Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater Prawn Macrobrachium rosenbergii (De Man) Postlarvae

Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL₁₅ giant freshwater prawn ( Macrobrachium rosenbergii ). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0–73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases.     

Cellulase degradation of shrimp chitosan for the preparation of a water-soluble hydrolysate with immunoactivity

ABSTRACT:   Samples of colloidal chitin and chitosans with deacetylation degrees (DD) of 50, 65, 75 and 95% (DD50, DD65, DD75 and DD95, respectively) were prepared from shrimp chitin. The hydrolytic activity of cellulase on these samples increased with the increasing DD of chitosan, with DD95 being the most easily hydrolyzed. Colloidal chitin was hardly hydrolyzed. The optimal reaction temperature and pH for cellulase digestion of chitosan were 55°C and pH 5.2, respectively. During cellulase digestion of chitosan, the 9-h hydrolysate had the highest enhancing effect on the proliferation of a human hybridoma cell, HB4C5. This hydrolysate is composed of low-molecular-weight chitosan (LMWC), with a molecular mass of 20.0 kDa, and chitooligosaccharides, which are composed of sugars with a degree of polymerization of 1–6. In vitro , the 9-h hydrolysate increased both cell proliferation and immunoglobulin (Ig)M secretion of HB4C5 because of the presence of chitooligosaccharides for the former activity and LMWC for the latter activity. In vivo , samples of the chitosan hydrolysate, chitooligosaccharide mixture and LMWC significantly increased the levels of serum IgG and IgM, and enhanced concanavalin A- and lipopolysaccharide-induced proliferation of mouse lymphocytes.     

TMAOase, trimethylamine-N-oxide demethylase, isa thermostable and active enzyme at 80°C

ABSTRACT:   Purified trimethylamine- N -oxide demethylase (TMAOase)from walleye pollack muscle is a thermostable protein that was notinactivated after heating at 80°C for 30 min.The heated enzyme was electrophoresed in the same manner as fornative enzyme. Circular dichroism (CD) spectra for purified enzymechanged reversibly in the temperature range of 10–80°C.As the enzyme was still active at 80°C, the CD spectralchange did not directly relate to enzyme activity. TMAOase activity inthe myofibrillar fraction decreased sharply above 30°C,but was extracted and recovered from the heated myofibrillar fraction,suggesting that the activity seemed to be interrupted and apparently inactivateddue to the thermal alteration of myofibrillar proteins or some unknownfactors. The complicated profile found in dimethylamine (DMA) formationfrom trimethylamine- N -oxide (TMAO) in walleye pollack muscleduring heating consisted of both enzymic and non-enzymic processes.Most DMA was produced enzymatically below 40°C and interruptedabove 40°C. Therefore, DMA and trimethylamine was formednon-enzymatically at high temperatures regardless of the presenceof native enzyme. A new, simple and easy purification method wasproposed based on the thermostable nature of the enzyme.     

Isolation and characterisation of two extracellular metalloproteases from Aeromonas salmonicida ssp. salmonicida

Two extracellular metalloproteases were purified from a culture filtrate derived from Aeromonas salmonicida ssp. salmonicida . One enzyme, leucine aminopeptidase (LAP), which had a molecular mass 37 kDa, hydrolysed aminoterminal l -leucine and l -phenylalanine. The activity was inhibited by 1,10-o-phenanthroline, but not by EDTA. The addition of excess Zn²⁺ to an o-phenanthroline-inhibited enzyme restored most of its activity. The peptidase was temperature stable, and had an optimum temperature and pH of 60 °C and 8, respectively. The other enzyme, metalloprotease 3 (MP3), which had a molecular mass 20 kDa, was an endoprotease, and hydrolysed azocoll and hide powder-azure, but not gelatine. The MP3 enzyme had an optimum temperature and pH of ≈40 °C and 7.5, respectively, and a cationic isoelectrical point.     

