Isolation, and identification of Thermophilic protein degrading Bacteria from traditional whey sample
Isolation, and identification of Thermophilic protein degrading Bacteria from traditional whey sample
Arezu Marefat,1Leila Sadeghi,2,*
1. Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran 2. Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
Introduction: Extremozymes are highly valuable in a variety of industrial processes, including those in the chemical, pharmaceutical, and agricultural industries. Researchers have become interested in the isolation and characterization of extremophiles since the available enzymes are insufficient to supply the majority of industrial demands. Thermophilic enzymes often exhibit strong catalytic activity at high temperatures due to great thermal stability, but their activities are lower at moderate temperatures than those of their mesophilic counterparts.
Due to their diverse biochemistry and numerous uses in the tannery and food industries, pharmaceutical formulations, detergents, and processes like waste treatment, silver recovery, and resolution of amino acid mixtures, thermophilic proteases of microbial origin have significant industrial potential. In household laundry, where over 13 billion tons of detergent are produced annually, thermophilic proteases are most commonly used. Industrial enzymes were produced from plants, animals, and microorganisms’ sources. For a number of reasons, microbial sources of proteases are favored to those from plants and animals. These enzymes are known to be produced by a wide range of microorganisms, including bacteria, fungus, yeast, and Actinomycetes. The single most common application of this enzyme is as an active component in laundry detergent. Isolation and characterization of new promising strains is a constant procedure for the synthesis of enzymes for industrial usage.
Whey, a milk-derived protein complex, is promoted as a functional food with several health advantages. Whey's biological constituents, including as lactoferrin, betalactoglobulin, alpha-lactalbumin, glycomacropeptide, and immunoglobulins, exhibit a variety of immune-stimulating qualities. Due to the release of a heat-resistant protease, psychrotrophic bacteria can have unfavorable effects on dairy goods like precipitation, gelatin, bitterness, and fat floating on top. Pseudomonas fluorescens is a representative of the genus Pseudomonas, and it is capable of secreting the common heat-resistant protease AprX, an alkaline metalloprotease of the serralysin family with a molecular weight of roughly 40–50 kDa. The aim of this research was identification of Thermophilic protein degrading Bacteria from whey and optimize the protease activity.
Methods: This study employed whey sample as a biologic sample that has several types of bacteria that degrade protein molecules. The whey bacteria were enriched in Luria-Bertani (LB) medium at first. The samples were cultivated for overnight with 120 rpm shaking at various temperatures (30, 40, and 50 °C). Then samples were isolated using cultured LB plate media and subsequently screened on casein- liquid medium, with casein consumption measured using a UV-VIS spectrophotometer after precipitation of intact proteins by using the trichloroacetic acid precipitation. Tyrosine is released into the environment when casein is digested by a protease enzyme, and tyrosine absorbs at a wavelength of 280 nm. In this study, the amount of produced tyrosine as a result of protein degradation was calculated as M of tyrosine liberated/g of protein. The typical tyrosine curve is shown in Fig.1.
Ammonium sulfate and protease enzyme from the sample were the materials used for precipitation. The enzyme was created and refined utilizing ammonium sulfate precipitation in a liquid media. The medium was placed on shaker overnight at 50 °C at a speed of 120 rpm. Medium color was changed to the yellow color at the following days which refer to protease enzyme reaction. Centrifugation at 3,500 rpm for 30 minutes at 4 °C was used to separate the precipitate from supernatant. Supernatant and pellet were separated during centrifugation. The pellet was eliminated because it was contained a Bacterium cell wall, whilst the supernatant is regarded as a crude enzyme and may be subjected to additional testing. Ammonium sulfate was used at different concentrations (50, 60, 70, and 80 %) to precipitate crude microbial enzymes during the optimization process. On the precipitate and supernatant of each level of treatment with ammonium sulfate, the specific activity of the protease enzyme and the protein content were seen. SDS-PAGE protein patterns are used for protein content determination. The Laemelli method for sodium-dodecyl sulphate polyacrylamide gel electrophoresis was used. With the help of sodium dodecyl sulfate, the total proteins were separated. The relative mobility of the common protein markers was used to estimate the molecular weight of the bands found in the sample. Based on the 16S rDNA gene sequence, bacteria with the best protease activity were finally found (Fig.2).
Results: Protease ability of bacteria was confirmed by UV-VIS spectrophotometer the method on Casein medium as substrate. The released of tyrosine by Samples in different temperatures were compared with each other. The most content of tyrosine liberated occurred in 50 °C which resulted to 389µM tyrosine liberated. The sample with the best protease activity was selected for 16S rRNA analysis. 16S rRNA gene amplified by PCR reaction and universal primers to sequencing and phylogenic analysis. The PCR product was analyzed by agarose gel electrophoresis Figure 2. Once a pure PCR product of the 16S gene was obtained, it was sequenced and aligned with the bacterial DNA database, thus identifying the bacteria. The sample with the best protease activity was Bacillus. Subtilis strain SAB6.
A 70% ammonium sulfate level treatment had the highest specific enzyme activity (76.245 U/mg) and highest protein content (2.359 mg/mL), according to the measurement of these two factors. In comparison to the specific enzyme activity before to purification treatment (21.42 U/mg), this specific enzyme activity was three times higher.
The purified protease by B. Subtilis was also confirmed to be single purified protein molecule by using SDS-PAGE and its molecular weight was determined as 22 kDa by using proteins of known molecular weight (Fig. 3).
Conclusion: In conclusion, the strain isolated in this work (Bacillus Subtilis strain SAB6) contains protease enzyme that extracts to out of the cells and could be cloned in further studied. The purification of Bacillus Subtilis strain SAB6 enzyme by ammonium sulfate purification can increase the specific enzyme activity.