fish viscera are one of the sources of digestive enzymes that may have some unique properties of fascinate with both basic research and industrial applications . their survival in waters required adaptation of their enzyme activity to low temperatures of their habitats . that is to say , fish proteinases have higher catalytic efficiency at low temperatures than those from warm - blooded animals [ 1 , 2 ] . in addition , the strong positive correlation between the habitat temperature of marine fish and the thermostability of its trypsin has been demonstrated [ 311 ] . high activity at low temperatures and instability against heat , low ph , and autolysis of fish proteinases are interesting for some industrial applications . cod trypsin is already practically used in food production and cosmetics [ 13 , 14 ] . furthermore , pacific cod and atlantic cod trypsins were utilized as catalyst of enzymatic peptide synthesis [ 9 , 15 ] . on the other hand , lipids in the tissue conventional methods for the removal of lipids from materials involve cooking , pressing , and liquid extraction .   on liquid extraction for enzyme preparation , it is usually used with organic solvents , such as hexane , ethanol , and acetone , and so forth [ 16 , 17 ] . however , the removal of lipids with organic solvents causes protein denaturation and/or loss of functional properties . organic solvents are also highly flammable and are toxic for human health .   consideration of such factors has led investigators to apply supercritical fluid extraction techniques to the lipid separation .   carbon dioxide ( co2 ) is a popular supercritical extractant particularly in food processing , flavor and aroma isolation , and pharmaceuticals manufacture , because co2 is nontoxic and does not leave a residue . supercritical co2 ( sco2 ) has been used for extraction of oils from some marine organisms [ 2022 ] . but , the aims of these studies were mainly the gain of oils rich in polyunsaturated fatty acids , especially epa and dha . so , the application of sco2 for isolation of enzymes and production of quality protein meal from different sources should be examined . recently , we prepared a defatted powder of squid viscera treating with sco2 and detected protease , lipase , and amylase activities in crude extract from the powder . next , we purified a phospholipase a2 from the starfish pyloric ceca defatted by sco2 extraction process .   in this study , with the aim of utilization of fish trypsin for food industry , we purified a trypsin ( sc - t ) from the mackerel viscera powder treated by sco2 defatting process and compared its enzymatic properties with those of other fish trypsins purified from the viscera defatted by acetone . mackerel ( scomber sp . ) were caught off busan , republic of korea . the mackerel viscera were collected from f & f co. , busan , republic of korea , and the visceral waste was brought to the laboratory in iced condition .   the co2 ( 99.99% pure ) was supplied by kosem , korea . n - p - tosyl - l - arginine methyl ester hydrochloride ( tame ) and ethylenediaminetetraacetic acid ( edta ) were obtained from wako pure chemicals ( osaka , japan ) . 1-(l - trans - epoxysuccinyl - leucylamino)-4-guanidinobutane ( e-64 ) , soybean trypsin inhibitor , n - p - tosyl - l - lysine chloromethyl ketone ( tlck ) , and pepstatin a were purchased from sigma chemical co. ( st . louis , mo , usa ) . the defatted powder of mackerel viscera was prepared as described by chun et al . the lipid extraction by sco2 was performed at temperature of 45c and pressure of 25  mpa . the total extraction time was 2.5  h.   the sco2 defatted powder was stored at 60c until further analysis . trypsin was extracted by stirring from 10.0  g of defatted powder in 50 volumes of 10  mm tris - hcl buffer ( ph 8.0 ) containing 1  mm cacl2 at 5c for 3  h.   the extract was centrifuged ( h-200 , kokusan , tokyo , japan ) at 10,000  xg for 10  min , and then the supernatant was concentrated by lyophilization and used as crude trypsin ( 50  ml ) . ten milliliters of crude trypsin was applied for four times to a column of sephacryl s-200 ( 3.9    64  cm ) pre - equilibrated with 10  mm tris - hcl buffer ( ph 8.0 ) containing 1  mm cacl2,and proteins were eluted ( 0.8  ml / min ) with the same buffer . each main trypsin fractions were gathered and concentrated by lyophilization .   