Characteristics of burnt meat in cultured yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis>

ABSTRACT:   In order to understand the characteristics of burnt meat in cultured yellowtail Seriola quinqueradiata , fish were kept at two different temperatures (13 and 30°C) and slaughtered by either spinal cord destruction (SCD) or suffocation in air (SA). Early postmortem changes during storage at 32°C were analyzed by rheological, biochemical, and histological methods. The burnt meat (with lightness parameter, L* ≥ 55) was observed at 1-h storage in the SA 30°C group, at 2 h in SCD 30°C, and at 4 h in SA 13°C; meat was normal for the SCD 13°C group until 6 h of storage. Breaking strength scores were higher for the normal meat (200 g/cm²) than burnt meat (70 g/cm²) at 4 h of storage. Expressible water content was higher for the burnt meat than for the normal meat. Adenosine triphosphate concentrations for the SCD groups were higher than for the SA counterparts. Moreover, pH decrease was much faster in the 30°C groups, showing pH 5.6 at 2 h of storage. A negative correlation between the pH and lactic acid contents in muscle ( P  < 0.001) was found. Histological analysis evidenced a larger pericellular area (40%) in the burnt samples than in the normal samples (16%). It was confirmed that a higher fish-keeping water temperature and a stressful slaughter method (faster glycolytic process) were determinative factors that influence the occurrence of burnt muscle in yellowtail, and that the effect of the former is larger than the latter.     

Effects of temperature, density and early weaning on the survival and growth of Atlantic ditch shrimp Palaemonetes varians larvae

This investigation examined the effects of temperature, density and early weaning on the survival and growth of Palaemonetes varians larvae. Survival of larvae raised at 17.5 °C was not significantly different (average + standard deviation) (94 ± 5%) from the survival of those raised at 19.5 °C (95 ± 5%) and at 21.5 °C (94 ± 4%). However, the duration of the larval stage was significantly longer for shrimp reared at 17.5 °C (17.3 ± 0.8 days) compared with shrimp reared at 19.5 °C (14.3 ± 0.7 days) and at 21.5 °C (11.3 ± 0.6 days). No significant differences ( P >0.05) were found in the survival rate, final weight and length of larvae reared at the densities of 5, 10, 20 and 50 larvae L⁻¹. The survival of P. varians larvae fed solely on Artemia was significantly higher ( P <0.05) than larvae weaned with an artificial practical diet from Zoea II stage (94 ± 4% and 82 ± 1%, respectively, for Artemia and artificial diet-fed larvae), but no significant differences ( P >0.05) were observed in the final larval weight or length between these two treatments. The survival and growth of the larvae fed with the practical diet tested is a promising step ahead in the development of the culture of this species as it eliminates both the need for Artemia throughout all larval stages, and the need for more expensive artificial diets.     

A comparative study of the effects of high pressure on proteolytic degradation of sardine and blue whiting muscle

ABSTRACT:   High-pressure technology is used as an alternative to heat processing because of its inactivating effect on microorganisms and enzymes. However, it can also alter the structure of other muscle proteins. The present study compares the effects of high pressure (300 MPa, 7°C, 20 min) on the proteolytic degradation and alterations in the myofibrillar proteins of sardine and blue whiting muscle. Also, muscle homogenates and enzyme extracts were pressurized in order to evaluate the high-pressure effects on unprotected proteolytic enzymes outside the whole muscle structure. Peak proteolytic activity was found to occur at 55°C in both species. The peak activity pH was pH 3 for the sardine and pH 8 for the blue whiting; the main enzyme families being aspartic proteases in the former and alkaline serine proteases in the latter. Pressurization lowered activity levels at the peak activity pH and temperature in the fish muscle (by 30.8% in the sardine and by 9.5% in the blue whiting) and also slightly in the enzyme extracts (by 16.8% in the sardine and by 19.4% in the blue whiting). The electrophoretic profiles disclosed higher protein degradation in the pressurized muscle. Overall, the observed changes in proteolytic activity can be attributed not only to the effect of high pressure on the enzymes, but also and mainly, to the effect on other muscle proteins.     

