• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.65-74
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• 2021

Serine proteases are the most versatile proteolytic enzymes with tremendous applications in various industrial processes. This study was designed to investigate the biochemical properties, critical residues, and the catalytic potential of alkaline serine protease using in-silico approaches. The primary sequence was analyzed using ProtParam, SignalP, and Phyre2 tools to investigate biochemical properties, signal peptide, and secondary structure, respectively. The three-dimensional structure of the enzyme was modeled using the MODELLER program present in Discovery Studio followed by Molecular Dynamics simulation using GROMACS 5.0.7 package with CHARMM36m force field. The proteolytic potential was measured by performing docking with casein- and keratin-enriched residues, while the effect of the inhibitor was studied using phenylmethylsulfonyl fluoride, (PMSF) applying GOLDv5.2.2. Molecular weight, instability index, aliphatic index, and isoelectric point for serine protease were 39.53 kDa, 27.79, 82.20 and 8.91, respectively. The best model was selected based on the lowest MOLPDF score (1382.82) and DOPE score (-29984.07). The analysis using ProSA-web revealed a Z-score of -9.7, whereas 88.86% of the residues occupied the most favored region in the Ramachandran plot. Ser327, Asp138, Asn261, and Thr326 were found as critical residues involved in ligand binding and execution of biocatalysis. Our findings suggest that bioengineering of these critical residues may enhance the catalytic potential of this enzyme.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.1-8
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• 2001

Spiders are carnivores that prey upon insects and other small arthropods through digestion of food outside the body. Although spider poison may contain proteolytic enzymes, these are thought to play an insignificant role in actual digestion. The source of active proteolytic enzymes can be either the digestive tract cells of spider, or natural microbial flora in the digestive tract of spider. In this study, digestive tracts from the spider, Nephila clavata, were screened for bacteria that have protease or lipase activity. A total of $10^3-10^5$ CFU was recovered from a spider and more than 90% of them showed protease and lipase activity respectively. Of the microbial isolates, 63.3% showed protease or lipase activity, and 50% of these showed both protease and lipase activity. Some of the isolates were characterized using a battery of chemical, phenotypic and genotypic methods. Eleven Gram negative bacteriaa (Acinetobacter calcoaceticus, A. haemolyticus, Alcaligenes faecalis, Cedecea davisae, C. neteri, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas fluorescens, Serratia marcescens, Stenotrophomonas maltophilia, Suttonella indologenes) and 11 Gram positive bacteria (Bacillus cereus, B. coagulans, B. pasteurii, B. thuringiensis, Cellulomonas flavigena, Corynebacterium martruchotii, Enterococcus durans, E. faecalis, Micrococcus luteus, Staphylococcus hominis, S. sciuri) were identified.

• Journal of Life Science
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• v.20 no.11
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• pp.1718-1724
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• 2010

Bioactive and chemical properties of silkworm powder (SP) degradation by fruit extract containing the proteolytic enzymes of kiwifruit, papaya, pineapple and pear were investigated. Silkworm powder was incubated with extracts from each fruit at $60^{\circ}C$ for 24 hr. Protein content was slightly higher in the SP treated with fruit extract than that in the control SP. Major minerals were K, Ca, Mg, and Zn. Major fatty acids were linolenic acid, oleic acid, and palmitic acid. When total protein patterns were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), silkworm protein was strongly degraded by the treatment of fruit extract from pineapple, papaya, and pear, but little silkworm degradation was observed in kiwifruit extract treatment. Fibriolytic activity was only detected in the SP by the fruit extract treatments from papaya and pear. DPPH radical scavenging activity was slightly stronger in the SP treated with fruit extract than that in silkworm powder. However, all these samples exhibiteda relatively low activity compared with the butylated hydroxytoluene (BHT). These results may provide the basic data for understanding the biological activities and chemical characteristics of SP treated with fruit extract for development of functional foods.

