, using 0-70% acetonitrile gradient in 0.1% trifluoroacetic acid (TFA). Elution of fragments was monitored at λ 214 nm, which is specific for detection of peptide bonds. The fragments are eluted according to their hydrophobicity, where more hydrophobic fragments are retained longer in the RP-HPLC column.
Chymotrypsin cleavage takes place on peptide bonds at the C-terminal part of preferably tyrosine, phenylalanine, tryptophan, and leucine residues, and with (much) less extent of methionine, valine, isoleucine, histidine, glycine and alanine residues . Due to this broad specificity, the cleavage may result in many fragments varying in size and hydrophobicity, as observed in the chromatographic profiles of proteolytically digested AMY and GLL1 (Fig. 3).
The fragmentation profile of AMY was significantly different from that of GLL1. Six sets of resolved peaks were recovered in both cases (Fig. 3) but their retention time, intensities, and peak distribution were different. Intensity of the peaks suggested much less materials were recovered from GLL1 digested samples than from AMY. This situation is likely contributed from highly hydrophobic or negative charged fragments that were not eluted from the RP-HPLC column due to their poor solubility in the solvent used . The amino acid sequences of AMY and GLL1 (see 3.5) showed that the charged amino acid distribution in the amino acid sequence of GLL1 (100 charged amino acid residues out of 494, ~20.2%) is higher than that of AMY (86 out of 468, ~18.4%), thus AMY contains more hydrophobic and non-charged amino acids. Unfortunately, correlation between the result from RP-HPLC and hydrophobic amino acid distribution cannot firmly be established because the nature of chymotrypsin digestion was unclear. The results may indeed indicate that hydrophobic amino acids in GLL1 are likely more clustered than in AMY, resulting in highly hydrophobic peptide fragments in GLL1. This hypothesis may be related to the structure of GLL1 (Fig. 8), which consists of one globular molecule that opposes two separable domains of AMY. However, possibility for the existence of highly negative charged peptide fragments from GLL1 can also not be excluded. Nevertheless, of the eluted fragments (Fig. 3), majority of AMY fragments are localized at peaks 5 and 6 whilst GLL1 are at peaks 3 and 4, suggesting different fragmentation had occurred. Thus, the RP-HPLC profiles of AMY and GLL1 indicated successful separation of the two enzymes.
4.4. Analysis of starch hydrolysis products to discriminate AMY and GLL1
Glucoamylase hydrolyzes starch at the non-reducing end of amylose or amylopectin to result in glucose therefore the enzyme activity assay is normally based on the release of reducing sugar [21, 29]. Unfortunately, α-amylase random digestion of starch may also result in reducing sugar i.e. maltose  therefore detection of glucoamylase activity in an α-amylase preparation can be anticipated. Glucoamylase can also, at lesser extent, act on 1,6-α-glycosidic bond of amylopectin , although its efficiency diverse greatly depending on the source organisms. Hydrolysis of the 1,6-α- bond may result in a less integrated starch molecule, hampering the formation of iodine-starch complex, which is the basis of standard α-amylase activity assay . Therefore, cross-detection of the two enzyme activity assays is inevitable. This phenomenon was notorious upon separation of AMY from GLL1 at pH 5.8 , where α-amylase activity was detected in GLL1 fractions.
One approach to discriminate α-amylase and glucoamylase is
Purified GLL1 was incubated with soluble starch substrate at 37oC and samples were taken after 5, 10, 15 and 45 minutes of incubation. Each sample was then applied onto a silica gel 60 TLC plate (20 cm x 20 cm, Merck, Darmstadt, Germany) using capillary glass tube. The plate was then placed in a TLC separation chamber that had been equilibrated with the mobile phase, which was the mixture of butanol : ethanol : water (at a ratio of 5:5:3, v/v/v). The plate was retrieved from the TLC separation chamber after the mobile phase migration reached three quarters of the length of the plate and then immediately short-submersed (few seconds) in a mixture of water : methanol : sulphuric acid (at a ratio of 45:45:10, v/v/v). The plate was then heated at 120oC for 15 minutes on a hot plate for visualization of the sugars.
