scholarly journals Enzymatic activity of alkaline protease from Bacillus cereus TD5B and its application as sheep skin dehairing agent

2021 ◽  
Vol 21 (2) ◽  
pp. 105-118
Author(s):  
Nanung Agus FITRIYANTO ◽  
MUSTHOFIYAH MUSTHOFIYAH ◽  
MUHLISIN MUHLISIN ◽  
Ambar PERTIWININGRUM ◽  
Novita KURNIAWATI ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Bacillus Cereus ◽  
Alkaline Protease ◽  
Protease Activity ◽  
Skim Milk ◽  
Protease Production ◽  
Activity Measurement ◽  
Randomized Design ◽  
Chrome Tanning ◽  
Agar Media

This study aims to determine the enzymatic activity of extracellular alkaline protease from Bacillus cereus TD5B and its potential application as a sheep skin dehairing agent. The B. cereus TD5B was screened for extracellular alkaline protease production on skim milk agar media, while its alkaline protease activity and the application were measured at 1%, 1.5%, and 2%. The application of alkaline protease from B. cereus TD5B as a sheep skin dehairing agent was observed through histological examination and physical properties measurement after chrome-tanning with lime and Na2S as control. The study was conducted in a completely randomized design, and the quantitative data were analyzed using Duncan’s Multiple Range Test. The results showed that a clear zone was seen surrounding B. cereus, indicating the bacteria’s proteolytic activity. The protease activity measurement showed that 2% of alkaline protease had the highest enzymatic activity at 144.75 U/mL/min. The highest tensile strength of sheep leather was obtained after dehairing at 1% alkaline protease concentration (350.26 kg/cm2), even though the highest elongation was obtained at 2% (34.92%). In contrast, different concentrations showed similar shrinkage temperatures at 90°C. This study concludes that the optimum alkaline protease concentration from Bacillus cereus TD5B as a sheep dehairing agent was 2%.

Production and characterisation of thermostable alkaline protease from Bacillus subtilis isolated from LA hot spring, Terengganu

2021 ◽  
Vol 16 (7) ◽  
pp. 84-91
Author(s):  
Maslinda Alias ◽  
Hakim Che Harun Mohammad ◽  
Ashraf Razali Nurul ◽  
Jasnizat Saidin ◽  
Nazaitulshila Rasit ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Alkaline Protease ◽  
Protease Activity ◽  
Hot Spring ◽  
Skim Milk ◽  
Industrial Applications ◽  
Protease Production ◽  
Significant Activity ◽  
Original Activity ◽  
Optimum Ph

This research aims to produce thermostable alkaline protease from Bacillus subtilis isolated from La Hot Spring, Terengganu, Malaysia. The study was also conducted to determine the optimum conditions for protease production and stability by considering several parameters including pH, temperature and salt concentration. All seven bacteria were screened on skim milk agar overnight at 37 °C. Three strains with the highest proteolytic activity were identified in protease specific medium. The thermostable alkaline protease had an optimum temperature of 60 °C which achieved 85.73, 82.90 and 83.05 U/mL of protease activity for the three strains respectively. Furthermore, the strains exhibited significant activity of more than 90% from their original activity. Meanwhile, the optimum pH for protease production was pH 9 with the protease activity of 76.76, 79.71 and 88.39 U/mL for TB4, TB6 and TB9 strains, respectively. Proteases were found stable at pH 9 where the loss did not exceed 30% of its original activity. Collectively, all of the data emphasised that proteases from B. subtilis were alkaline thermostable proteases in accordance with a recent report. The finding highlights the viability of the proteases for biotechnological and industrial applications.

scholarly journals Production of alkaline protease from Aspergillus oryzae isolated from seashore of Bay of Bengal

2018 ◽  
Vol 10 (4) ◽  
pp. 1210-1215 ◽  
Author(s):  
Raghu Ram M ◽  
Suman Kumar Yepuru
Keyword(s):  
Aspergillus Oryzae ◽  
Wheat Bran ◽  
Bay Of Bengal ◽  
Alkaline Protease ◽  
Protease Activity ◽  
Skim Milk ◽  
Protease Production ◽  
Chemical Parameters ◽  
Oil Cakes ◽  
Physical And Chemical

