Abstract
The present study aim was to isolate bacterial strains, producing protease, from soil samples of different industrial wastes and dumping areas of slaughter houses. For this, 25 bacterial isolates forming clear zones in skimmed milk agar plates as a result of casein hydrolysis were selected. One bacterial isolate showing the highest protease activity (400 U/ml) was identified as Bacillus subtilis through 16S rRNA gene sequencing. Full-length alkaline protease gene was amplified, sequenced, cloned in expression vector pT7-7, and transformed in E. coli BL21C. Over-expression of protease gene was determined in isopropyl β-d-1-thiogalactopyranoside (0.5 mM) at 37 °C for 3 h. Expressed alkaline protease was purified and 10 folds increased enzyme activity was determined as compared to the wild type B. subtilis DMA-09. The purified protease was detected on SDS-PAGE as a single band of 31 kDa and was stable at 90 °C and 85% enzyme activity was retained at 100 °C. The optimum enzyme activity was obtained at pH 10 and was enhanced in the presence of 5 mM each Mg2+ and Ca2+ while phenylmethylsulfonyl fluoride (PMSF) inhibited its activity at 0.5 mM. The expressed purified alkaline protease may find potential applications in food and biotechnological industries.
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Abbreviations
- SK:
-
Skimmed milk
- LBUV:
-
Luria–Bertani
- NaCl:
-
Sodium chloride
- DNA:
-
Deoxy ribonucleic acid
- IPTG:
-
Isopropyl-β-D-thiogalactopyranoside
- dNTPs:
-
deoxyribonucleotide triphosphates
- PCR:
-
Polymerase chain reaction
- NCBI:
-
National center for biotechnology information
- BLAST:
-
Basic local alignment search tool
- OD:
-
Optical density
- CM:
-
Carboxymethyl
- NaOH:
-
Sodium hydroxide
- KCl:
-
Potassium chloride
- MgCl2 :
-
Magnesium chloride
- EDTA:
-
Ethylenediaminetetraacetic acid
- PMSF:
-
Phenylmethylsulfonyl fluoride
- pCMB:
-
p-chloromercuribenzoate
- DFP:
-
Di-isopropyl fluorophosphates
- kDa:
-
Kilodalton
- ORF:
-
Open reading frame
- SDS:
-
Sodium dodecyl sulphate
- PAGE:
-
Polyacrylamide gel electrophoresis
- mL:
-
Milliliter
- M:
-
Molar
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DAB designed, performed experiments, and analyzed the results. AB helped in conducting experiments. AR supervised the study and helped in manuscript preparation.
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Bukhari, D.A., Barkat, A. & Rehman, A. Expression, purification, and molecular characterization of a full-length thermostable alkaline protease gene from Bacillus subtilis DMA-09. Biologia 76, 741–750 (2021). https://doi.org/10.2478/s11756-020-00608-6
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DOI: https://doi.org/10.2478/s11756-020-00608-6