Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24
The current study characterized β-galactosidase-producing bacteria isolated from dairy products, in Lahore, Pakistan. Biochemical tests and the 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) hydrolysis test identified 50 Lactobacillus isolates. Fifteen isolates with blue color on X-gal plates, L...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2024-01-01
|
| Series: | Current Research in Biotechnology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S259026282400087X |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850173218231418880 |
|---|---|
| author | Arifullah Dilara Abbas Bukhari Zuhra Bibi Habiba Ramzan Samina Younas Abdul Rehman |
| author_facet | Arifullah Dilara Abbas Bukhari Zuhra Bibi Habiba Ramzan Samina Younas Abdul Rehman |
| author_sort | Arifullah |
| collection | DOAJ |
| description | The current study characterized β-galactosidase-producing bacteria isolated from dairy products, in Lahore, Pakistan. Biochemical tests and the 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) hydrolysis test identified 50 Lactobacillus isolates. Fifteen isolates with blue color on X-gal plates, L. acidophilus MR-24 displayed the highest enzyme activity (905.15 U/l), characterized based on 16S rRNA gene sequencing. The enzymatic activity was enhanced 10-fold by using a Sephadex G-75 column. The highest enzyme production was obtained at pH 7.0 and a temperature of 37 °C. The β-galactosidase (lac M, L, and LM with molecular weight 951 bp, 1887 bp, and 2.8 kb respectively), was extracted from L. acidophilus MR-24. It was ligated to the pTZ57R cloning vector after Polymerase chain reaction (PCR) and agarose gel analysis. The confirmation of cloning was done via colony PCR and restriction digestion analysis. Sequencing data indicated that the enzyme consists of two overlapping regions; lac L and M encoding 70 kDa and 35 kDa protein respectively. The β-galactosidase shows significant homology with the gene from other Lactobacillus sp. Compared to crude enzymes, the co-expression of lac L and M in E. coli produced active proteins with a 30-fold increase in activity after purification via ion-exchange chromatography. The purified enzyme revealed its maximal activity at pH 7 while pH 3.0 and 9.0 showed minimal activity. The optimum temperature was 60 °C for native and 45 °C for expressed enzyme, remaining 40 % activity at 90 °C. The enzyme’s 3-dimensional (3D) structure showed Domain N, A, and C. The L. acidophilus MR-24 strain, as a probiotic in dairy products, can provide benefits to individuals with lactose intolerance. |
| format | Article |
| id | doaj-art-315d02d0dae24fc8a673d7efe07bcaf5 |
| institution | OA Journals |
| issn | 2590-2628 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Current Research in Biotechnology |
| spelling | doaj-art-315d02d0dae24fc8a673d7efe07bcaf52025-08-20T02:19:54ZengElsevierCurrent Research in Biotechnology2590-26282024-01-01810026110.1016/j.crbiot.2024.100261Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24 Arifullah0Dilara Abbas Bukhari1Zuhra Bibi2Habiba Ramzan3Samina Younas4Abdul Rehman5Department of Zoology, Government College University, Lahore, PakistanDepartment of Zoology, Government College University, Lahore, PakistanDepartment of Zoology, Government College University, Lahore, PakistanDepartment of Zoology, Government College University, Lahore, PakistanDepartment of Zoology, Government College University, Lahore, PakistanInstitute of Microbiology and Molecular Genetics, University of Pujnab, Quaid-e-Azam Campus, 54590, Lahore, Pakistan; Corresponding author at: Department of Microbiology and Molecular Genetics, University of Punjab, New Capmus, Lahore 54590, Pakistan.The current study characterized β-galactosidase-producing bacteria isolated from dairy products, in Lahore, Pakistan. Biochemical tests and the 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) hydrolysis test identified 50 Lactobacillus isolates. Fifteen isolates with blue color on X-gal plates, L. acidophilus MR-24 displayed the highest enzyme activity (905.15 U/l), characterized based on 16S rRNA gene sequencing. The enzymatic activity was enhanced 10-fold by using a Sephadex G-75 column. The highest enzyme production was obtained at pH 7.0 and a temperature of 37 °C. The β-galactosidase (lac M, L, and LM with molecular weight 951 bp, 1887 bp, and 2.8 kb respectively), was extracted from L. acidophilus MR-24. It was ligated to the pTZ57R cloning vector after Polymerase chain reaction (PCR) and agarose gel analysis. The confirmation of cloning was done via colony PCR and restriction digestion analysis. Sequencing data indicated that the enzyme consists of two overlapping regions; lac L and M encoding 70 kDa and 35 kDa protein respectively. The β-galactosidase shows significant homology with the gene from other Lactobacillus sp. Compared to crude enzymes, the co-expression of lac L and M in E. coli produced active proteins with a 30-fold increase in activity after purification via ion-exchange chromatography. The purified enzyme revealed its maximal activity at pH 7 while pH 3.0 and 9.0 showed minimal activity. The optimum temperature was 60 °C for native and 45 °C for expressed enzyme, remaining 40 % activity at 90 °C. The enzyme’s 3-dimensional (3D) structure showed Domain N, A, and C. The L. acidophilus MR-24 strain, as a probiotic in dairy products, can provide benefits to individuals with lactose intolerance.http://www.sciencedirect.com/science/article/pii/S259026282400087XLactobacillus acidophilus MR-24β-galactosidaseLactoseGene cloningGene expressionEnzyme characterization |
| spellingShingle | Arifullah Dilara Abbas Bukhari Zuhra Bibi Habiba Ramzan Samina Younas Abdul Rehman Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24 Current Research in Biotechnology Lactobacillus acidophilus MR-24 β-galactosidase Lactose Gene cloning Gene expression Enzyme characterization |
| title | Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24 |
| title_full | Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24 |
| title_fullStr | Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24 |
| title_full_unstemmed | Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24 |
| title_short | Heterodimeric studies of β-galactosidase genes as biocatalyst of lactose from Lactobacillus acidophilus MR-24 |
| title_sort | heterodimeric studies of β galactosidase genes as biocatalyst of lactose from lactobacillus acidophilus mr 24 |
| topic | Lactobacillus acidophilus MR-24 β-galactosidase Lactose Gene cloning Gene expression Enzyme characterization |
| url | http://www.sciencedirect.com/science/article/pii/S259026282400087X |
| work_keys_str_mv | AT arifullah heterodimericstudiesofbgalactosidasegenesasbiocatalystoflactosefromlactobacillusacidophilusmr24 AT dilaraabbasbukhari heterodimericstudiesofbgalactosidasegenesasbiocatalystoflactosefromlactobacillusacidophilusmr24 AT zuhrabibi heterodimericstudiesofbgalactosidasegenesasbiocatalystoflactosefromlactobacillusacidophilusmr24 AT habibaramzan heterodimericstudiesofbgalactosidasegenesasbiocatalystoflactosefromlactobacillusacidophilusmr24 AT saminayounas heterodimericstudiesofbgalactosidasegenesasbiocatalystoflactosefromlactobacillusacidophilusmr24 AT abdulrehman heterodimericstudiesofbgalactosidasegenesasbiocatalystoflactosefromlactobacillusacidophilusmr24 |