Aspergillus oryzae isolatedon  Potato dextrose agar  from soil samples of kottakoduru seashore of Bay of Bengal, Andhra Pradesh, India seashore of Bay of Bengal by spread plate method and was screened for alkaline protease production on Skim milk containing agar plates and identified by clear zones of protein hydrolysis around colonies. Different physical and chemical parameters such as pH, temperature, substrate concentration and incubation time were optimized for the better production of alkaline protease. The maximum protease activity was found at pH of 8 containing 10% wheat bran at 300C, after 72 hours of fermentation.ZnSO4was effective activator for protease activity and sodium dsulphate had shownmore than 50% inhibition of enzyme activity. Among the different oil cakes used for the production of enzyme the Sesame  oil cake proved to be suitable substrate after wheat bran for the production of protease by Aspergillus oryzae.

scholarly journals Isolation, Screening, Characterization and Identification of Alkaline Protease Producing Bacteria from Leather Industry Effluent

2021 ◽  
Author(s):  
Chandran Masi ◽  
Getachew Gemechu ◽  
Mesfin Tafesse
Keyword(s):  
Alkaline Protease ◽  
Protease Activity ◽  
Bacterial Species ◽  
Skim Milk ◽  
Industrial Effluents ◽  
Leather Industry ◽  
Skim Milk Agar ◽  
Secondary Screening ◽  
Agar Media ◽  
Industry Effluent

Abstract BackgroundA wide variety of Bacterial species produces protease enzyme and the application of same enzyme have been manipulated precisely and used in various biotechnological areas including industrial and environmental sectors. The main aim of this research study was to isolate, screen and identify protease producing bacteria which were sampled from leather industry effluent present in the outer skirts of Addis Ababa, Ethiopia.PurposeTo isolated alkaline protease producing bacteria from leather industrial effluents and to characterization (Secreening and identification).MethodsSample collected from Modji leather industrial effluents and stored in the microbiology lab. After isolated bacteria from effluent using serial dilution and followed by isolate protease producing bacteria using skim milk agar media. After studying Primary and secondary screening using zonal inhibition methods to select potential protease producing bacteria using skim milk agar media. Finally to characterization and identification of potential bacteria using biochemical methods, protein estimation, biomass, protease assay and gene sequencing (16S rRNA) method to finalized best protease producing bacteria. ResultsTwenty-eight different bacterial colonies were isolated initially from the leather industry effluent sample situated at Modjo town of Ethiopia. The isolated bacteria were screened using primary screening method with skim milk agar medium. Three isolates namely MS12, ML5 and ML12 showing highest zone of proteolysis as a result of casein degradation on the agar plates were selected and subjected to secondary screening. Further secondary secreening confirmed that MS12, ML5 and ML12 has efficient proteolytic activity and can be considered as potent protease producer. The three isolates were then subjected to morphological and biochemical tests to identify probably bacterial species and all the three bacterial isolates were found out to be of Bacillus species. Shake flask method was carried out to identify the most potent one having greater biomass production capabilities, protein quantity and protease activity. ML12 isolated from leather effluent waste showed highest Protein(170mg/ml), Protease activity(19U/ml), high biomass production and the same was subjected to molecular identification using 16s sequencing and a Phylogenetic tree was constructed to identify the closest neighbor. The isolate ML12 is 97.87% homologous to Bacillus cereus strain (KY995152.1) and 97.86% homologous to Bacillus cereus strain (MK968813.1).ConclusionsThis study has revealed that the leather industry effluent site has significant feature of housing potent bacterial species producing protease of commercial value. Being one among the most widely used enzyme, comparatively. Protease holds a larger scope for research and commercialization any other type of enzymes. There is a need to develop novel protease enzymes for further necessary applications of these enzymes. Moreover, enzyme produced by bacteria which are present in effluents are a greater boon to establish the significance of converting industrial wastes to a highly valuable enzymes especially like proteases.

scholarly journals Isolation, screening, characterization, and identification of alkaline protease-producing bacteria from leather industry effluent

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Chandran Masi ◽  
Getachew Gemechu ◽  
Mesfin Tafesse
Keyword(s):  
Bacillus Cereus ◽  
Alkaline Protease ◽  
Protease Activity ◽  
Bacterial Species ◽  
Skim Milk ◽  
Industrial Effluents ◽  
Leather Industry ◽  
Skim Milk Agar ◽  
Secondary Screening ◽  
Industry Effluent