The TLC profile (Fig. 4) shows that the product of starch hydrolysis by GLL1 was solely monosaccharide i.e. glucose (G1). The intensity of the G1 spot was increasing from 5 to 45 minutes of incubation, indicating that glucose was produced over time. The spots at the sample application points were from the un-hydrolyzed soluble starch substrate. Apparently, most of the starch molecules were hydrolyzed within 15-45 minutes of incubation. Most importantly, no higher oligomeric sugar forms were detected, even during the first 5-10 minutes of incubation, suggesting the absence of α-amylase. This result provided the undisputed proof that GLL1 had successfully been separated from AMY despite of α-amylase activity was detected in the GLL1 fraction. Thus, the detected α-amylase activity was not originated from AMY.
4.5. Properties of AMY and GLL1
Since the purity of AMY and GLL1 was firmly established, each enzyme could now be characterized. AMY was found active at a pH range of 5.0-7.5 and a temperature range of 30-60oC, with an optimum at 5.5 and 50oC, respectively . GLL1 was also found active a pH range of 4.6-6.8 and a temperature range of 30-65oC, with an optimum at 5.6-6.4 and 50oC, respectively . These results suggest that the two enzymes are active at similar conditions. Moreover, their characters are similar to most other
Recently, the amino acid sequences of AMY (GenBank accession code HQ172905) and GLL1 (HQ415729) were successfully elucidated as derived from their chromosomal DNA [16, 33]. The amino acid sequence of AMY is similar to that of published earlier by Itoh and coworkers , having mutations at six amino acid residues (Asp>Asn153, Ile>Val159, Ser>Asn190, Ser>Xaa216, Asp>Asn239, and Ser>Thr295). The six deviating residues in AMY comprise an additional predicted glycosylation site (Asn153), which according to a structural modelling, is highly plausible because it resides in a long surface loop .
The amino acid sequence of GLL1 is similar to glucoamylases from strain HUT 7212 (GLU) and KZ (GLA) . GLU and GLA share high sequence identity , with only seven amino acid residues different. However, these seven residues are responsible for differences in their characteristics, where GLA exhibits potential thermal stability and GLU has higher affinity towards substrate. The amino acid sequence of GLL1 also differs to both GLU and GLA at precisely those particular seven residues, which four are being identical to GLU and three to GLA. Interestingly, these mutations result in GLL1 to adopt the potential thermal stability and higher affinity towards substrate. Thus, GLL1 behaves as a hybrid of GLU and GLA.
The calculated theoretical pI values  of AMY and GLL1 (based on the amino acid sequences of their mature protein sequences: AMY sequence starts at residue Glu27 of the full-length protein containing signal and pre-peptides as encoded by
The activity of AMY is diminished in the presence of ethylene diamine tetra acetate (EDTA), a chelating agent. Inactivation of α-amylase by chelating agent is well known, as the enzyme requires calcium ion for its activity and integrity . This inactivation by chelating agent was not observed with GLL1. However, the activities of both AMY and GLL1 were increased in the presence of calcium or magnesium ions (data not shown). Further, AMY demonstrated lower activity in phosphate-citrate buffer. This phenomenon may arise from the citrate, which is reported to interact with calcium ion . The activity of AMY decreased concomitantly with the increase of the citrate buffer concentration but was fully recovered upon back-titration with calcium chloride (data not shown). Based on this observation, citrate buffers [1, 5] should only be used with considerable reservation and the use of tris buffer is recommended. This recommendation is in line with the necessity to perform purification at basic pH for HIC. In addition, this finding may also explain higher glucoamylase activity detected in AMY fractions when the purification was carried out using phosphate citrate buffer, pH 5.8 . Since the buffer does not influence the glucoamylase activity as it does to AMY, the disparity between the two enzyme activities was much less pronounced in comparison to the one presented in Fig. 2.
