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Effect of temperature on protease production by B. subtilis. The results shown are the mean of triplicate experiment.
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Molasses was used as a sole carbon source for the protease production from Bacillus subtilis EFRL 01 in batch wise submerged condition. The bacterial culture was grown on mineral medium and maximum production was noted after 8 h of incubation. The effect of different variable such as carbon sources (0.5 and 1.0%), nitrogen sources (0.75), sodium ch...
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... production was investigated in a temperature range of 25 to 55°C by B. subtilis when grown on mineral medium incorporated with 1.0% molasses, 0.75% peptone and 2.0% sodium chloride and incubated for 8 h with initial pH of medium 8.5. Maximum proteases activities were produced at 45°C (Figure 9). Many investigators have studied the correlation between protease secretions with temperature but this depends on Afr. ...
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Citations
... Enzymes are currently in high demand due to their multifaceted applications spanning various sectors, including food and feed production, pharmaceuticals, textiles, and biofuels (Research and Markets, 2018). Notably, enzymes like pectinase, xylanase, protease, amylase, and cellulase find significant biotechnological applications, as they play a pivotal role in converting mixed feedstocks into sugar syrups suitable for the production of diverse chemicals, enzymes, and biofuels (Papadaki et al., 2020;Shrestha et al., 2022;Simair et al., 2017a, b;Simair et al., 2018;Qureshi et al., 2011, Qureshi et al., 2012Qureshi et al., 2016;Qureshi et al., 2023;Tunio et al., 2024). Furthermore, there is a burgeoning interest in cost-effective substrates that can be degraded by a single microorganism to concurrently produce multiple enzymes or enzyme combinations (Papadaki et al., 2020;Shrestha et al., 2022;Devos et al., 2024;de de de Oliveira Simas et al., 2024). ...
Lignocellulose recalcitrance essentially dictates high cost and low efficiency of its enzymatic saccharification. The complex lignocellulosic structure is a major obstacle to enzymatic saccharification and green ionic liquid pretreatment methods face difficulty of water washing prior enzymatic hydrolysis. Ionic liquid tolerant lignocellulolytic enzymes overcome the inhibition of enzymes during enzymatic saccharification and eliminated water washing step after pretreatment of lignocellulosic biomass. The cell wall composition cellulose, hemicellulose, and lignin, three main wall polymers of various agricultural residues and food waste lignocellulose substrates also affected the enzyme activities secreted by the B. subtilis strain. This study explores the potential of the halophilic, alkalophilic, and ionic liquid (IL)-tolerant strain Bacillus subtilis BC-001 for the simultaneous production of hydrolytic enzymes essential for lignocellulosic biorefinery processes. BC-001 produced cellulase, amylase, xylanase, pectinase and protease are 70.41, 87.14, 65.50, 122.55 and 66.48 U/mL, respectively under optimized fermentation conditions. Cellulase produced by BC-001 retained more than 80% activity after 72 hours in 20% w/w of different ILs tested and enzymes retained more than 68% activities after 12 h in 50% w/w ILs. Employing such IL-stable cellulase, enzymatic saccharification is conducted without water washing on rice straw (RS) that has been pretreated with various ILs, including 1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium chloride, and choline chloride. This research is novel as the study marks the first instance of multi-hydrolytic enzyme production from a novel alkalophilic, halophilic, thermophilic, and IL-tolerant bacterial strain using a mixture of untreated agricultural residues.
... Subsequently, the mixture was cooled to room temperature, and the activity was measured at an absorbance of 540 nm. The measurement of amylase enzyme activity was conducted using a method that combines elements from the procedures outlined by Naguib and Qureshi (Naguib et al 1964, Qureshi et al 2013. ...
The optimization of extracellular α-amylase production by six different Aeromonas and Bacillus species from different soil samples was conducted. The bacterial strains Aeromonas jandaei (accession no. OR136166), Aeromonas hydrophila (Accession no. OR136168), Aeromonas veronii (accession no OR136284), Bacillus subtilis (accession no. OR357659), Bacillus cereus (accession no. OR136189) and Bacillus amyloliquefaciens (accession no. OR244384) underwent biochemical characterization. In this study we have found that among all the three species of Bacillus, Bacillus subtilis showed the maximum enzyme activity and for Aeromonas species it is Aeromonas veronii which displayed the maximum enzyme activity. Bacillus subtilis displayed its maximum α-amylase activity of 8.96 µmol/min at pH 7, and temperature at 35 o C after 24 hours of incubation. And Aeromonas veronii displayed the maximum α-amylase activity of 7.58 µmol/min at pH 7, 35 0 C temperature after 24 hours of incubation.
