Partial purification and characterization of protease extracted from kinema
Proteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attrac...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-03-01
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| Series: | Heliyon |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844024032043 |
| _version_ | 1797259753764683776 |
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| author | Dambar Bahadur Khadka Tikaram Pahadi Sunil Aryal Dhan Bahadur Karki |
| author_facet | Dambar Bahadur Khadka Tikaram Pahadi Sunil Aryal Dhan Bahadur Karki |
| author_sort | Dambar Bahadur Khadka |
| collection | DOAJ |
| description | Proteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attracted more attention as a source of microbial protease. The present study aimed to extract protease from kinema, partially purify the extracted protease following dialysis after precipitation with ammonium sulfate, and determine general characteristics of protease. The kinema having highest proteolysis activity after three days of control fermentation (Temperature 30±2 °C, RH 66 ± 2%) was taken for the study. About 2.45 fold of purification with overall recovery of 63.21% was achieved after precipitation with ammonium sulfate at 30–70% saturation level followed by dialysis of crude extracted protease. The dialysed kinema protease had specific activity of 7.90 U/mg. The enzyme remained actively functional across a wider pH (5–9) and temperature (40-60 °C) range. SDS-PAGE and Zymogram confirmed the presence of three major active bands respectively of 29.04 kDa, 36.09 kDa and 46.35 kDa in the kinema protease extract. The enzyme kinetics data on casein, fitted to Mechaelis Mentens’ plots showed the protease had Vmax of 1.001 U/ml with corresponding Km value of 0.825 mg/ml. Metal ions such as iron, mercury and aluminium showed the inhibition effect whereas presence of sodium, zinc, and calcium shows the activation effect on protease performance. The enzyme was active over various natural substrates; showing maximal activity on casein, and subsequent to bovine serum albumin, gelatin, hemoglobin and whey protein respectively. Furthermore, molecular weight distribution of the protease extract and activity inhibition with ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride, suggesting the protease from kinema could be a metal dependent serine protease or mixture of them. |
| first_indexed | 2024-03-07T14:00:40Z |
| format | Article |
| id | doaj.art-001d0f71bfb4411aa6d3a05c5b65ef40 |
| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-04-24T23:14:27Z |
| publishDate | 2024-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Heliyon |
| spelling | doaj.art-001d0f71bfb4411aa6d3a05c5b65ef402024-03-17T07:57:33ZengElsevierHeliyon2405-84402024-03-01105e27173Partial purification and characterization of protease extracted from kinemaDambar Bahadur Khadka0Tikaram Pahadi1Sunil Aryal2Dhan Bahadur Karki3Central Department of Food Technology, Tribhuvan University, Dharan, Nepal; Central Campus of Technology, Tribhuvan University, Dharan, Nepal; Corresponding author. Central Department of Food Technology, Tribhuvan University, Dharan, Nepal.Central Campus of Technology, Tribhuvan University, Dharan, NepalCentral Department of Food Technology, Tribhuvan University, Dharan, NepalCentral Department of Food Technology, Tribhuvan University, Dharan, NepalProteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attracted more attention as a source of microbial protease. The present study aimed to extract protease from kinema, partially purify the extracted protease following dialysis after precipitation with ammonium sulfate, and determine general characteristics of protease. The kinema having highest proteolysis activity after three days of control fermentation (Temperature 30±2 °C, RH 66 ± 2%) was taken for the study. About 2.45 fold of purification with overall recovery of 63.21% was achieved after precipitation with ammonium sulfate at 30–70% saturation level followed by dialysis of crude extracted protease. The dialysed kinema protease had specific activity of 7.90 U/mg. The enzyme remained actively functional across a wider pH (5–9) and temperature (40-60 °C) range. SDS-PAGE and Zymogram confirmed the presence of three major active bands respectively of 29.04 kDa, 36.09 kDa and 46.35 kDa in the kinema protease extract. The enzyme kinetics data on casein, fitted to Mechaelis Mentens’ plots showed the protease had Vmax of 1.001 U/ml with corresponding Km value of 0.825 mg/ml. Metal ions such as iron, mercury and aluminium showed the inhibition effect whereas presence of sodium, zinc, and calcium shows the activation effect on protease performance. The enzyme was active over various natural substrates; showing maximal activity on casein, and subsequent to bovine serum albumin, gelatin, hemoglobin and whey protein respectively. Furthermore, molecular weight distribution of the protease extract and activity inhibition with ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride, suggesting the protease from kinema could be a metal dependent serine protease or mixture of them.http://www.sciencedirect.com/science/article/pii/S2405844024032043KinemaProteaseEnzyme extractionPartial purificationCharacterization |
| spellingShingle | Dambar Bahadur Khadka Tikaram Pahadi Sunil Aryal Dhan Bahadur Karki Partial purification and characterization of protease extracted from kinema Heliyon Kinema Protease Enzyme extraction Partial purification Characterization |
| title | Partial purification and characterization of protease extracted from kinema |
| title_full | Partial purification and characterization of protease extracted from kinema |
| title_fullStr | Partial purification and characterization of protease extracted from kinema |
| title_full_unstemmed | Partial purification and characterization of protease extracted from kinema |
| title_short | Partial purification and characterization of protease extracted from kinema |
| title_sort | partial purification and characterization of protease extracted from kinema |
| topic | Kinema Protease Enzyme extraction Partial purification Characterization |
| url | http://www.sciencedirect.com/science/article/pii/S2405844024032043 |
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