The hydrophobicity profile of AMY and GLL1 was analyzed based on the amino acid sequence of the mature enzymes , using an online analysis service at ExPASy (http://web.expasy.org/protscale/) . Another handful online service is also available at http://www.vivo.colostate.edu/molkit/hydropathy/index.html. The window size values for the frame normalization were scanned from 3 to 21 and compared to determine the significance of the regions that represent the hydrophobic character. The profile at window frame 9 (recommended for hydrophilic protein) and 19 (for hydrophobic protein) are presented in Fig. 5. The prediction points were linearly weighted with 100% relativity at the window edges. The score for hydrophobicity is ranged from -4.5 (hydrophilic) to 4.5 (hydrophobic), where the curve above the midpoint (zero) is interpreted as regions with hydrophobic character. As shown in Fig. 5, regions with hydrophobic character in AMY are at the residues 50-75, 210-230, 310-330, 375-410, and 425-440, whilst for GLL1 are at 225-230, 375-380, 425-445, and 475-480. This
Another computational study was performed using the on-line hydrophobic cluster analysis (HCA)program . This approach has previously been done to compare the hydrophobic clusters in α-amylases . The sequence of AMY and GLL1 were submitted to the drawcha server (http://bioserv.impmc.jussieu.fr/hca-form.html) and the resulted profiles were analyzed manually following Gaboriaud
5. AMY recombinant behaviour on purification in chromatography columns
The biochemical characteristics of AMY and GLL1 are to be improved to meet specific conditions for application, such as resistance to high temperature, chemical inactivation, and proteases . This can be achieved by engineering at both gene and protein levels, which require convenient access to the genetic information and protein structure. Although the genes encoding for AMY or GLL1 are successfully elucidated, commencing an educated and directed genetic engineering entails the structure of the enzymes. The amino acid sequence of GLL1 is nearly identical to that of GLU , which its structure has been reported (in complex with amylases inhibitor acarbose, PDB accession code 2F6D). Therefore, structural study of GLL1 was performed employing the structure of GLU. The structure of
Overexpression of soluble and functional AMY in
5.1. AMY recombinant behavior upon purification in AEX columns
Likewise AMY, AMY recombinant was secreted into overproduction medium therefore it was harvested by cold centrifugation at 6000
The dialyzed AMY recombinant was then loaded onto a resource-Q AEX column (GE Healthcare Europe GmbH, Diegem, Belgium), which had been equilibrated with that respective buffer. Purification was performed in a cold cabinet (~7oC) using a fast protein liquid chromatography (FPLC) Äkta system (GE Healthcare Europe GmbH, Diegem, Belgium) and monitored on-line with the Unicorn program. The enzyme was recovered from the column upon an elution with an increasing gradient of sodium chloride 0-1 M. As the control, purified AMY was also subjected to purification with the same column.
Purification of AMY in the resource-Q column showed that minor contaminants were still present in the purified sample (Fig. 6). The major contaminant has, however, higher absorbance at λ 260 nm, suggesting that it may not be protein. This contaminant appeared yellowish in colour, which may be originated from the overproduction medium component that was co-purified in HIC, AEX, and SEC columns. Further, the elution profile of AMY recombinant was very similar to that of AMY, except for an additional large protein peak upon sample application and washing step prior to the sodium chloride salt gradient. These additional peaks unmistakably are originated from other proteins and components of overproduction medium because the AMY recombinant sample applied was not purified prior to this column. AMY recombinant was not pre-purified with the HIC because no glucoamylase was co-produced. Nevertheless, similarity of their elution profiles in the resource-Q AEX column suggests that AMY and AMY recombinant share similar surface charge distribution.
Further, AMY recombinant was subjected to purification in DEAE-52 cellulose AEX column (Whatman Nederland BV, s’Hertogenbosch, The Netherlands) following the purification of
Supernatant from cold centrifugation was mixed with DEAE-52 cellulose AEX resin that had been equilibrated with 50 mM sodium acetate buffer, pH 5.2. After 1.5 hours of incubation at 4oC, the resin was allowed to settle and the unbound proteins were carefully decanted. The resin was washed three times with and then suspended in the respective buffer. The DEAE-52 cellulose AEX suspension was poured into an empty chromatographic column and eluted with the respective buffer. The proteins were recovered from the column by a sodium chloride salt gradient 0-1 M.