Abstract Background A wide variety of bacterial species produces protease enzyme, and the application of the same enzyme has been manipulated precisely and used in various biotechnological areas including industrial and environmental sectors. The main aim of this research study was to isolate, screen, and identify alkaline protease-producing bacteria that were sampled from leather industry effluent present in the outer skirts of Addis Ababa, Ethiopia. Purpose To isolate and characterize the alkaline protease-producing bacteria from leather industrial effluents. Methods Samples are collected from Modji leather industrial effluents and stored in the microbiology lab. After isolated bacteria from effluent using serial dilution and followed by isolated protease-producing bacteria using skim milk agar media. After studying primary and secondary screening using zonal inhibition methods to select potential protease-producing bacteria using skim milk agar media. Finally, to identify the potential bacteria using biochemical methods, bacterial biomass, protease activity, and gene sequencing (16S rRNA) method to finalize the best alkaline protease producing bacteria identified. Results First twenty-eight different bacterial colonies were isolated initially from the leather industry effluent sample situated at the Modjo town of Ethiopia. The isolated bacteria were screened using the primary and secondary screening method with skim milk agar medium. At the primary level, we selected three isolates namely ML5(14 mm), ML12(18 mm), and MS12 (15 mm), showing the highest zone of proteolysis as a result of casein degradation on the agar plates were selected and subjected to primary screening. Further secondary screening confirmed that the zone of inhibition methods ML5 (14.00±0.75 mm), ML12 (19.50±0.66 mm), and MS12 (15.00±1.32 mm) has efficient proteolytic activity and can be considered as effective protease producer. The three isolates were then subjected to morphological and biochemical tests to identify probably bacterial species, and all the three bacterial isolates were found out to be of Bacillus species. The shake flask method was carried out to identify the most potent one having greater biomass production capabilities and protease activity. ML12 isolated from leather effluent waste showed the highest protease activity (19 U/ml), high biomass production, and the same was subjected to molecular identification using 16s sequencing and a phylogenetic tree was constructed to identify the closest neighbor. The isolate ML12 (Bacillus cereus strain -MN629232.1) is 97.87% homologous to Bacillus cereus strain (KY995152.1) and 97.86% homologous to Bacillus cereus strain (MK968813.1). Conclusions This study has exposed that from twenty-eight different bacterial samples isolated from leather industry effluent; further primary and secondary screening methods were selected three potential alkaline protease strains. Finally, based on its biochemical identification, biomass, and protease activity, ML12 (Bacillus cereus strains) is the best strain identified. The alkaline protease has the significant feature of housing potent bacterial species for producing protease of commercial value.

scholarly journals Bacteria Associated with the Tannery Effluent and their Alkaline Protease Activities

2012 ◽  
Vol 21 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Mihir Lal Saha ◽  
K. J. M. Hashina Begum ◽  
Mahbubar Rahman Khan ◽  
Donald James Gomes
Keyword(s):  
Alkaline Protease ◽  
Skim Milk ◽  
Bacterial Count ◽  
Protease Production ◽  
Tannery Effluent ◽  
Leather Processing ◽  
Proteolytic Activities ◽  
Key Words Bacteria ◽  
Good Number ◽  
Number Of Bacteria

Samples collected from different stages of the tannery processing were found to be alkaline. A good number of bacteria were found to be associated with the different stages of leather processing. The aerobic heterotrophic bacterial count ranged in between 11.9 × 106 and 46.7 × 106. The highest count was observed in the soaking stage and the minimum was found with the bating stage. Among 40 isolates, 31 showed positive proteolytic activities on different protein based media. Identified organisms were Bacillus subtilis (9), B. licheniformis (6), B. alcalophilus (2), B. badius (2), B. cereus (2), B. circulans (2), B. pumilus (2), B. alvei (1), B. brevis (1), B. coagulans (1), B. megaterium (1), B. polymyxa (1) and Micrococcus varians (1). Proteolytic activity was measured as zone ratio on skim milk agar which was found to be in between 1.5 and 5.8. Higher zone ratio was observed in B. subtilis (TS/1/E), B. pumilus (TS/1/S1/A3 and TD/S2/C3), B. licheniformis (TS/1/Q) and B.  badius (TD/21-D). The alkaline protease production by the nine selected isolates ranged in between 7.1 and 119.3 U/ml. Two isolates of B. pumilus (TS/1/S1/A3 and TD/S2/C3) were found to be good alkaline protease producers (119.3 and 94.8 U/ml) among the tested organisms. Biotechnologically these two isolates or their enzymes could be utilized in the tannery industry.   Key words: Bacteria, tannery, effluent, alkaline protease   D. O. I. 10.3329/ptcb.v21i1.9563   Plant Tissue Cult. & Biotech. 21(1): 53-61, 2011 (June)

scholarly journals Immobilization of Pseudomonas Aeruginosa in Different Matrices for the Production of Alkaline Protease

2020 ◽  
Vol 9 (1) ◽  
pp. 956-959
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzymatic Activity ◽  
Alkaline Protease ◽  
Calcium Alginate ◽  
Protease Production ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Microbial Cells ◽  
Protease Enzyme

