Isolation of lysozyme producing Bacillus subtilis Strains, identification of the new strain Bacillus subtilis BSN314 with the highest enzyme production capacity and optimization of culture conditions for maximum lysozyme production

Naveed, Muhammad; He, Tao; Wang, Fenghuan; Yin, Xian; Chan, Malik Wajid Hussain; Ullah, Asad; Xu, Baocai; Aslam, Sadar; Ali, Nawazish; Abbas, Qamar; Hussain, Ishtiaq; Khan, Ali Nisar; Khan, Abdul Majeed

doi:10.1016/j.crbiot.2022.06.002

Cited by 14 publications

(10 citation statements)

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“…Researchers are trying to find techniques/methods to purify proteins secreted/produced by B. subtilis without denaturation. In this study, a multi-step methodology was used to purify the lysozyme product (protein) biosynthesized from the previously isolated strain Bacillus subtilis BSN314 [ 34 ]. This multistep methodology included ammonium sulfate precipitation, dialysis, ultrafiltration, and gel column chromatography.…”

Section: Discussionmentioning

confidence: 99%

“…For activation, the culture was taken, spread on Luria agar (LA) medium and allowed to grow at 37 °C for 24 h. A single colony was selected for further inoculation in 4 mL of Luria broth (LB) medium in a 10 mL shaking tube, and incubated for 24 h at 37 °C and at 220 rpm. To obtain a second generation of cultures, 1 mL of the inoculum was transferred to 100 mL of the LB medium and incubated for another 24 h under the same growth conditions [ 34 ].…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme activity was calculated by the following formula:

where I = enzyme activity, A1 = absorbance at 450 nm, A2 = absorbance after 1 min, A1 − A2 = the change in absorbance per minute at 450 nm, 0.001 = decrease the absorbance value at 450 nm by 0.001 per minute as an activity unit. Ew = 0.5 mL detection enzyme solution contains the mass (mg) or volume (mL) of the original enzyme solution [ 34 ].…”

Section: Methodsmentioning

confidence: 99%

“…Following fermentation, various parameters were applied to the Bacillus species to determine the maximum lysozyme production. It was found that Bacillus subtilis BSN314 was able to achieve the highest lysozyme production [ 34 ]. In the present study, the lysozyme obtained from Bacillus subtilis BSN314 was purified by salt (ammonium sulfate) precipitation and dialysis methods and characterized by the bactericidal activity of lysozyme.…”

Section: Introductionmentioning

confidence: 99%

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Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria

et al. 2023

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In the present study, lysozyme was purified by the following multi-step methodology: salt (ammonium sulfate) precipitation, dialysis, and ultrafiltration. The lysozyme potential was measured by enzymatic activity after each purification step. However, after ultrafiltration, the resulting material was considered extra purified. It was concentrated in an ultrafiltration centrifuge tube, and the resulting protein/lysozyme was used to determine its bactericidal potential against five bacterial strains, including three gram-positive (Bacillus subtilis 168, Micrococcus luteus, and Bacillus cereus) and two gram-negative (Salmonella typhimurium and Pseudomonas aeruginosa) strains. The results of ZOI and MIC/MBC showed that lysozyme had a higher antimicrobial activity against gram-positive than gram-negative bacterial strains. The results of the antibacterial activity of lysozyme were compared with those of ciprofloxacin (antibiotic). For this purpose, two indices were applied in the present study: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus (AMI/PAI; 1.01/101) and Bacillus subtilis 168 (AMI/PAI; 1.03/103), compared to the antibiotic (ciprofloxacin) used in this study. Atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified protein was further processed by gel column chromatography and the eluate was collected, its enzymatic activity was 21.93 U/mL, while the eluate was processed by native-PAGE. By this analysis, the un-denatured protein with enzymatic activity of 40.9 U/mL was obtained. This step shows that the protein (lysozyme) has an even higher enzymatic potential. To determine the specific peptides (in lysozyme) that may cause the bactericidal potential and cell lytic/enzymatic activity, the isolated protein (lysozyme) was further processed by the SDS-PAGE technique. SDS-PAGE analysis revealed different bands with sizes of 34 kDa, 24 kDa, and 10 kDa, respectively. To determine the chemical composition of the peptides, the bands (from SDS-PAGE) were cut, enzymatically digested, desalted, and analyzed by LC-MS (liquid chromatography-mass spectrometry). LC-MS analysis showed that the purified lysozyme had the following composition: the number of proteins in the sample was 56, the number of peptides was 124, and the number of PSMs (peptide spectrum matches) was 309. Among them, two peptides related to lysozyme and bactericidal activities were identified as: A0A1Q9G213 (N-acetylmuramoyl-L-alanine amidase) and A0A1Q9FRD3 (D-alanyl-D-alanine carboxypeptidase). The corresponding protein sequence and nucleic acid sequence were determined by comparison with the database.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The enzyme activity was calculated by the following formula:

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria

et al. 2023

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“…Glucose as a quick-acting carbon source was convenient for microorganism utilization. Therefore, the addition of glucose caused the strain growing too fast so that the nutrient was insufficient for subsequent MCE production (Naveed et al 2022 ). Soluble starch was a polysaccharide and called as “slow-acting carbon source”.…”

Section: Discussionmentioning

confidence: 99%

Isolation and production optimization of a novel milk-clotting enzyme Bacillus velezensis DB219

Zhang

Liu

et al. 2022

AMB Expr

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The milk-clotting enzyme (MCE) is a crucial ingredient in cheese manufacture. Due to the limits of traditional MCE, finding viable substitute is a pressing issue. This study aims to isolate and identify a wild strain with high milk-clotting activity (MCA) and low proteolytic activity (PA) and optimize the fermentation conditions for MCE production. A strain of Bacillus velezensis DB219 with high MCA/PA value (9.2) was isolated from dairy soil (Wuchang, Heilongjiang, China) and identified through 16S rRNA from 40 strains. The optimal wheat bran, carbon, nitrogen, inoculum size, volume and initial pH were 60 g/L, soluble starch 12.5 g/L, corn steep liquor 3 g/L, 5%, 40 mL and 6.15, respectively for improving DB219 MCE production through single factor experiment. The wheat bran concentration, corn steep liquor concentration and volume were the most critical factor and their changed range was determined through Plackett–Burman design and the steepest ascent/descent experiments. The response surface analysis experiment of three factors and three levels was conducted by Box–Behnken design. The theoretical optimal fermentation conditions for DB219 MCE were as follows: wheat bran concentration 60.14 g/L, soluble starch 12.5 g/L, corn steep liquor 3 g/L, inoculum size 5%, volume 40.08 mL and initial pH 6.15. DB219 MCE achieved the maximal MCA (3164.84 SU/mL) that was 101.9% of the predicted value (3104.49 SU/mL) and 4.3-fold higher than the control.

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Characterization of heat, salt, acid, alkaline, and antibiotic stress response in soil isolate Bacillus subtilis strain PSK.A2

Kadapure,

Dalbanjan,

S K

2024

Int Microbiol

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Isolation of lysozyme producing Bacillus subtilis Strains, identification of the new strain Bacillus subtilis BSN314 with the highest enzyme production capacity and optimization of culture conditions for maximum lysozyme production

Cited by 14 publications

References 91 publications

Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria

Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria

Isolation and production optimization of a novel milk-clotting enzyme Bacillus velezensis DB219

Characterization of heat, salt, acid, alkaline, and antibiotic stress response in soil isolate Bacillus subtilis strain PSK.A2

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