Surprisingly, AMY recombinant was not bound to DEAE-52 cellulose AEX resin, as judged from both SDS PAGE analysis and enzymatic assay. Reflecting back to purification on the resource-Q AEX column at pH 8.0, AMY and AMY recombinant were eluted at 30-35% of B solution (1 M sodium chloride, thus 300-350 mM), suggesting that the enzymes were not strongly bound to the AEX matrix. Thus, to observe no interaction between AMY recombinant and AEX resin at pH 5.2 is logical, although the pH is still higher than the pI of the enzyme. However, AMY demonstrated binding to DEAE-52 cellulose matrix in that condition, suggesting the recombinant protein has different protein surface character.
5.2. AMY recombinant behavior upon purification in sugar affinity columns
Another approach to purify AMY recombinant is the use of α-, β-, or γ-cyclodextrin (CD) columns, which was reported previously for the purification of amylolytic enzymes . CD is cyclic polymer of D-glycopyranosyl that is linked by α-D-(1-4) glycosidic bond and has no non-reducing or reducing ends. The α-, β-, or γ- variant of cyclodextrin matrices differs only on the number of glucose residue that builds up the dextrin ring, being six, seven, and eight, respectively . The protein target is bound to the interior of the CD molecule
The use of CD affinity columns is lucrative because the purification can directly be carried out after the harvesting step. After cold centrifugation, the supernatant that contained AMY recombinant was directly loaded onto α-, β-, or γ- CD affinity columns, which had already been equilibrated with 10 mM sodium acetate buffer, pH 5.5. After an extensive elution with the same buffer to remove unbound proteins, the column was eluted with 1% (w/v) α-, β-, or γ- CD in 10 mM sodium acetate buffer, pH 5.5, respective to the type of the column. Samples taken during the elution were subjected to an analysis with SDS PAGE. As the control, this purification procedure was also applied to the purified AMY.
Purification of AMY on the CD columns showed an equally strong interaction with both α- and β-CD matrices variant but weakly to γ-CD, as judged from the amount of AMY eluted from the respective CD column (Fig. 7). The result is in agreement with the reported use of CD column to separate α- from β-amylase from the amylolytic complex in higher plants . Surprisingly, AMY recombinant was not bound onto any of the CD matrices. This result set further doubt on the surface character of AMY recombinant as being different to AMY.
Excessive amount of contaminants from the production medium was one of the suspects for this altered AMY recombinant behaviour upon purification with AEX DEAE-52 cellulose or CD columns, but the result from the latter challenged that possibility. Instead, different glycosylation pattern emerged as the prime suspect because glycosylation has indeed been reported as the main drawback for heterelogous expression of protein in
Overexpression of amylolytic enzymes in
6. Structure function study of AMY in the absence of the structure
The domain organization of AMY was studied by means of limited proteolysis by trypsin-TPCK, which has specific activity to cleave lysine and arginine residues. This procedure was employed to study the domain organization of bacterial cellulase . The fragments recovered from the limited proteolytic digestion of AMY were separated in a SEC column and subjected onto functional analysis .
Benefiting from the availability of amino acid sequences of AMY and GLL1, structural study
The protein surface of AMY and GLL1 is mainly composed by negatively charged amino acid residues (Fig. 8). AMY does have more hydrophobic residues but they are concentrated at the interface between A/B to C domains. Discounting these domain interface hydrophobic residues in AMY, GLL1 has more hydrophobic patches on its surface. The large hydrophobic patch on the surface of AMY (Fig. 8, top right) is interrupted by positively charged residues (lysine) and putative additional glycosylation site, which increases the overall hydrophilicity of that hydrophobic patch. The surface representation suggests that the surface profile of GLL1 is more hydrophobic, thus the
6.1. Proteolytic digestion of AMY
Purified AMY was incubated with trypsin treated with tosyl phenylalanyl chloromethyl ketone (TPCK) for 72 hours at 37oC at a substrate to protease mol ratio of 15:1. The reaction was carried out in 25 mM Tris-HCl buffer, pH 8.3 containing 20 mM calcium chloride. The reaction mixture was then transferred to -20oC for storage prior to further analysis in SDS PAGE analysis, or immediately applied to a sephadex G50 SEC column for the enzyme’s domain separation .