Numerous enzymes are used in various types of industries, and one such enzyme used in several of these industries is proteases. Aforementioned, industries such as dairy, detergent, leather, fermentation and several other industries are benefitted with protease enzyme. In the present study, the efficiency of protease production was studied by enriching and immobilizing several matrices by a gram negative bacteria known as Pseudomonas aeruginosa .The immobilization was carried out by four different matrices under two different concentrations of 3% and 4%.Yielded results revealed highest enzymatic activity of 240 (U/ml) in 3% calcium alginate. Still, second highest enzymatic activity of 230 (U/ml) was seen in 4% calcium alginate. On the contrary, free microbial cells showed an enzymatic activity of 100 (U/ml). The peak activities for other methods area as follows: 4% calcium alginate - 133 (U/ml), 3% agar-agar - 100 (U/ml), 4% agar-agar - 91 (U/ml), 3% Gelatin - 85 (U/ml), 4% Gelatin - 88 (U/ml) and Polyacrylamide – 104 (U/ml). The most optimum matrix for the cellular entrapment of Pseudomonas aeruginosa is seen in 3% calcium alginate for alkaline protease production.

scholarly journals OPTIMASI JENIS DAN KADAR SUMBER NITROGEN SERTA pH MEDIUM UNTUK PRODUKSI PROTEASE DARI ISOLAT HTcUM6.2.2 DARI TAUCO SURABAYA

2017 ◽  
Vol 2 (2) ◽  
pp. 98 ◽  
Author(s):  
Monika Rahayu ◽  
Evi Susanti
Keyword(s):  
Nitrogen Source ◽  
Protease Activity ◽  
Skim Milk ◽  
Protease Production ◽  
Production Medium ◽  
Medium Ph ◽  
Ph Optimum ◽  
Research Stage ◽  
Nitrogen Percentage

AbstrakPenelitian ini bertujuan untuk  mengetahui sumber nitrogen, kadar sumber nitrogen dan pH optimum untuk produksi protease isolat HTcUM6.2.2. Penelitian ini merupakan penelitian eksperimental laboratoris menggunakan Rancangan Acak Lengkap (RAL) satu faktorial masing-masing yaitu jenis, kadar sumber nitrogen, serta pH medium dengan tahap penelitian terdiri dari peremajaan, validasi kemurnian isolat HTcUM6.2.2, produksi ekstrak kasar protease isolat HTcUM6.2.2,  uji aktivitas protease. Hasil penelitian menunjukkan sumber nitrogen optimum untuk menghasilkan protease dari isolat HTcUM6.2.2 adalah susu skim dan limbah cair tahu. Kadar limbah cair tahu optimum untuk produksi protease dari isolat HTcUM6.2.2 sebesar 10 %. Produksi protease cukup tinggi dan  relatif konstan antara pH 6 sampai 8.  Aktivitas protease tertinggi sebesar 0,817 U/ml dicapai dengan penggunaan 10 % limbah cair tahu pada medium produksi, pH = 7 selama 3 hari. Kata kunci: protease, tauco, isolat, nitrogen, limbah cair tahu   AbstractThis study aims to determine the optimum of the nitrogen source, percentage of nitrogen source and pH for the production of protease from isolate HTcUM6.2.2. This research is a laboratory experimental with a research stage comprising inoculation and validation of the purity of the isolates HTcUM6.2.2, production of crude extract of protease to determine source of nitrogen, percentage of nitrogen source and pH optimum, determination of protease activity. The results showed that the optimum source of nitrogen to produce proteases from the HTcUM6.2.2 isolate was skim milk and tofu wastewater. Percentage of nitrogen source optimum to produce protease of  HTcUM6.2.2 isolate was 10 % of tofu wastewater. Protease production is relatively high and constantance at pH 6 to 8. The highest protease activity was achieved by the use of 10 % tofu wastewater at production medium, pH = 7 for 3 days was 0,817 U/mL. The keywords: protease, tauco, isolate, nitrogen, tofu wastewater

scholarly journals CULTIVATION CONDITIONS FOR PROTEASE PRODUCTION BY A THERMO-HALOSTABLE BACTERIAL ISOLATE PLS A

2019 ◽  
Vol 19 (1) ◽  
pp. 18-23
Author(s):  
Teuku M. Iqbalsyah ◽  
Malahayati Malahayati ◽  
Atikah Atikah ◽  
Febriani Febriani
Keyword(s):  
Protease Activity ◽  
Hot Springs ◽  
Skim Milk ◽  
Dry Weight ◽  
Protease Production ◽  
Cultivation Conditions ◽  
Thermostable Enzymes ◽  
Cultivation Period ◽  
Protease Enzyme ◽  
Nacl Concentration