• Korean Journal of Food Science and Technology
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• v.33 no.6
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• pp.676-681
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• 2001

This study was carried out to evaluate the quality characteristics of frozen soy yogurts prepared with different proteolytic enzymes and starter culture. The viable cell counts of lactic acid bacteria in frozen soy yogurts were measured $10^8$ CFU/g by the single culture method, while $10^9$ CFU/g by the mixed culture method except ${\alpha}-chymotrypsin$ treatment. The viable cell counts of lactic acid bacteria did not decrease after freezing for 30 min in ice cream maker. The lactic acid bacteria from the mixed culture showed better bile acid tolerance than those from the single culture. The lactic acid bacteria from the frozen soy yogurt prepared with ${\alpha}-chymotrypsin$ and mixed culture of Bifidobacterium bifidum and Lactobacillus bulgaricus showed better acid tolerance and bile acid tolerance. The highest(73.45%) overrun was observed in the frozen soy yogurt treated with ${\alpha}-chymotrypsin$ and mixed culture of B. bifidum and L. bulgaricus. The melt-down percent was higher in the single culture than the mixed culture. In sensory test, the frozen soy yogurt prepared with ${\alpha}-chymotrypsin$ and mixed culture of B. bifidum and L. bulgaricus was the most desirable, the highest scores in sourness, bitterness and mouthfeel.

• Korean Journal of Food Science and Technology
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• v.16 no.2
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• pp.211-217
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• 1984

To study affinity of proteolytic enzymes to soy proteins, the physicochemical and functional properties of enzymatically modified protein products, kinetic parameters and degree of hydrolysis were measured using trypsin, alcalase (serine type protease) and pronase. Bacterial alcalase and pronase showed much greater affinity to soy protein than animal intestinal trypsin. This effect was very significant when unheated soy isolate was used as a substrate. Specific activities of these enzymes decreased with the increment of substrate concentration (over 2.0%, w/v) when heat denatured soy protein was used as a substrate. However, the decrease in specific activity was negligible at substrate concentrations lower than 2.0%. Polyacrylamide gel electrophoretic results showed that the pattern of 2S protein band changed distinctly in alcalase hydrolysis as compared with those of trypsin and pronase. Protein solubilities of alcalase and pronase hydrolyzates increased by 25-30%, at their pI (pH 5.0) over the control. Virtually no change was observed in solubility by trypsin hydrolysis. Heat coagulability and calcium-tolerance of the protein increased by enzymatic hydrolysis. No clear tendency, however, was observed for emulsion properties, foam expansion and the amount of free -SH groups. The enzyme treatment considerably decreased foam stability.

• Korean Journal of Food Science and Technology
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• v.33 no.5
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• pp.603-610
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• 2001

The quality characteristics of soy yogurt prepared with different proteolytic enzymes and starter culture were evaluated. In order to facilitate the growth of lactic acid bacteria and subsequent production of lactic acid, soy protein isolate(SPI) was hydrolyzed using three kinds of proteases; one extracted from Aspergillus oryzae, bromelain and ${\alpha}-chymotrypsin$. The cultural systems employed thereafter for lactic fermentations were: 1) Bifidobacterium bifidum, 2) B. bifidum and Lactobacillus acidophilus, 3) B. bifidum and Lactobacillus bulgaricus. In soy yogurt, pH was more decreased by mixed culture method than single culture method with the accumulation of lactic acid. Viable cells of lactic acid bacteria in soy yogurts were measured $10^8$ CFU/g by the single culture method while $10^9$ CFU/g by the mixed culture method except ${\alpha}-chymotrypsin$ treatment. The amount of free amino acids in soy yogurts were substaintially increased by enzyme treatment. Viscosity was decreased by enzyme treatment, resulting in higher viscosity by ${\alpha}-chymotrypsin$ treatment. Water holding capacity was found to be higher in the single culture method in case of enzyme treatment. Among the various volatile flavor components isolated and identified after enzyme hydrolysis, acetaldehyde, ethanol, diacetyl, butyl alcohol contents tended to increase by lactic fermentation.

