1. The document describes the extraction and purification of lactase (β-galactosidase) from a local isolate of Lactobacillus acidophilus. 2. Four L. acidophilus strains were screened for lactase production, with strain Lac4 showing the highest activity and selected for further study. 3. The enzyme was purified using ammonium sulfate precipitation, dialysis, gel filtration chromatography, and polyacrylamide gel electrophoresis, increasing its specific activity and purification fold at each step.

1. Extraction and Purification of Lactase (-galactosidase) from a
Local Isolate of Lactobacillus acidophilus
Rafal Ahmad, Mohammed A. Jebor, Anwara, Abdulla & Rasha K. Mahdi, 2014
Presented By,
Saiful Islam
Jahangirnagar University
BGE-503
Enzyme Technology
Lactase

2. Introduction (Lactase EC 3.2.1.23)
 Hydrolytic enzyme
 Has two enzymatic activity
 Hydrolysis of lactose and cleaves cellotetrose and
cellotriose
 Splitting of -glycosides
 Hydrolyzes lactose into glucose and galactose
 Caters to lactose intolerance people
 Two-third people of this world suffering from
lactose intolerance

3. Commercial applications of Lactase
 Food
 Texture and flavor of food products
 Pharmaceutical industries
Commercial Lactase is produced from
microorganisms including
1. Bacteria
2. Yeast
3. Mold

4. Materials and Methods
 Bacterial Strains and Growth Conditions
 Detection of Lactase Produced by Lactobacillus acidophilus
 Preparation of O-Nitrophenol Standard Curve
 Enzyme Extraction
 Measurement of Lactase Activity
 Determination of Protein Concentration
Purification of Lactase Enzyme
• Precipitation with Ammonium Sulfate
• Dialysis
• Enzyme Separation through Gel Filtration
Chromatography
• Protein Analysis and Gel Electrophoresis

5. Results and Discussions
 Four lactobacillus acidophilus (Lac1, Lac2, Lac3 and Lac4) were screened
 All the strains were found to be lactose producing
 But the strain that has the highest activity (Lac4) was selected for this study.
 The enzyme was purified using precipitation by ammonium sulfate, dialysis, gel
filtration chromatography, and polyacrylamide gel electrophoresis.
Table1: Enzyme Activity and Protein Concentration for Lactobacillus acidophilus

6. 1. Precipitation with Ammonium Sulfate
• In order to concentrate the crude extract
• 80% saturation was the best ratio
• depends on the salting out phenomenon
Table 2: Ratios of Ammonium Sulfate Precipitation of Lactase Enzyme

7. • Specific activity reached to (0.00000108) U/mg
• Purification fold (4.7)
• Yield (73%) as shown
• The ammonium sulfate was used in enzyme precipitation because of its
• high solubility
• low cost compared with other salts
• did not hamper pH and enzyme stability
Table 3: Purification Steps of Lactobacillus acidophilus Lactase Enzyme

8. 2.Dialysis
• Dialyzed overnight against phosphate buffer
• Specific activity reached to (0.00000154) U/mg
• Purification fold of 6.7
• Yield of 55.7%
3. Gel Filtration Chromatography
• Gel filtration on sephadex G-200 column (15*1 cm)
• Produced one peak of purified enzyme
• The specific activity of the enzymes from the harvested peak was (0.0000075)
U/mg
• Purification fold of 6.9
• Yield of 33.9%

9. Figure 1: Gel Filtration Chromatography Using DEAE-Cellulose Sephadex G-200 Column
(15×1) Cm with Phosphate Buffer (PH 7), Flow Rate12 Ml/Hr and Fraction Volume 3ml for
Purification Lactase

10. 4.Gel Electrophoresis of Lactase
• In order to investigate the purity of the lactase
• There was one band with molecular weight 93 KDa
93 kDa

11. Lactase
From
Lactobacillus
acidophilus
Precipitation with ammonium sulphate
Dialysis
gel filtration using sephadex
G-200 chromatography
MW 93 KDa using the SDS-PAGEExtraction and Purification
Applications
Lactose intolerance
Food industry Dairy industry
Pharmaceutical
industries
Conclusion

12. THANK YOU

13. ANY QUESTIONS
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