Digestion of AMY by trypsin-TPCK resulted in two fragments with molecular masses of ~39 kDa (f39) and ~10 kDa (f10) , as judged from an SDS PAGE analysis. Based on the size of the fragments and proteolytic cleavage prediction according to its amino acid sequence, the f39 is designated as the N-terminal domain whilst f10 as the C-terminal. α-Amylases structure comprises of an (α/β)8-TIM barrel structural motif that is built up from the N-terminal part (domain A and B) and of the C-terminal part (domain C) . These two major domains are linked by a long surface loop. The integrated domain A/B is assigned as the catalytic domain whilst domain C is postulated as the starch-binding domain. As the two major domains of AMY were apparently separated upon proteolytic digestion, the functioning of f39 and f10 were evaluated.
6.2. Separation of f39 and f10 in an SEC column
The separation of f39 and f10 was performed in a Sephadex G50 SEC column (1.3 x 50 cm, bed volume ~48 ml) with gravity flow, in 20 mM phosphate citrate buffer, pH 5.8. Fractions of 5 ml were collected and the protein elution profile was measured by absorbance at λ 280 nm (UV-160, Shimadzu Corp., Tokyo, Japan). Only the collected protein absorption peak fractions were used for further analysis. As the control, purified AMY was also applied to the column and eluted with the same conditions for the proteolytically digested sample. The amount of AMY applied was also kept similar to that of used in the proteolytic reaction for fair comparison.
Two distinct protein peaks were recovered from the proteolytic digestion reaction mixture (Fig. 9) as oppose to one peak from the purified AMY. The use of SEC column at a 50 kDa cut off allowed a clear separation because AMY was eluted right at the end of the void volume retention whilst digested AMY was eluted after the void volume. Trypsin (~23 kDa) was not detected because its amount was very small (out numbered by f39 and f10, having an AMY to trypsin mass ratio of 34:1). Should trypsin be detected, it may contribute to a small increase of absorbance at fraction 7 of the digested AMY. Fractions 3 (of AMY), 4 and 8 (of f39 and f10, respectively) were selected for further analysis.
Only AMY and f39 demonstrated α-amylase activity, confirming the assignment of f39 function as the catalytic domain. However, the KM value of f39 suggested lower affinity towards starch substrate. Interestingly, lower f39 KM value was not followed by the decrease of the
Further, AMY was pre-treated under various conditions that resulted in denatured and partially denatured enzymes prior to proteolysis. Similar experiments were also carried out using a chemically modified AMY . The results were employed to assess the domain organization and assignment of AMY as well as to predict the precise location of trypsin cleavage and the nature of the catalytic domain. These results are being reported elsewhere .
Amylolytic enzymes from
This paper is dedicated to Prof. Oei Ban Liang (1930-2010), the founder of the Inter-University Centre for Biotechnology, Bandung Institute of Technology, where most of the works presented in this chapter were carried out.