... Wang et al. [11] also found activation by Zn ++ but inhibition by Al +++ and Fe +++ in purified nattokinase isolated from natto, a product similar to kinema. Metal ion has an essential role in enzymatic activity and enzyme production; B. subtilis when grown on NaCl and ZnCl 2 has been shown to increase protease production but in contrast KCl reduced the production [84]. Similarly, divalent metal ions such as cobalt and zinc were stated to increase the activity in many commercial neutrase and alkaline protease from microbial sources too [3]. ...
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.
... (Dianli et al., 2019) . (Sattar & Umar, 2011) . (Sikora & Hoffman, 1992) . ...
The root–knot nematode (Meloidogyne incognita) on tomato (Solanum lycopersicum Linn.) is one of the most
important plant pathogens, causing economic damage worldwide and in Iran. In order to biologically control this
nematode, after nematode multiplication on the susceptible tomato variety (Early Orbana Y) and obtaining a
pure and abundant nematode population, the plants were irrigated with a solution of 3 per thousand Probi 96
(containing Bacillus subtilis) and Chitoplus (containing chitosan). After 45 days of applying the treatments, the
seedlings were transferred from the greenhouse to the laboratory, and growth indices such as fresh and dry shoot
weight, shoot length, fresh and dry root weight, root length. Also, disease indices such as the average number of
galls, the number of nematode egg masses in the root, the number of nematode eggs in the root, the number of
second–stage juveniles in the soil, and the gall index were measured. The results showed that the use of B.
subtilis and chitosan, either alone or in combination, led to an increase in plant growth indices and a reduction in
nematode pathogenicity indices. Therefore, based on the results obtained in this study, it can be concluded that
the use of these compounds is suggested as one of the effective strategies in an integrated management program
for root–knot nematodes in tomatoes.
... The production of extracellular protease from microorganisms is greatly influenced by media components and physicochemical factors. Thus, various carbon and nitrogen substrates, divalent metal ions, environmental and fermentation parameters such as pH, incubation temperature, time, agitation speeds, inoculums ages and density have significant effect on protease production [24]. ...
Proteases are one of the predominant groups of industrial enzymes and it represents for about 65% of the total global enzyme market. Proteases of microbial origin have great importance over plant sources because they minimize industrial production costs, increase characteristics of the desired products and widely used in biotechnological process. Among the protease enzymes, aspartic proteases are the most important groups of proteolytic enzymes which are mainly produced by plants, animals and many microorganisms to degrade large polypeptides into peptides and amino acids. Microorganisms are also mainly preferred in the production of aspartic protease since they have most of the characteristics desired for biotechnological application rather than plant protease. Aspartic proteases produced from microbial sources are widely used in pharmaceutical, protein hydrolysis, detergent, cheese-making, photographic, baking, meat, leather, food and beverage industries. Although acid protease is vital to enhance the demands of many food and other industries, there are factors affecting the production of aspartic protease. Hence, aspartic protease production using microorganisms is highly affected by various carbon and nitrogen substrates, divalent metal ions, pH, incubation temperature, time, agitation speeds, age of inoculum and density. This review highlights on the production and applications of microbial aspartic proteases.
... The activity of amylase enzymes was measured using a combined method from Naguib and Qureshi [16], [17]. Substrates containing 1 mL of amylase enzymes were mixed into a reaction containing 1 mL of 0.5% soluble starch in an acetate buffer (pH 5.6) then incubated at 30°C for 30 minutes [16], [14]. ...
... Then the reaction was stopped by adding 2 mL of reagent DNS solution, then heated for 5 minutes. The tubes were cooled to room temperature and absorbance was measured at 540 nm against empty substrates and enzymes [17]. Furthermore, the obtained data calculated the value of enzyme activity and glucose levels produced by comparing with the standard glucose curve. ...
... Then the reaction was stopped by adding 2 mL of reagent DNS solution, subsequently it was heated for 5 minutes. The tubes are cooled to room temperature and the absorbance was measured at 540 nm against empty substrates and enzymes [17]. ...