• International Journal of Industrial Entomology and Biomaterials
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• v.22 no.2
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• pp.83-93
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• 2011

Biochemical and enzymatic characterization for extracellular protease isolated from Cordyceps militaris cultivated on rice bran medium was investigated. C militaris produced proteolytic enzymes from 10 days after inoculation, maximum enzyme production was found at 25 days. The optimum temperature and pH of proteases production was at $25^{\circ}C$ and pH 7.0, respectively. The protease activity was observed in the four peaks (Pro-I, Pro-II, Pro-III, and Pro-IV) separated through Sephadex G-100 column chromatography. The separated protease was optimally active at $25^{\circ}C$. Optimum pH of the protease was between 7 and 8. Enzyme was also stable over at $30-80^{\circ}C$. The enzyme was highly stable in a pH range of 4-9. Protease activity was found to be slightly decreased by the addition of $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, $Fe^{2+}$ and $Cu^{2+}$, whereas inhibited by the addition of $Ca^{2+}$ and $Co^{2+}$ Protease activity was inhibited by protease inhibitor PMSF. On the other hand, the partially purified protease was investigated on proteolytic protease activity by zymogram gel electrophoresis using three substances (casein, gelatin and fibrin). Four active bands (F-I, FII, F-III, and F-IV) of fibrin degradation were revealed on fibrin zymogram gels. Both of F-II and FIII showed caseinolytic, fibrinolytic and gelatinolytic activities in three gels. Thermostability, pH stability, and pH-thermostability of the enzyme determined the residual fibrinolytic activity also displayed on fibrin zymogram gel. The only one enzyme (F-II) displayed over a broad range of temperature at $30-90^{\circ}C$. The FII displayed fibrinolytic activity in the pH range 3-5, but was inactivated in the range of pH 6-11. The F-I and F-III showed enzyme activity in the pH range of 6-11. In the pH-thermostability, the F-II only kept fibrinolytic activity after heating at $100^{\circ}C$ for 10, 20 and 30 min at pH 3 and pH 7, respectively. On the other hand, the F-II was retained activity until heating for 10 min under pH 11 condition. By using fibrin zymogram gel electrophoresis, extracellular fibrinolytic enzyme F-II from C. militaris showed unusual thermostable under acid and neutral conditions.

• Animal Bioscience
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• v.34 no.9
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• pp.1514-1524
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• 2021

Objective: This study investigated the meat quality characteristics, endogenous proteolytic enzymes, collagen content, and myosin heavy chain (MyHC) isoforms of different muscles of Thai native cattle (TNC). Methods: Infraspinatus (IF), Longissimus thoracis (LT), and Supraspinatus (SS) muscles were obtained from two TNC breeds, Kho-Lan (KL, n = 7) and Kho-Isaan (KI, n = 7). The muscle and meat characteristics of TNC breeds and their relationship with MyHC expression were examined. Results: Three MyHC isoforms namely MyHC I, MyHC IIa, and MyHC IIx were detected in the muscles. The KL had higher (p<0.05) MyHC IIx than the KI. The IF muscle had higher (p<0.05) MyHC I compared to other muscles. The LT muscle had the least MyHC I. The LT had higher (p<0.05) MyHC IIx than the IF and SS muscles. The IF presented the least MyHC IIx. The KL had higher (p<0.05) lightness and moisture content and lower crude protein, redness, cooking loss, shear force, and calpastatin than the KI. The glycogen, total collagen, soluble collagen, crude protein, ash contents, and troponin T degradation product of IF and SS were lower (p<0.05) than that of LT. Ether extract in LT was lower (p<0.05) than that of IF and SS. The percentage of MyHC I, MyHC IIa, and MyHC IIx were significantly correlated with muscle and meat characteristics of TNC. Conclusion: These results suggest that the differences in the MyHC isoforms may partly account for the variation in meat quality between breeds and among muscles of TNC.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.4
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• pp.587-594
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• 2010