The work has been supported by Indonesian Ministry of Research and Technology (RUT 1993-1996 to S.S), Indonesian Ministry of National Education (HB 2000-2003 to W.T.I), Royal Netherlands Academy of Arts and Science (KNAW SPIN mobility program 2004 to S.S), Indonesian Toray Science Foundation (STRG 2006 to K.H.), and Institut Teknologi Bandung (RU-ITB 2007-present to D.N.). We thank Prof. J.J. Beintema (Rijks
AEX, anion exchange chromatography; AMY, α-amylase from
Study on amyloglucosidase of a newly isolated Saccharomycopsis sp. TJ-1 from the Indonesian fermented food (tape). Annal Bogor. Sukara E. Kumagai H. Yamamoto K. 1998 5 2 77 83
Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol Adv. Zhenming C. Chi Z. Liu G. Wang F. Ju L. Zhang T. 2009 27 423 431
Kennedy JF, Cabalda VM, White CA.Enzymic starch utilization and genetic engineering. Trends Biotech. 1988 6 8 184 189
Amylolytic enzymes produced by the yeast Saccharomycopsis fibuligera. Biologia. Hostinova E. 2002 57 11 247 251
Proteolysis of alpha-amylase from Saccharomycopsis fibuligera: characterization of digestion products. Biologia. Hasan K. Ismaya W. T. Kardi I. Andiyana Y. Kusumawidjaya S. Ishmayana S. et al. 2008 63 6 1044 1050
Rodriguez-Sanoja R. Oviedo N. Sanchez S. Microbial starch-binding. domain Curr. Opin Microbiol. 2005 8 260 267
Starch-binding domains in the post-genome era. Cell Mol Life Sci. Mahovic M. Janecek S. 2006 63 2710 2724
Properties and applications of starch-converting enzymes of the alpha-amylase family. J Biotechnol. van der Maarel M. J. E. C. van der Veen B. Uitdehaag J. C. M. Leemhuis H. Dijkhuizen L. 2002 94 137 155
Nielsen JE, Borchert TV.Protein engineering of bacterial alpha-amylase. Biochim Biophys Acta. 2000 1543 253 274
Application of enzymes for textile fibres processing. Biocatal Biotransform. Araujo R. Casal M. Cavaco-Paulo A. 2008 26 5 332 349
Direct production of ethanol from raw starch corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase. Appl Environ Microbiol. Shigechi H. Koh J. Fujita Y. Matsumoto T. Bito Y. Ueda M. et al. 2004 70 5037 5040
Recent advances in microbial raw starch degrading enzymes. Appl Biochem Biotechnol. Sun H. Zhao P. Ge X. Xia Y. Hao Z. Liu J. et al. 2010 160 4 988 1003
Process Biochem. Mc Cue P. P. Shetty K. A. role for. amylase peroxidase-linked polymerization. in phenolic. antioxidant mobilization. in dark-germination. soybean implications for. health 2004 39 1785 1791
Kumar P. Satyanarayana T. Microbial glucoamylases. characteristics applications Crit. Rev Biotechnol. 2009 29 3 225 255
Amylolytic bacterial lactic acid fermentation- A review. Biotechnol Adv. Reddy G. Altaf M. Naveena B. J. Venkateshwar M. Kumar E. V. 2008 26 1 22 34
Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64. Biologia. Natalia D. Vidilaseris K. Satrimafitrah P. Purkan Ismaya. W. T. Permentier H. et al. 2011 66 1 27 32
Glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form. J Mol Biol. Lawson C. L. van Montfort R. Strokopytov B. Rozeboom H. J. Kalk K. H. de Vries G. E. et al. 1994 236 590 600
Chromatographic media for bioseparation. J Chromatogr A. Alois J. 2005 1065 1 3 12
Hydrophobic chromatography on homologous series of alkylagaroses : A comparison of charged and electrically neutral column materials. J Chromatogr A. Halperin G. Breitenbach M. Tauber-Finkelstein M. Shaltiel S. 1981 215 0 211 228
J Biochem (Tokyo). Fuwa H. A. new method. for microdetermination. of amylase. activity by. the use. of amylose. as the substrate. 1954 41 583 603
Notes on sugar determination. J Biol Chem. Somogyi M. 1952 195 19 23
Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera. Int J Biochem. Gasperik J. Kovac L. Minarikova O. 1991 23 1 21 25
Hydrophobic interaction chromatography of proteins and peptides on spheron Strop P. Mikes F. Chytilova Z. 300J Chromatogr A. 1978
Webster J. Oxley D. Peptide Mass. Fingerprinting Chemical. genomics reviews. protocols Heidelberg. Germany Springer. 2005 227 240
Fresen J Anal Chem. Rittinghaus K. Franzen K. H. H. P. L. C. in protein. analysis an. alternative to. gel-filtration gel-electrophoresis 1980 301 2 144 144
Demonstration by mass spectrometry that pseudo-hevein and hevein have ragged C-terminal sequences. Biochim Biophys Acta- Prot Struct Mol Enzymol. Soedjanaatmadja U. M. S. Hofsteenge J. Jeronimus-Stratingh C. M. Bruins A. P. Beintema J. J. 1994 1209 1 144 148
Clin Biochem. Walter A. Chymotrypsin Molecular. catalytic properties. 1986 19 6 317 322
Tarr GE, Crabb JW.Reverse-Phase High-Performance Liquid Chromatography of hydrophobic proteins and fragments thereof. Anal Biochem. 1983 131 99 107
Colorimetric quantification of carbohydrate. Curr Protoc Food Anal Chem. Fourier E. 2001E1.1. 3E.1.1.4.