View the article online for updates and enhancements. You may also like Production and characterization of cellulase from the newly isolated Bacillus subtilis A8 on rice bran and corncob Y S Soeka and Sulistiani-The addition of both Ca 2+ and Na + metal ions to prebiotic productions from cassava peels to enhance the quality of broilers A S Agustina, S Wahyuni, H Natsir et al.-Abstract. Amylase has an important role in biotechnology development and occupies an important position in the world enzyme market, as a biocatalyst in various industrial fields. This study has the goal to find microbial isolates that have the ability to produce amylase enzymes. The study was conducted in two stages, namely: 1) Isolation and selection of microbes that can produce amylase enzymes using starch as substrate, was incubated for 4-7 days at 30°C. Microbial isolates that can produce amylase enzymes are characterized by the presence of clear zones around the colony after the addition of an iodine solution of 1% in the overgrown media of microbes, 2) Test the activity of amylase enzymes using a dinitrosalicylic acid reagent test. The activity of the amylase enzyme is determined by measurement using a spectrophotometer at a wavelength of 540 nm. The sample used comprised of 7 types of ragi tape and 2 samples from cassava tape that has been fermented for 5-7 days. The results obtained in the first stage were 65 microbial isolates, 16 of which had clear zones, consisting of 7 isolates from ragi tape samples and 9 isolates from cassava tape samples. In the enzyme activity test, there are several isolates that have the potential to produce amylase enzymes, these include R5I4 (0.897 ± 0.018 U/mL), R2I5.1 (0.814 ± 0.011 U/mL), R5I3 (0.727 ± 0,042 U/mL) (derived from cassava ragi tape samples) and T2I2.2 (0.812 ± 0.013 U/mL), T2I6.1 (0.817 ± 0.010 U/mL), T2I2.1 (0.735 ± 0.023 U/mL), T1I4 (0.755 ± 0.020 U/mL) (derived from cassava tape samples). The isolate with the highest enzyme activity is the R5I4 which has the value enzyme activity of 0.897 ± 0.018 U/mL and with a fairly high or moderate category, while the lowest enzyme activity is the T1I1.1 isolate of 0.284 ± 0.020 U/mL.
... The activity of amylase enzymes was measured using a combined method from Naguib and Qureshi [16], [17]. Substrates containing 1 mL of amylase enzymes were mixed into a reaction containing 1 mL of 0.5% soluble starch in an acetate buffer (pH 5.6) then incubated at 30°C for 30 minutes [16], [14]. ...
... Then the reaction was stopped by adding 2 mL of reagent DNS solution, then heated for 5 minutes. The tubes were cooled to room temperature and absorbance was measured at 540 nm against empty substrates and enzymes [17]. Furthermore, the obtained data calculated the value of enzyme activity and glucose levels produced by comparing with the standard glucose curve. ...
... Then the reaction was stopped by adding 2 mL of reagent DNS solution, subsequently it was heated for 5 minutes. The tubes are cooled to room temperature and the absorbance was measured at 540 nm against empty substrates and enzymes [17]. ...
Amylase has an important role in biotechnology development and occupies an important position in the world enzyme market, as a biocatalyst in various industrial fields. This study has the goal to find microbial isolates that have the ability to produce amylase enzymes. The study was conducted in two stages, namely: 1) Isolation and selection of microbes that can produce amylase enzymes using starch as substrate, was incubated for 4-7 days at 30°C. Microbial isolates that can produce amylase enzymes are characterized by the presence of clear zones around the colony after the addition of an iodine solution of 1% in the overgrown media of microbes, 2) Test the activity of amylase enzymes using a dinitrosalicylic acid reagent test. The activity of the amylase enzyme is determined by measurement using a spectrophotometer at a wavelength of 540 nm. The sample used comprised of 7 types of ragi tape and 2 samples from cassava tape that has been fermented for 5-7 days. The results obtained in the first stage were 65 microbial isolates, 16 of which had clear zones, consisting of 7 isolates from ragi tape samples and 9 isolates from cassava tape samples. In the enzyme activity test, there are several isolates that have the potential to produce amylase enzymes, these include R5I4 (0.897 ± 0.018 U/mL), R2I5.1 (0.814 ± 0.011 U/mL), R5I3 (0.727 ± 0,042 U/mL) (derived from cassava ragi tape samples) and T2I2.2 (0.812 ± 0.013 U/mL), T2I6.1 (0.817 ± 0.010 U/mL), T2I2.1 (0.735 ± 0.023 U/mL), T1I4 (0.755 ± 0.020 U/mL) (derived from cassava tape samples). The isolate with the highest enzyme activity is the R5I4 which has the value enzyme activity of 0.897 ± 0.018 U/mL and with a fairly high or moderate category, while the lowest enzyme activity is the T1I1.1 isolate of 0.284 ± 0.020 U/mL.
... Prakasham et al. [60] reported the use of a complex organic nitrogen compound resulting in an increase of protease amount secreted compared to that when using an inorganic nitrogen compound. Similarly, Qureshi et al. [61] reported an increase in the amount of protease produced by B. subtilis EFRL 01 when using organic nitrogen sources from yeast extract. ...