To develop commercially available food protein hydrolysates, the effects of different types of enzymes and substrates on bitterness and solubility of partially hydrolyzed food proteins were investigated. Four types of proteins (casein, isolated soy protein (ISP), wheat gluten, and gelatin) and five types of proteolytic enzymes (a microbial alkaline protease (alcalase), a microbial neutral protease (neutrase), papain, bromelain, trypsin) were used. To profile the pattern of hydrolysis, the degree of hydrolysis (DH) were monitored during 180 min of reaction time by pH-stat method. Casein showed the highest susceptibility to hydrolysis for all five proteases compared to those of ISP, gluten, and gelatin. In addition, the bitter intensity and solubility (nitrogen soluble index, NSI) of each protein hydrolysate were compared at DH 10%. Bitterness and solubility of protein hydrolysates were highly affected by DH and the types of enzymes and substrates. At DH=10%, casein hydrolysate by trypsin, ISP and gluten hydrolysates by either bromelain or neutrase, and gelatin hydrolysates by the five proteases tested in this study were highly soluble and less bitter.

• Korean Journal of Veterinary Research
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• v.29 no.4
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• pp.445-455
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• 1989

The proteins of the ruminant erythrocyte membranes were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and their relations to the slow erythrocyte sedimentation rate(ESR) of the ruminants were investigated by treating the erythrocytes with proteinases such as trypsin, chymotrypsin and pronase, and glycosidases such as neuraminidase and galactosidase. Protein content in the erythrocyte membrane was $2.85{\pm}0.28$ in human, $3.60{\pm}0.41$ in Korean cattle, $3.71{\pm}0.36$ in Holstein, $4.13{\pm}0.83$ in Korean native goat and $3.94{\pm}0.56mg/ml$ in sheep, showing higher in ruminant animals than in human(p<0.01). Although the general protein profiles of the ruminant erythrocyte membranes were almost similar to that of human, all the ruminant erythrocyte membranes showed one additional protein band, called band-Q in the previous report on proteins of bovine erythrocyte membrane, which migrated electrophoretically to the mid position between band-2 and band-3 in human erythrocyte membranes. The glycoprotein profiles of ruminant erythrocyte membranes revealed by periodic acid Schiff(PAS) stain showed a marked difference from that of human. The PAS-1(glycophorin) and PAS-2(sialoglycogrotein) present in human erythrocyte membranes were almost absent from the ruminant animals. Instead, a strong PAS-positive band near the origin of the electrophorograms, which was named as PAS-B in the previous report on proteins of bovine erythrocyte membranes, was shown in the ruminant animals except sheep. In addition, the erythrocyte membranes of Korean native goat and sheep showed a moderate PAS-negative band near the tracking dye of the electrophorograms, which was named as PAS-G in this study. In the erythrocyte treated with the enzymes, the migration of each protein fracture of erythrocyte membranes in response to each enzyme was diverse according to different species or breed of ruminant animals. Among others, band-Q present in ruminants was slightly or moderately decreased by trypsin-, chymotrypsin-, and pronase- treatments of the erythrocytes, but not only in sheep. It was particularly noticeable that PAS-B, a fraction of glycoprotein, present in ruminants except sheep, was better digested by proteinases than by glycosidases, showing remarkable increase(p<0.01) of the ESR in accord with complete digestion(disappearance) of the PAS-B band by pronase, trypsin or chymotrypsin treatment of erythrocytes. In sheep, there was almost no any response to the various enzymes in general protein and glycoprotein profiles of the erythrocyte membranes except PAS-G, which was markedly decreased by pronase treatment of the erythrocytes. Nevertheless, the ESRs were accelerated in erythrocytes treated with pronase, trypsin, chymotrypsin and neuraminidase. Erythrocyte osmotic fragility was increased in erythrocytes treated with only pronase among five enzymes in all the human and ruminant animals used in this study.