Ramesh MV, Lonsane BK.End product profiles of starch hydrolysis by bacterial alpha-amylases at different temperature and pH values. Biotech Lett. 1989 11 9 649 652
Biotechnol Lett. Joutsjoki V. V. Parkkinen E. E. M. Torkkeli T. K. A. novel glucoamylase. preparation for. grain mash. saccharification 1993 15 3 277 282
Saccharification of cassava starch by Saccharomycopsis fibuligera YCY1 isolated from Loog-Pang (rice cake starter). Songklanakarin J Sci Technol. Saelim K. Dissara-Kittikun Y. H. A. 2008supp. 1)): 65 EOF 71 EOF
Chemical modification of Saccharomycopsis fibuligera R64 alpha-amylase to improve its stability against thermal, chelator, and proteolytic inactivation. Appl Biochem Biotech. submitted. Ismaya W. T. Hasan K. Kardi I. Zainuri A. Rahmawaty R. I. Permanahadi S. et al.
Nucleotide-sequence of the alpha-amylase gene (Alp1) in the yeast Saccharomycopsis fibuligera. FEBS Lett. Itoh T. Yamashita I. Fukui S. 1987 219 2 339 342
Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. The proteomics protocols handbook. New York: Humana Press; Gasteiger E. Hoogland C. Gattiker A. Duvaud S. Wilkins M. R. Appel R. D. et al. 2005 571 607
Alpha-amylases and approaches leading to their enhanced stability. FEBS Lett. Janecek S. Balaz S. 1992 304 1 3
Davies CW, Hoyle BE. 842.The Interaction of calcium ions with some phosphate and citrate buffers. J Chem Soc. 1953 1953 4134 4136
J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. Kyte J. Doolittle R. F. A. simple method. for displaying. the hydropathic. character of. a. protein 1982 157 1 105 132
Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives. Cell Mol Life Sci. Callebaut I. Labesse G. Durand P. Poupon A. Canard L. Chomilier J. et al. 1997 53 621 645
Hydrophobic cluster analysis of the primary sequences of α-amylases. Int J Biol Macromol. Raimbaud E. Bullion A. Perez S. Henrissat B. 1989August): 217 225
FEBS Lett. Gaboriaud C. Bissery V. Benchetrit T. Mornon J. P. Hydrophobic cluster. analysis an. efficient new. way to. compare analyse amino. acid sequences. F. E. B. 1987 224 1 149 155
Ulmer KM. Protein Engineering. Science. 1983 219 4585 666 671
Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain. FEBS J. Ševčík J. Hostinová E. Solovicová A. Gašperík J. Dauter Z. Wilson K. S. 2006 273 10 2161 2171
Roles of the aromatic residues conserved in the active center of Saccharomycopsis fibuligera alpha-amylase for transglycosylation and hydrolysis activity. Biochem. Matsui I. Yoneda S. Ishikawa K. Miyairi S. Fukui S. Umeyama H. et al. 1994 33 189 196
Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem. Sahdev S. Khattar S. Saini K. 2008 307 1 249 264
The enzymatic and molecular characteristics of Saccharomycopsisα-amylase secreted from Saccharomyces cerevisiae. Agric Biol Chem. Matsui I. Matsui E. Ishikawa K. Miyairi S. Honda K. 1990 54 8 2009 2015
Improving protein crystallizability by modifications and engineering. In: Bergfors TE, editor. Protein Crystallization. 2nd ed. La Jolla, CA, USA: International University Line; Qiu X. CA Janson 2009p.