Major progress in the fields of agriculture, industry, and biotechnology over the years has influenced the quest for a potent microorganism with favorable properties to be used in scientific research and industry. This study intended to isolate a new thermophilic-protease-producing bacterium and evaluate its growth and protease production under cultural conditions. Protease producing bacteria were successfully isolated from Sungai Klah Hot Spring Park in Perak, Malaysia, and coded as SKF4; they were promising protease producers. Based on microscopic, morphological, and 16S rRNA gene analysis, isolate SKF4 was identified as Geobacillus thermoglucosidasius SKF4. The process of isolating SKF4 to grow and produce proteases under different cultural conditions, including temperature, pH, NaCl concentration, carbon and nitrogen sources, and incubation time, was explored. The optimum cultural conditions observed for growth and protease production were at 60 to 65 • C of temperature, pH 7 to 8, and under 1% NaCl concentration. Further, the use of casein and yeast extract as the nitrogen sources, and sucrose and fructose as the carbon sources enhanced the growth and protease production of isolate SKF4. Meanwhile, isolate SKF4 reached maximum growth and protease production at 24 h of incubation time. The results of this study revealed a new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time. The high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.
... Bacillus licheniformis ALW1 (Accession number LC315920) was found to be the most powerful isolate able to produce alkaline protease under submerged fermentation condition in 250 mL Erlenmeyer flasks holding 50 mL of the production medium. The production medium described by Qureshi et al. (2011) composed of (%) glucose; 1, peptone; 0.5, MgSO 4 ⋅7H 2 O; 0.2, KH 2 PO 4 ; 0.3, NaCl; 0.2 and adjusted by 1 N NaOH and HCL to pH 7, was cultured by 2.5 mL of 24 h inoculum (pre-cultured on tryptic soy broth) and incubated at 37 � C, 180 rpm for 2 days. At the end of the fermentation period, the cultured medium was centrifuged at 4 � C for 10 min at 5000 rpm and the clear supernatant was used further as the crude enzyme. ...
The growing industrial applications of alkaline proteases urged the production of highly active stable enzymes. In the current study, the enzyme production was achieved by submerged fermentation using the bacterial strain Bacillus licheniformis ALW1 that was further optimized to reach 22.903U/mL. The overall optimization fold was 46.7, achieved under the optimized fermentation medium composed of (%) molasses; 8, (NH4)2SO4; 0.45, wheat bran; 5, MgSO4·7H2O; 0.2, KH2PO4; 0.4, NaCl; 0.2 adjusted at pH 8 and incubated for 7days at 37 °C and 280 rpm. Additionally, the enzyme activity after partial purification was optimized by studying the effect of pH and temperature as well as the substrate concentration. The results indicated that the enzyme optimum activity was achieved at pH 9 and 70 °C with Km, Vmax and Kcat values of 3.846 mg/mL, 76.923U/mL/min and 1.206min⁻¹ respectively. Thermal stability study of the partial pure enzyme indicated the half live times of the enzyme as 693.15, 231.05 and 57.76 min⁻¹ at 55, 60 and 65 °C respectively, confirming its thermo-stability. Finally, the efficacy of the partial pure enzyme as a detergent additive was examined. The enzyme retained more than 80% of its activity with an efficient washing performance in the removal of blood stain after 30 min at 50 °C in addition to a commercial detergent.
... But on the basis of various economical, technological and ethical issues, microorganisms are considered as the best sources of proteases (Sharma et al. 2019). Many past works have been done for the production of proteases by strains of Bacillus (Sattar et al. 2011), Rhizopus (Benabda et al. 2019Pandey et al. 2016) and Penicillium (Djamel et al. 2009;Omrane et al. 2018). Fungi elaborate a wider variety of enzymes than bacteria. ...
Abstract In order to achieve high yield of fungal protease in a very cost effective way and to meet its increased market demand, current study deals with the screening of various agro-wastes as carbon source for the production of protease from Rhizopus oryzae (SN5)/NCIM-1447 under solid state fermentation. Substrates and culture parameters such as wheat bran, soybean meal, black-gram husk, rice husk, mixture of wheat bran, soybean meal, nitrogen sources, pH, temperature and incubation time were first optimized with one factor at time strategy and then EVOP factorial and yield of alkaline protease was achieved 412.8 U/gds at 28 °C and pH = 6 after 72 h of fermentation taking wheat bran and soybean as a substrate in 4:1 ratio. Further artificial neural networks (ANN), was trained with data of EVOP and yield of protease was enhanced up to 422.6 U/gds with wheat bran: soyabean in ratio of 70:30, pH 6.2 at 30 °C. The evolved process and Rhizopus oryzae (SN5)/NCIM-1447 strain would be promising for protease production at industrial scale at low cost.
