starch is a major storage product of   many economically important crops such as wheat , rice , cassava , and potato .
a large variety of microorganisms employ extracellular or intracellular enzymes to hydrolyze starch thus enabling its utilization as a source of energy .
one of the most important groups of enzymes that process starch is represented by the -amylase family or family 13 glycosyl hydrolases [ 2 , 3 ] .
amylases ( ec 3.2.1.1 , -1,4-glucan - glucanohydrolase ) are enzymes that hydrolyze starch polymers yielding diverse products including dextrins and smaller polymers of glucose .
these enzymes are of great biotechnological interest with many applications ranging from food industry to the production of biofuels .
since each different application requires amylases with unique properties it is often necessary to search the biodiversity for new sources of these enzymes .
several amylases isolated from yeasts such as candida antarctica , candida japonica ,   lipomyces kononenkoae , saccharomycopsis fibuligera , schwanniomyces alluvius , trichosporon pullulans ,   and filobasidium capsuligenum    have been described .
we have previously reported the characterization of an -amylase ( amy1 ) from the basidiomycetous yeast cryptococcus flavus which exhibited important biochemical properties for its industrial utilization such as high stability at ph 5.5 and optimal temperature at 50c . the gene encoding this -amylase ( amy1 ) was cloned and successfully expressed in saccharomyces cerevisiae . in order to assess the use of s. cerevisiae as a host for the heterologous production of amy1 we sought the purification and enzymatic characterization of recombinant enzyme produced in this system .

 s. cerevisiae cenpk2 ( mata/ura3 - 52/ura3 - 52 leu 2 - 3,112 trp1 - 289/trp1 - 289 his3 - 1/his3 - 1 ) .
a colony of s. cerevisiae cenpk2 harboring yepamy was cultured in sd medium ( 0,62% ynb , 2% glucose , uracil , tryptophan , and histidine ) , and 2  ml of this preculture was transferred to 1  l conical flask containing 200  ml of the same medium following incubation at 28c for 60  h ( 200  rpm ) .
s. cerevisiae cells were harvested by centrifugation ( 5,000  g/10  min ) , and the supernatant was dialyzed overnight against water and loaded on a q - sepharose fast flow column ( 0.9  18  cm ) previously equilibrated in 50  mm sodium acetate ( ph 5.5 ) .
the column was washed with the same buffer , and proteins were eluted by a linear gradient of 00.5  m nacl .
four fractions of 3  ml were collected and monitored for the presence of proteins and amylase activity .
these samples were collected , dialyzed , lyophilized , and resuspended in 1  ml of water and loaded on a sephacryl s-200 hr ( 2.5  85  cm ) column previously equilibrated with 50  mm sodium acetate containing 0.1  m nacl .
the column was washed with the same buffer at a flow rate of 12  mlh .
fractions containing amylase activity were pooled , dialyzed against water , lyophilized , and stored   at   20c . in chromatography experiments , the protein content of each fraction
protein integrity and the molecular mass calculation were performed by running samples on 12% sodium dodecyl sulfate - polyacrilymide gels .
molecular mass markers ( fermentas life sciences ) were as follows : -galactosidase ( 116  kda ) , bovine serum albumin ( 66.2  kda ) , ovalbumin ( 45  kda ) , lactate dehydrogenase ( 35  kda ) , rease bsp98i ( 25  kda ) , -lactoglobulin ( 18.4  kda ) , and lysozyme ( 14.4  kda ) by manufacturer .
gels were washed with distilled water , incubated with 50  mm sodium acetate ( ph 5.5 ) for 60  min , and then incubated at 4c for 12  h in a solution containing 0.5% of starch ( in 50  mm sodium acetate buffer [ ph 5.5 ] ) .
the gel was then incubated at 37c for 2  h , and bands with amylase activity were detected after staining with iodine solution ( 1  mm i2 in 0.5  m ki ) . 