Romanos MA, Scorer CA, Clare JJ.Foreign gene expression in yeast: a review. Yeast. 1992 8 423 488
Cloning and expression of the Saccharomycopsis fibuligera glucoamylase gene in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. Yamashita I. Itoh T. Fukui S. 1985 23 2 130 133
Cloning and expression of the Saccharomycopsis fibuligera alpha-amylase gene in Saccharomyces cerevisiae. Agric Biol Chem. Yamashita I. Itoh T. Fukui S. 1985 10 3089 3091
Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the α-amylase and glucoamylase genes from lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol Bioeng. Eksteen J. M. van Rensburg P. Cordero Otero. R. R. Pretorius I. S. 2003 84 6 639 646
Affinity purification of amylases on cyclodextrin-sepharose columns. Stärke. Subbaramiah K. Sharma R. 1989 41 9 357 359
Hamilton LM, Kelly CT, Fogarty WM. Review: cyclodextrins and their interaction with amylolytic enzymes.Enzyme Microb Tech. 2000 26 8 561 567
Sauer Jr Sigurskjold. B. W. Christensen U. Frandsen T. P. Mirgorodskaya E. Harrison M. et al. Glucoamylase structure/function. relationships protein engineering. Biochim Biophys. Acta-Prot Struct. Mol Enzymol. 2000 1543 2 275 293
Gilkes NR, Kilburn DG, Miller RC, Warren RAJ.Structural and functional-analysis of a bacterial cellulase by proteolysis. J Biol Chem. 1989 264 30 17802 17808
Nuc Acids Res. Goujon M. Mc William H. Li W. Valentin F. Squizzato S. Paern J. et al. A. new bioinformatics. analysis tools. framework-E at. E. M. B. L. B. I. 2010W 695 9
Bioinformatics. Arnold K. Bordoli L. Kopp J. Schwede T. The-M S. W. I. S. S. workspace O. D. E. L. web-based a. environment for. protein structure. homology modelling. 2006 22 195 201
Chen VB, Arendall III WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: all-atom structure validation for macromolecular crystallography.Acta Crystallogr D. 2010 66 12 21
Features and development of Coot. Acta Crystallogr D. Emsley P. Lohkamp B. Scott W. Cowtan K. 2010 66 486 501
DeLano WL. The PyMOL molecular graphics system, Delano Scientific LLC, Palo Alto, CA-USA. 2008
Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup, execution, and analysis of Poisson-Boltzmann electrostatics calculations.Nuc Acids Res. 2004W 665W667.
Proc Natl Acad Sci USA. Baker N. A. Sept D. Joseph S. MJ Holst Mc Cammon. J. A. Electrostatics of. nanosystems application. to microtubules. the ribosome. 2001 98 10037 10041
The evolution of starch binding domain. FEBS Lett. Janecek S. Sevcik J. 1999 456 119 125
Raw-starch digesting and thermostable alpha-amylase from yeast Cryptococcus sp. S2: purification, characterization, cloning, and sequencing. Biochem J. Iefuji H. Chino M. Kato M. Limura Y. 1996 318 989 996
Prot J. Hostinová E. Janeček Š. Gašperík J. Gene sequence. bioinformatics enzymatic characterization. of α-amylase. from Saccharomycopsis. fibuligera K. Z. 2010 29 5 355 364