Production of recombinant a-amylase from Bacillus subtilis by Kluyveromyces lactis
Nascimento, Bruno Motta
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Abstract: Amylolytic enzymes catalyze the hydrolysis or the modification of starch structure. Many organisms are able to produce these enzymes, but only a few of them exhibit satisfactory characteristics for industrial application. The most common commercial a-amylases with desirable industrial characteristics are produced by bacteria from the genus Bacillus. Although the yield of a-amylase produced by these bacterial strains can be increased with culture optimization, the recombinant production of the enzyme could offer some advantages such as easier genetic manipulation/mutation, inducible expression, faster production, higher yields and easier purification steps. The present work proposes the production of a recombinant a-amylase in Kluyveromyces lactis. A Bacillus subtilis NRRL B-4212 strain provided the a-amylase gene, which was amplified and inserted in a pKLAC2 vector in four different ways: (i) with both the B. subtilis signal sequence and K. lactis leader sequence; (ii) with only the yeast leader or (iii) only the B. subtilis signal, and (iv) without any signal or leader sequence. The final constructs were linearized and the cassettes were used for K. lactis CBS 2359 cells transformation. After growth in a selective medium, the positive yeast colonies were assayed for enzymatic activity on agar plates and liquid medium. It was expected that cells with construct (i) and cells with construct (ii) would present extracellular enzymatic activity. On the other way, transformants with constructs (iii) and transformants with construct (iv) should present an intracellular activity. However, only constructs (ii) and (iv) behaved as expected. Construct (i) could not secrete the enzyme, indicating that the presence of both signals might interfere in the secretion machinery, and construct (iii) presented a high extracellular activity (1200 U/L), even though it did not have the yeast leader sequence, indicating that the yeast cell probably can also recognize the bacterial signal peptide. One sample from each construct was selected to determinate the enzyme’s optimal temperature and pH, obtaining results between 50 an 55 °C, which is consistent with other observed values for Bacillus amylases. Transformant LAC-EXAMY 4 was also cultured in different carbon sources to observe cellular growth and enzyme production. The recombinant yeast was able to quickly grow in the presence of glucose, sucrose and lactose. Even without the induction with lactose or galactose the enzyme was produced with a basal level (0.44 U/mL). However, the presence of these carbon inducers greatly enhances the production yield, achieving 2.21 U/mL with lactose and 2.55 U/mL with galactose as the carbon source.
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