-amylase dextrinizing activity was assayed in a reaction system containing 100  l 0.5% ( w / v ) soluble starch , 40  l 0.5 m acetate buffer ( ph 5.5 ) , enzyme solution ( 060  l ) , and water to a total volume of 200  l .
after 10  min at 60c , the reaction was stopped with 200  l 1.0  m acetic acid .
saccharifying activity was determined by measuring the production of reducing sugars from starch by using the dinitrosalicylic acid ( dns ) method .
one unit of dextrinizing activity was defined as the amount of enzyme necessary to hydrolyse 0.1  mg starch / min . one unit of saccharifying activity was defined as the amount of enzyme necessary to produce 1  mg glucose equivalent / min .
the optimum ph of the recombinant amylase was determined by varying the ph of the reaction mixtures using the following buffers ( 50  mm ) : glycine - hcl ( ph 1.03.0 ) , sodium acetate ( ph 4.05.5 ) , sodium phosphate ( ph 6.0 - 7.0 ) , and tris - hcl ( ph 8.010 ) at 60c . in order to determine ph stability ,
the residual enzyme activity was assayed in 50  mm sodium acetate buffer ( ph 5.5 ) .
the optimum estimated temperature of the enzyme was evaluated by measuring amylase activity at different temperatures ( 30c to 70c ) in 50  mm sodium acetate ( ph 5.5 ) .
the effect of temperature on enzyme stability was determined by measuring the residual activity after 15 , 30 , 45 , and 60  min of preincubation in 50  mm sodium acetate ( ph 5.5 ) at 55 and 60c . in order to determine the effect of metal ions ,
the assay was performed after preincubation of the enzyme with various metal ions at a final concentration of 4  mm .
the effect of 10  mm cacl2 and dtt ( 510  mm ) was also evaluated .
km values for the purified enzyme were determined by incubating the enzyme with 00.8  mgml soluble starch in 50  mm sodium acetate ( ph 5.5 ) at 60c .
the data obtained were fitted to a standard lineweaver - burk model using linear least squares regression .
briefly , purified amylase was incubated with 0.5% starch , glycogen , pullulan , amylase , and amylopectin , in 50  mm sodium acetate ( ph 5.5 ) at 40c .
aliquots ( 10  l ) were removed after incubation for 6 and 12  h. the chromatogram was developed with n - butanol / methanol / h2o ( 4  :  2  :  1 ) . sugars were detected by thin - layer chromatography .
circular dichroism ( cd ) assays were carried out using jasco j-815 spectropolarimeter ( jasco , tokyo , japan ) equipped with a peltier type temperature cuvette holder .
proteins ( 0.085 to 0.100  mg / ml ) were analyzed in 2  mm glycine ph 3.0 and 4.0 , 2  mm sodium acetate ph 5.5 , 2  mm tris - hcl ph 7.0 and 8.0 .
we used cd data over the wavelength range 260200  nm to ensure a satisfactory cd signal and to prevent the high signal - noise ratio .
four consecutive measurements were accumulated , and the mean spectra were corrected for the baseline contribution of the buffer .
the observed ellipticities were converted into molar ellipticity ( [  ] ) based on molecular mass per residue of 115  da .
protein structure and thermal unfolding curves were tracked by changes in [  ] at 222 or 206  nm .
the temperature dependence of the secondary structure was estimated from the far - uv cd curves adjustments using the cdnn deconvolution software ( version 2.1 , bioinformatik.biochemtech.uni-halle.de/cdnn ) .
amy1 was purified from the supernatant of s. cerevisiae cultures in a two - step chromatographic procedure .
elution profiles of both q - sepharose and sephacryl - s200hr chromatography showed one peak with amylase activity ( data not shown ) .
the enzyme was purified to homogeneity with 3.79-fold increase in specific activity with a yield of   ~10.3% as compared to the supernatant ( table 1 ) . comparing with the recombinant enzyme ,
wild - type amy1 was purified from c. flavus cultures in only a single purification step .
sds - page analysis of the purified recombinant amylase showed a single protein band corresponding to ~67  kda ( figure 1(a ) ) which showed -amylase activity after zymogram analyses ( figure 1(b ) ) . among the amylase - producing yeasts and filamentous fungi , the -amylases from cryptococcus sp s-2 , aspergillus fumigatus , lipomyces starkeyi , and lipomyces kononenkoae   showed similar molecular masses as amy1 produced in s. cerevisiae .
the optimum ph for the recombinant enzyme was determined as ph 5.5 ( figure 2(a ) ) .
amy1 was tested for ph stability by preincubating the purified enzyme in appropriate buffers for 1 hour .
the data suggest that amy1 is tolerant to wide ph range ( figure 2(b ) ) .