Some properties of the intestinal proteases of the rabbitfish,Siganus canaliculatus (Park)

Some properties of the intestinal proteases of the rabbitfish were examined. At 25°C, both trypsin and chymotrypsin showed pH optima of 8.0. Leucine aminopeptidase, however, displayed maximum activity in the pH range, 7.0–9.0. Leucine aminopeptidase had the highest optimum temperature (60°C), and chymotrypsin, the lowest (30°C). The optimum temperature of trypsin was 55°C. The activation energy, E_a, was found to be 8.24 for trypsin and 8.50 kcal mol^–1 for chymotrypsin. The E_a for leucine aminopeptidase was 6.29 kcal mol^–1 above 40°C and 1.73 kcal mol^–1 below 40°C. Substrate concentration-velocity plots showed that all three enzymes followed Michaelis-Menten kinetics; the K_m and V_max were estimated for the three enzymes. The effects of various protease inhibitors on enzyme activity were also examined and confirmed the protease classes to which each enzyme belonged. The three proteases examined have similar properties to proteases in other fishes.     

Effects of copper on survival and growth of larval false clown anemonefish <Emphasis Type="Italic">Amphiprion ocellaris</Emphasis>

ABSTRACT:   Larval false clown anemonefish of about 12 h after hatching were fed newly-hatched brine shrimp nauplii in natural (control) and copper-added seawater (40–640 µg–Cu/L) at 26.5 ± 0.5°C for 14 days. Survival rate of the fish increased with increasing copper concentrations up to 160 µg–Cu/L. The rates at 80 and 160 µg–Cu/L were 65 and 80%, respectively, and were significantly higher than that of the control (30%). A positive effect of copper addition on the survival rate was also observed in the other rearing experiment with the fish from seven different spawning chances. Growth rate of the survival fish was not affected by copper concentrations. The copper concentration of the survived fish increased with increasing copper concentrations in the rearing water.     

Peptidase compensation in the digestive system of whiteleg shrimp Penaeus vannamei against dietary Kunitz‐type soybean trypsin inhibitor

The aim of this study was to assess the regulatory process of digestive peptidases of crustaceans in the presence of soybean trypsin inhibitor (SBTI). This naturally occurring inhibitor in soybean meal was used to inhibit the activity of digestive serine peptidases of the whiteleg shrimp Litopenaeus vannamei. In vitro, SBTI inhibited the total proteolytic activity and chymotrypsin activity by 65%. Trypsin activity was reduced by SBTI from 40% to 15% from 2 to 4 hr of incubation, which is the average time of residence of feed in the shrimp digestive system. During the bioassays, experimental groups were fed with increasing concentration of supplemental SBTI (1 g kg^?1 and 2 g kg^?1) and digestive gland and faeces of individual specimens were collected daily. At the end of the bioassay, peptidase activity of digestive gland and faeces was shown, revealing differential inhibition after feeding for 5 days. Several serine peptidases were observed in zymograms, showing a compensation effect on the digestive gland through the activation of peptidases from different catalytic type. These results provide evidence that the shrimp digestive gland can overcome the effect of SBTI by two adaptive mechanisms: synthesis of additional peptidases of the serine class and other unidentified peptidases.     

Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated <Emphasis Type="Italic">Octopus maya</Emphasis>

Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9–10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction on enzyme activity of 70 and 20% was observed in HP and GJ extracts, respectively when protease inhibitor Pepstatin A was used. That result suggests that the main proteases in the HP were aspartic acid proteinases type (possibly Cathepsin D) and some of them were present in the GJ. Dissociating discontinuous polyacrylamide gel electrophoresis showed activity bands between 20 and 28, 30 and 34, 35 and 45, 60 and 70 kDa, and a last one between 75 and 100 kDa. We concluded that extracellular digestion of O. maya takes place in an acid environment, around pH 6. In contrast, intracellular digestion in the HP is developed at pHs between 3 and 4, where cathepsin D could be the most important enzyme for O. maya.     