then the concentrated fraction ( 10  ml ) was applied to a sephadex g-50 column ( 3.9    64  cm ) pre - equilibrated with the above buffer , and proteins were eluted ( 0.7  ml / min ) with the same buffer . a single trypsin fraction was pooled and used as purified trypsin ( sc - t ) . trypsin activity was measured by the method of hummel   using tame as a substrate . one unit of enzyme activity was defined as the amount of the enzyme hydrolyzing one micromole of tame in a minute . the effect of inhibitors on trypsin was determined by incubating trypsin with an equal volume of proteinase inhibitor solution to obtain the final concentration designated ( 0.1  mm e-64 , 1  mg / ml soybean trypsin inhibitor , 5  mm tlck , 1  mm pepstatin a and 2  mm edta ) . after incubation of the mixture at 25c for 15  min , the remaining activity was measured , and percent inhibition was then calculated . the ph dependencies of trypsin were determined in 50  mm buffer solutions [ acetic acid - sodium acetate ( ph 4.07.0 ) , tris - hcl ( ph 7.09.0 ) , and glycine - naoh ( ph 9.011.0 ) ] at 30c . the temperature dependencies of trypsin were determined at ph 8.0 and at various temperatures . the temperature and ph stabilities of trypsin were found by incubating enzyme at ph 8.0 for 15  min at a range of 2080c and by incubating the enzyme at 30c for 30  min at a range of ph 4.011.0 , respectively . the effect of cacl2 on trypsin activity was found by incubating the enzyme at 30c and at ph 8.0 in the presence of 10  mm edta or 10  mm cacl2 . sds - page was carried out using a 0.1% sds-13.75% polyacrylamide slab gel by the method of laemmli . the gel was stained with 0.1% coomassie brilliant blue r-250 in 50% methanol-7% acetic acid , and the background of the gel was destained with 7% acetic acid . to analyze the n - terminal amino acid sequence of sc - t , the enzyme was electroblotted to a polyvinylidene difluoride ( pvdf ) membrane after sds - page . the amino acid sequence of the enzyme was analyzed by using a protein sequencer , procise 492 ( perkin elmer , foster city , calif , usa ) . the viscera of mackerel ( scomber sp . ) were treated by sco2 to separate lipids on the condition of 40c , 25  mpa , and 2.5  h.   since sco2 extracted almost all oil from the squid viscera in the previous study , we adopted the condition to remove lipids from the mackerel viscera . trypsin was then extracted from 10.0  g of defatted powder by sco2 , and the crude enzyme was prepared .   as shown in table 1 , the crude enzyme contained 1,390  mg of total protein and 1,049  u of total trypsin activity . previously , we extracted trypsin from the pyloric ceca powder ( 13.9  g ) of spotted mackerel defatted by acetone , and 3,633  mg of total protein and 3,270 u of total trypsin activity were detected in the crude enzyme solution . although these data were not compared directly , the yields of protein and trypsin activity per weight of acetone powder were approximately two times higher than those of sco2 powder . however , the specific activity ( 0.8  u / mg ) of crude enzyme in this study is almost the same as that ( 0.90  u / mg ) in the previous study . so , it is thought that the difference of total activity might come from the variation of specimen , and the defatting condition with sco2 in this study would not cause significant denaturation of fish trypsin . the sc - t was purified from the crude enzyme solution by two steps of chromatographies including sephacryl s-200 and sephadex g-50 , which is the same purification procedure as that in the previous study . the sc - t was consequently purified 48-fold with a high recovery ( 50% ) from the crude enzyme solution ( table 1 ) and had a specific activity of 36  u / mg which is fairly higher than that of spotted mackerel trypsin . in addition , the sc - t was found nearly homogeneous on sds - page ( figure 1 ) , and its molecular weight was estimated as approximately 24,000 , which is similar to that of spotted mackerel trypsin . furthermore , the n - terminal twenty amino acids sequence of sc - t was analyzed to be ivggyectpysqpwtvslns that accords with that of spotted mackerel trypsin . these results also show that the sco2 defatting process in this study removes lipids in fish viscera as effectively as acetone defatting process for preparation of fish trypsin . crude enzyme extract usually contains various proteins , and sometimes fish trypsin is composed of some isozymes . in general , the purification of fish trypsin was carried out by the combination of some types of chromatography [ 2830 ] .   