this result was similar to that found for -amylases from a variety of yeast strains [ 20 , 21 ] .
the optimum ph for yeast -amylase activity is usually in the range of 4.0 and 6.0 [ 5 , 7 , 16 , 21 ] .  
amylase activity measured as a function of temperature from 30 to 70c shows that the activity was the highest at 60c ( figure 2(c ) ) .
this optimum temperature is in agreement with the -amylases from cryptococcus sp s-2 treated with 1  mm cacl2   and aureobasidium pullulans n13d .
thermostability is considered an important and useful criterion for industrial application of -amylase from microorganisms . as shown in figure 2(d ) , the residual amylase activity is practically constant at 55c after 1  h of incubation . however , at 60c 50% of the residual activity was lost .
proposed that the c - terminal raw starch binding motifs present in cryptococcus sp s-2 amylase ( amy - cs2 ) might be related to the thermostability presented by this amylase . because amy1 has 97% sequence identity with amy - cs2   it is possible that the same explanation applies to amy1 .  
the results   presented on table 2 indicate that recombinant amy1 is poorly affected by most of the 4 mm ions tested ( mg , mn , and ca )   since the relative activity was higher than 70% . in addition , it was also found that in the presence of 10  mm cacl2 , amy1 still maintained its original activity ( data not shown ) which shows that this enzyme is not affected by ca .
however , takeuchi et al .   related that the pichia burtonii-amylase was activated by ca ( 135% ) .
these authors proposed that the activation of enzyme by calcium ions probably happened during the purification procedure when the enzyme lost activity .
the bacillus halodurans 38c-21 -amylase also had its activity increased in presence of ca   .
the presence of nh4 ions had the most prominent activating effect ( 111% ) on recombinant amy1 .
however , zn , cu , and hg acted as inhibitors of amylase activity , with cu   and hg showing a complete inhibition ( table 2 ) .
the inhibition by hg    may indicate the importance of indole group of amino acid residues in enzyme function [ 4 , 24 ] .
the wild - type amylase is inhibited by hg , ag , cu , and mg    while that from l. kononenkoae cbs 5608 is inhibited by ag   and cu   .
interestingly , the amylase from yeast aureobasidium pullulans n13d is not inhibited by cu which had an activating effect on the purified enzyme .
the enzyme 's activity was practically constant when dtt ( 425  mm ) was used ( data not shown ) .
this result was different from that observed with the thermotoga maritima msb8 amylase expressed in e. coli which showed increased activity in presence of ddt ranging from 5 to 10  mm .
reported that the km for wild - type amy1 was 0.056  mgml which is considerably smaller than the km observed for the recombinant amylase ( 0.37  mg  ml ) which showed michaelis - menten type kinetics with soluble starch as substrate .
the km of the recombinant enzyme is similar to other yeast amylases such as schwanniomyces alluvius   and thermomonospora curvata .   in order to establish the specificity of recombinant amy1 ,
the enzyme was incubated with starch , amylopectin , amylose , glycogen ,   and pullulan .
these substrates consist of different glucose polymers containing -1,4 linkages or a mixture of -1,4- and -1,6-glucosidic linkages .
amy1 showed the   highest activity towards soluble starch ( 100% ) amylopectin ( 97.9% ) , amylose ( 50.6% ) but no activity was observed on pullulan .
this result is similar to that observed for the bacillus subtilis truncated -amylase produced in e. coli , but different from that observed for wild - type amy1 which showed the highest activity towards starch ( 100% ) , glycogen ( 18.9% ) , amylopectin ( 16.7% ) , and amylose ( 7.1% ) .
the recombinant enzyme displayed typical -amylase properties based on the evidence that oligosaccharides from several sizes were the main products from the digestion of glycogen , amylose , amylopectin , and starch ( figure 3 ) .  
structural characterization of   recombinant ( amy1 ) and wild - type ( amyw ) amylase from c. flavus was carried out by circular dichroism spectroscopy for comparison .
far - uv cd spectra of amy1 and amy1w at ph 7.0 and 25c are quite different showing typical features of beta / unordered and beta/-helix secondary structure pattern , respectively ( figure 4(a ) ) .