Investigation of humoral immune factors from selected groups of southern bluefin tuna, Thunnus maccoyii (Castelnau): implications for aquaculture

Serum immunoglobulin, lysozyme and classical and alternative complement activity were analysed in different groups of wild and captive southern bluefin tuna (SBT), Thunnus maccoyii (Castelnau), from ambient water temperatures of 12 ± 1 and 20 ± 1 °C. Groups held captive for the longest time were found to have the highest levels of these humoral immune mediators, despite a drop in ambient temperature from 20 ± 1 to 12 ± 1 °C during the captivity period. Therefore, it may be that the immune response in these endothermic fish is not inhibited by low temperature to the extent seen in poikilothermic fish. Also, length of time in captivity appears to be associated with increased antigen exposure to maintain high levels of humoral immune mediators in these groups. Lysozyme activity was optimal at pH 5.8 and 6.2, suggesting that two isoforms, with different pH optima, are present. The SBT serum was found to lyse sheep erythrocytes by both classical and alternative complement pathways. Classical pathway activity occurred in the absence of prior sensitization with antiserum to sheep red blood cells, suggesting that natural antibodies may be present (or lectin or C-reactive protein mediated activation). Complement activity was relatively resistant to freezing at −20 °C but heating at between 45 and 50 °C for 20 min destroyed all complement activity.     

Effects of bioflocs on water quality,and survival,growth and digestive enzyme activities of Litopenaeus vannamei (Boone) in zero‐water exchange culture tanks

A 30‐day experiment was performed to investigate the effects of bioflocs on water quality, and survival, growth and digestive enzyme activities of the white shrimp Litopenaeus vannamei. Altogether 28 shrimp (7.4 ± 0.1 g) were stocked in each 150 L tank. Two bioflocs treatments and one control were managed: ‘bioflocs 1’ and ‘bioflocs 2’ based on two different densities of the bioflocs, and clean water control without the bioflocs. Brown sugar was added to the bioflocs 1 and bioflocs 2 treatment tanks accounting for 28% and 80% of the shrimp feed respectively (corresponding to proximate C/N ratios of 10 and 14 in daily additions of organic matter respectively), so as to promote bioflocs production and approximately 14 mL L^?1 in treatment bioflocs 1 and 20 mL L^?1 in treatment bioflocs 2 were maintained from day 15. Monitoring of selected water quality parameters throughout the whole experiment period showed that all parameters remained within recommended levels for shrimp culture in the bioflocs treatments at zero‐water exchange, especially low total ammonia nitrogen and nitrite nitrogen levels. By the end of the experiment, shrimp survival rates were above 86%, with no significant differences (P > 0.05) among the three groups. Both weight gain rate and special growth rate tended to increase in the bioflocs treatments compared to those in the control. Meanwhile, the overall specific activities of protease, amylase, cellulase and lipase of the shrimp in the bioflocs treatments were all higher than those in the control; and for the specific activity of the same digestive enzyme, the differences between the bioflocs treatments and the control performed inconsistently among different organs: hepatopancreas, stomach and intestine. Present results suggest that the bioflocs can not only maintain favourable water quality conditions for shrimp culture and help shrimp grow well in zero‐water exchange culture systems, but may also have a positive effect on digestive enzyme activities of the shrimp.     

诺氟沙星对中国明对虾鳃和血清ECOD、APND和GST活性的影响

诺氟沙星药饵每天2次投喂中国明对虾(Fenneropenaeus chinensis),给药剂量分别为0 mg/kg(对照组)、15mg/kg(LD组)、30 mg/kg(MD组)和60 mg/kg(HD组),连续投喂7 d后,分析诺氟沙星对中国明对虾鳃和血清7-乙氧基香豆素-O-脱乙基酶(ECOD)、氨基比林-N-脱甲基酶(APND)和谷胱甘肽-S-转移酶(GST)活性的影响。结果表明,不同浓度诺氟沙星对中国明对虾鳃和血清GST活性呈现显著抑制作用(P<0.05),且GST活性随着取样时间推移表现出先下降后上升的趋势。其中MD组鳃GST活性在4 h时达到最低,是对照组的8.87%;LD组血清GST活性在24 h达到最低值12.01 U/mL,仅为对照组的25.02%。与对照组相比较,给药组中国明对虾鳃和血清APND和ECOD活性均受到显著抑制(P<0.05),且呈现出一定的时间和剂量效应,随着浓度升高,APND和ECOD活性逐渐降低;而随着时间推移,APND和ECOD活性呈现先下降后上升的趋势。本研究通过探讨诺氟沙星对上述酶类的影响,为诺氟沙星的合理使用及与其他药物的联用提供理论参考。     