however , we achieved a high purification of a trypsin from the mackerel viscera powder defatted by acetone using only two steps of gel filtration . so , in this study , we purified the sc - t by gel filtration according to the previous study . the sc - t was strongly inhibited by specific trypsin inhibitors ( soybean trypsin inhibitor and tlck ) , but e-64 ( cysteine proteinase inhibitor ) , pepstatin a ( aspartic proteinase inhibitor ) , and edta ( metalloproteinase inhibitor ) had no inhibitory effect on the activity of sc - t ( table 2 ) . the influence of ph on the sc - t activity is shown in figure 2(a ) . the enzyme hydrolyzed tame substrate effectively between ph 7.0 and 9.0 , with an optimum around ph 8.0 . the optimum ph of sc - t was the same as those of other fish trypsins [ 311 , 3138 ] , but lower than those of bluefish ( ph 9.5 )   and atlantic bonito ( ph 9.0 ) . the sc - t was active over a broad temperature range ( 2070c ) with the optimum at about 60c . because mackerel is a temperate - zone fish , the sc - t possesses similar optimum temperature with other trypsins from temperate - zone fish , such as anchovy , true sardine , yellow tail , and jacopever . the optimum temperature sc - t is slightly lower than those of tropical - zone fish ( around 65c ) [ 3335 , 39 , 40 ] but is evidently higher than those of frigid - zone fish trypsins ( around 50c ) [ 4 , 610 ] . the ph stability of sc - t is shown in figure 3(a ) . the sc - t was stable at 30c for 30  min in the ph range from ph 6.0 to 11.0 .   unlike mammalian trypsins , diminished stability of the trypsin was more pronounced after exposure at acidic ph . instability at acidic ph was also observed for other fish trypsins [ 1 , 311 , 3235 , 37 , 39 , 40 ] .   for temperature stability , the sc - t was stable below 40c , but the activity quickly fell over 50c ( figure 3(b ) ) .   while the sc - t and other temperate - zone fish trypsins are relatively less stable than tropical - zone fish trypsins , they are obviously stable than frigid - zone fish trypsins [ 8 , 10 ] .   as described previously , there is a strong relationship between habitat temperature of marine fish and thermostability of their trypsins [ 8 , 10 ] . the effect of calcium ion on the stability of sc - t was then investigated . the stability of sc - t was enhanced by calcium ion ( figure 4 ) . similar results have been reported for various fish trypsins [ 1 , 311 , 3335 , 39 , 40 ] . bovine trypsinogen has two calcium - binding sites , and the primary site , with a higher affinity for calcium ions , is common in trypsinogen and trypsin and the secondary site is only in the zymogen [ 41 , 42 ] . occupancy of the primary calcium - binding site stabilizes bovine trypsin toward thermal denaturation or autolysis [ 41 , 42 ] . in the previous paper , we described trypsin of arabesque greenling which also has the primary calcium - binding site . the sc - t was stabilized by calcium ion from denaturation in this study . so , the result suggests that the sc - t may possess the primary calcium - binding site like bovine and arabesque greenling trypsins . with the aim of utilization of fish trypsin for food industry , we purified a trypsin ( sc - t ) from mackerel ( scomber sp . ) viscera powder treated by the sco2 defatting process and compared its enzymatic properties with those of other fish trypsins purified from the viscera defatted by acetone .   in this study , we adopted the condition of 40c , 25  mpa , and 2.5  h to separate lipids from the viscera . consequently , we could remove most of the lipids from the viscera and could extract considerable amount of trypsin from the defatted powder . the characteristics of purified sc - t were nearly the same as those of other fish trypsins , especially spotted mackerel trypsin . therefore , we concluded that the sco2 defatting process is useful as a substitute for the organic solvent defatting process .
viscera of mackerel ( scomber sp . ) 
 were defatted by supercritical carbon dioxide ( sco2 ) treatment . 
 trypsin ( sc - t ) was then extracted from the defatted powder and purified by a series of chromatographies including sephacryl s-200 and sephadex g-50 . 
 the purified sc - t was nearly homogeneous on sds - page , and its molecular weight was estimated as approximately 24,000  da . 
 n - terminal twenty amino acids sequence of sc - t was ivggyectahsqphqvslns . 
 the specific trypsin inhibitors , soybean trypsin inhibitor and tlck , strongly inhibited the activities of sc - t . 
 the ph and temperature optimums of sc - t were at around ph 8.0 and 60c , respectively , using n-p - tosyl - l - arginine methyl ester as a substrate . 
 the sc - t was unstable below ph 5.0 and above 40c , and it was stabilized by calcium ion . 
 these enzymatic characteristics of sc - t were the same as those of other fish trypsins , especially spotted mackerel ( s. borealis ) trypsin , purified from viscera defatted by acetone . 
 therefore , we concluded that the sco2 defatting process is useful as a substitute for organic solvent defatting process .