Polyextremophiles have increasingly been utilised to produce thermostable enzymes with better stability in multiple extreme conditions. This study reports the screening results of four new bacterial isolates (PLS A, PLS 75, PLS 76 and PLS 80), isolated from an under water hot springs, in producing thermo-halostable protease enzyme. Optimum cultivation conditions for the protease production were also studied. Screening of protease-producing isolates was conducted using Thermus solid medium enriched with 3% skim milk and 0.5% casein. The growth of the isolates showing protease activity was monitored by measuring the cell dry weight and protease activity during 24 h cultivation period. The activity was also measured at various cultivation conditions, i.e. temperature, pH and salt concentrations. Amongst the four isolates, only PLS A showed the ability to produce protease. The optimum cultivation conditions for protease production were observed at 65°C, pH 7 for 18 h incubation. The activity increased with the addition of 1% NaCl concentration (0.085 Unit/mL). The ability of PLS A isolate to produce thermo-halostable protease was encouraging as they could potentially be used in industries requiring the enzyme with multiple extremes. 

Optimization of Nutritional parameters by One-Factor-At-A-Time method for the Biosynthesis of Alkaline Protease from isolated strain Alternaria alternata TUSGF1

2021 ◽  
Vol 10 ◽  
Author(s):  
Tapasi Polley ◽  
Uma Ghosh
Keyword(s):  
Particle Size ◽  
Solid State ◽  
Solid State Fermentation ◽  
Alternaria Alternata ◽  
Alkaline Protease ◽  
Carbon Sources ◽  
Skim Milk ◽  
Protease Production ◽  
Agricultural Residue ◽  
Nutritional Parameters

Background: Alkaline protease essential enzymes that have several applications in our industry. Objective: The aim was optimization of nutritional parameters by one-factor-at-a-time (OFAT) method in solid-state fermentation. Method: Production of protease employing our laboratory new isolate, Alternaria alternata TUSG1 (strain accession number- MF401426) under solid-state fermentation was optimized. The nutritional factors was investigated and only one agricultural residue (cauliflower leaves) with different particle size was checked. Results: Highest enzyme production was obtained with medium particle size of cauliflower leaves (610 U/gds) followed by coarse waste (603U/gds) and fine waste (596 U/gds) using 106 spores/ml as inoculum at 30° C for 7 days. The organism utilized carbon sources 0.5 % (w/w) dextrose, fructose, maltose, sucrose, lactose and starch. Among them maltose was found to be the best carbon source. A variety of inorganic and organic media components were investigated for nitrogen sources 0.3 % (w/w) and skim milk turned out to the best. Conclusion: The maximum enzyme activity was obtained with 1% maltose, 0.5% skim milk and 0.05% MgSO4. With optimized media 1.53 fold increase in the protease production at agricultural residue cauliflower leaves was obtained.

Casein, Fluorescein Isothiocyanate as a Substrate for Measuring Protease Production by Psychrotrophic Bacteria

1987 ◽  
Vol 50 (9) ◽  
pp. 744-749 ◽  
Author(s):  
GENEVIEVE L. CHRISTEN ◽  
M. SENICA
Keyword(s):  
Protease Activity ◽  
Free Amino ◽  
Skim Milk ◽  
Fluorescein Isothiocyanate ◽  
Enzyme Concentration ◽  
Protease Production ◽  
Amino Groups ◽  
Bacterial Populations ◽  
Psychrotrophic Bacteria ◽  
Bacterial Protease

Fluorescein isothiocyanate-labelled casein was evaluated as a substrate for measuring bacterial protease activity in milk. Using protease from Bacillus amyloliquefaciens, effects of substrate type, pH, temperature and enzyme concentration were determined. The procedure was sensitive to .012 unit of enzyme activity. The procedure was then used as a method to detect protease production by psychrotrophic bacteria growing in pasteurized skim milk at 7°C for 5 d. Three psychrotrophic bacteria were added at three different initial populations. Bacterial population, protease activity and cumulative free amino groups were monitored daily. Bacterial populations were not correlated with either protease activity or cumulative free amino groups. Pro-tease activity and cumulative free amino groups were correlated (r=.46, p=.001). Both protease activity and cumulative free amino groups peaked as the microorganisms entered the late log phase (approximately 108 colony forming units per ml). This occurred after incubation for 2 d at 7°C in two replications and after 3 d in the third. Both values decreased with continued storage.

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