Partial characterization of digestive proteases of the three‐spot cichlid Cichlasoma trimaculatum (Günter 1867)

Partial characterization of digestive proteases in the three‐spot cichlid Cichlasoma trimaculatum juveniles was conducted. It was determined that there is higher alkaline proteases activity (3.95 ± 0.32 IU mg^?1 protein) compared to acidic proteases (2.01 ± 0.57 IU mg^?1 protein). Optimal temperature for alkaline proteases is 60 °C which resulted in more thermostability to temperature changes. On the other hand, optimal temperature for acidic proteases is 50 °C. Optimal pH for acidic proteases was pH 2, while for alkaline proteases, it was pH 10, which resulted in more stability in relation to pH changes than acidic protease. The use of specific inhibitors and the SDS‐PAGE electrophoresis analysis revealed seven types of bands for alkaline proteases, which make evident the main presence of serine proteases. In acidic proteases, more than 98 g kg^?1 of the activity was inhibited with pepstatin A inhibitor. Therefore, it is evident that C. trimaculatum digestion is composed by acidic and alkaline proteases; thus, it should be considered an omnivorous fish.     

Digestive protease activity in juvenile Farfantepenaeus californiensis as a function of dissolved oxygen and temperature

Total protease, trypsin and chymotrypsin activities were examined in postlarval Farfantepenaeus californiensis (Holmes) after they had been reared for 50 days at two dissolved oxygen concentrations (5.8 and 2.6 mg L⁻¹) and three temperatures (19, 23 and 27 °C). Three replicated experiments were performed with a 12-h light/dark photoperiod. Two-way analysis of variance indicated a significant effect of temperature and oxygen (P < 0.05) on total protease and chymotrypsin activities, but no effect of oxygen was found on trypsin activity. A tendency towards increased protease, trypsin and chymotrypsin activities with acclimation temperature was observed. Total protease activity (units per mg protein) varied from 0.26 in shrimp held at 19 °C and low oxygen concentration to 1.41 in shrimp held at 23 °C and high oxygen concentration. Trypsin activity (units per mg protein) varied from 0.16 in shrimp held at 19 °C and low oxygen concentration to 0.86 in shrimp held at 27 °C and high oxygen concentration. Chymotrypsin activity (units per mg protein) varied from 0.014 in shrimp held at 19 °C and low oxygen concentration to 0.15 (units per mg protein) in shrimp held at 27 °C and low oxygen concentration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymograms of hepatopancreas from each of the treatment groups showed numerous bands. The results suggest that different digestive protease enzymes arise as an adaptation mechanism to temperature and dissolved oxygen variations at this particular stage of life.     

Cryoprotective effects of shrimp head protein hydrolysate on gel forming ability and protein denaturation of lizardfish surimi during frozen storage

ABSTRACT:   The effects of shrimp head protein hydrolysate (SHPH) from three species of shrimp (northern pink shrimp [ Pandalus eous ], endeavour shrimp [ Metapenaeus endeavouri ], black tiger shrimp [ Penaeus monodon ]) on gel forming ability and protein denaturation of lizardfish surimi during frozen storage at −25°C were evaluated. The quality of lizardfish surimi with 5% (dried matter) of any of the three SHPH or sodium glutamate (Na-Glu) was examined in terms of gel strength, whiteness, Ca-ATPase activity and the amount of unfrozen water, comparing with those of surimi without additive as the control. The residual Ca-ATPase activity and gel strength of surimi with SHPH were higher than those of the control throughout 180 days of frozen storage, regardless of shrimp species. The highest effect was found in surimi with Na-Glu. The gel strength and Ca-ATPase activity found a high positive correlation. The addition of SHPH to surimi also increased the amount of unfrozen water by approximately 1.29–1.36 fold higher than the control, however kamaboko gels of the control was significantly whiter. From these results, freeze-induced denaturation of lizardfish muscle protein could be lessened by the addition of SHPH, resulting in a high gel strength and Ca-ATPase activity.     