while the 222  nm dichroic band is predominantly associated with -helical structure , the dichroic band at 198 and 206  nm may arise from changes in other secondary structure elements in the protein , as unordered or beta structure .
the secondary structure content of both proteins slightly differs on helical ( amy1 , 5.7% and amy1w , 9.5% ) and antiparallel -sheet ( amy1 , 42.1% and amy1w , 32.4% ) and is similar for other secondary structures .  
thermal stability of the proteins at ph 7.0 was assessed upon raising the temperature from 20 to 95c ( figures 4(b ) and 4(d ) ) .
the thermal denaturation curves analyzed from far - uv cd measurements at 206 and 222  nm at ph 7.0 strongly indicated the amy1w   and amy1 as thermally stable proteins since no pattern of protein denaturation could be verified ( data not shown ) . indeed , although the cd spectra of the amy1w in this condition show a gradual increase ( downward ) of the dichroic bands at 206  nm and slight at 222  nm ( figure 4(d ) ) , no secondary structure changes were detected suggesting only    conformational changes of protein or secondary structure rearrangement as a function of temperature .
in contrast , cd spectra of the amy1 show a gradual increase and decrease of the dichroic bands at 222 and 200  nm , respectively , suggesting the alteration of helical and -sheet structures content ( figure 4(b ) ) .
a slight increase was observed in terms of -helix ( from 5.7% to 6.8% ) and decrease of -sheet ( from 42.1% to 38.8% ) when raising the temperature from 25 to 95c .
altogether , these results revealed the high thermal stability of both proteins and the difference in folding pattern of amy1 compared with wild - type amylase from c. flavus reflecting in decrease ( 10-fold ) of enzymatic activity ( km values ) of recombinant protein using a soluble starch as substrate , as discussed above .   besides significant distinction in ph region where the maximum enzymatic activity of amy1 occurred ( figure 2(a ) ) , the protein achieved similar secondary structure content ( helical 5.7%6.5% and -sheet ~42% ) in a broad range of ph ( ph 3.0 , 5.5 , and 7.0 ) , as indicated by the cd spectra ( figure 4(c ) ) and cdnn estimative . at ph 8.0 as well as ph 4.0 ,
it was verified the mainly increase in the cd signal at 200  nm ( figure 4(c ) ) , compatible with minor -sheet structures content ( ~37% ) compared with ph 3.0 , 5.5 , and 7.0 ( ~42% ) .
noteworthy , although no secondary structure changes have been observed , analogous or different conformation of amy1 side chains could   occur   as a function of those phs , as judged by the relative activity of the enzyme in these condition ( figures 2(a ) and 2(b ) ) . in these cases ,
some important structural changes related with side chains ionization of aspartate , glutamate , and histidine residues at ph 4.0 and 5.5 could occur and are fundamental to enzyme activity . in this work
we have observed some significant biochemical and slight structural discrepancies between wild - type and recombinant amy1 especially with respect to km values , enzyme specificity , and helical and -sheet structure content .
these could be explained by small folding alterations in the structure of recombinant amylase during its expression .
different patterns of protein glycosylation between c. flavus and s. cerevisiae could also account for these differences .
work is under way to assess the role of glycosylation on the activity of amy1 .
because recombinant amy1 showed considerable thermostability ,   its use in biotechnological processes should be considered .
in conclusion , the enzymatic and structural features of amy1 , the highest activity towards soluble starch , amylopectin , and amylase , in contrast with the lowest activity of amy1w , points out this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels .
 an extracellular alpha - amylase ( amy1 ) whose gene from cryptococcus flavus was previously expressed in saccharomyces cerevisiae was purified to homogeneity ( 67  kda ) by ion - exchange and molecular exclusion chromatography . 
 the enzyme was activated by    nh4 + and inhibited by   cu+2 and hg+2 . 
 significant biochemical and structural discrepancies between wild - type and recombinant -amylase with respect to km values , enzyme specificity , and secondary structure content were found . 
 far - uv cd spectra analysis at ph 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of amy1 compared with wild - type amylase from c. flavus , which reflected in decrease ( 10-fold ) of enzymatic activity of recombinant protein . despite the differences , the highest activity of amy1 towards soluble starch , amylopectin , and amylase , in contrast with the lowest activity of amy1w , points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels . 
1. Introduction
2. Material and Methods
3. Results and Discussion
4. Conclusion