Effect of pH-shifting on the gel forming characteristics of salt-ground meat from walleye pollack

ABSTRACT:   In order to elucidate the mechanism of the changes in gel forming characteristics of fish meat by pH-lowering, the gelation-temperature curve and the gelation-moisture content curve were examined using the acidified walleye pollack surimi or neutralized one after acidification. In the gelation-temperature curve, the gel strength was highest at 30°C and lowest at approximately 50–60°C, irrespective of pH shifting. The gel strength at 30°C and 80°C decreased with the decrease in pH value. The neutralization of acidified surimi improved the gel strength, but it was considerably lower than the original gel strength. The gel strength at 50–60°C was not affected by pH lowering. The gel strength at 80°C could not be revived to the original by pH readjustment, either in the presence or in the absence of EDTA. These results suggest that irreversible changes of meat protein take place under the low pH, and the oxidation ability of sulfhydryl (SH) groups of protein molecule is not affected by pH-shifting.     

Comparative growth performances of diploid and triploid Chinese shrimp Fenneropenaeus chinensis (Osbeck, 1765) under different salinities

This study aimed at evaluating the ploidy effects on growth performances of Chinese shrimp ( Fenneropenaeus chinensis Osbeck, 1765) reared in different salinities under laboratory conditions. In the acute salinity experiment, there was no difference ( P >0.05) in tolerance observed in triploid and diploid shrimp due to abrupt salinity changes. The lethal salinity for 50% of the individuals in 96 h at 23–25 °C was about 2 g L⁻¹ in both triploids and diploids. While for the chronic salinity experiment, statistical analyses confirmed that the differences in growth performances including the specific growth rate (SGR), the feeding rate (FR), feed conversion efficiency (FCE) and intermoult period (IP) between triploid and diploid were related to salinity. Diploid shrimp reared in 20 g L⁻¹ exhibited highest SGR ( P <0.05), while triploids performed well in 20 and 30 g L⁻¹ salinities ( P <0.05). Based on the survival and growth data, the optimal salinity for the culture of diploid F. chinensis should be 20 g L⁻¹ and for triploids it should be between 20 and 30 g L⁻¹.     

Digestive proteinases and peptidases in the hepatopancreas of the southern brown shrimp (Farfantepenaeus subtilis) in two sub‐adult stages

The aim of this study was to examine proteinases and peptidases from the hepatopancreas of two sub‐adult stages of Farfantepenaeus subtilis: SAS₆ (5.93 ± 0.69 g wet weight) and SAS₁₃ (13.26 ± 0.60 g wet weight). Trypsin and chymotrypsin activity was higher in the extract from the SAS₆ individuals (P < 0.05). The highest activity among aminoacyl‐β‐naphthylamide substrates was found using alanine‐, arginine‐, leucine‐ and lysine‐β‐naphthylamide. There was a positive correlation between the recommended concentration of essential amino acids in penaeid shrimp feed and aminopeptidase activity in both sub‐adult stages. Proteolytic activity of F. subtilis was strongly inhibited by specific trypsin inhibitors. The optimal temperature for trypsin, chymotrypsin and leucine aminopeptidase activity was between 45 and 55 °C. Six and seven bands were found in caseinolytic zymograms for SAS₆ and SAS₁₃ respectively. All bands were inhibited by phenylmethylsulfonyl fluoride in both sub‐adult stages. The use of tosyl‐lysine‐chloromethyl‐ketone and benzamidine caused strong inhibition of the proteolytic bands. Trypsin and chymotrypsin activity was the main difference observed between the protease pattern of SAS₆ and SAS₁₃F. subtilis.