| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | Saturation mutagenesis was performed to study the relationship between the structure and function of the recombinant LOXs using the overlap extension PCR (SOE-PCR) method [17] and the primers listed in Table 1. PCR was performed through three separate PCR reactions and the whole process was divided into two steps. First, two separate PCR reactions were implemented at the same time to get two fragments (upstream and downstream fragments with mutations) by using two pairs of primers. Second, the third PCR reaction was performed to obtain the full length gene using the purified products from the previous two PCRs as templates. Clones were picked with sterile toothpicks and transferred into separate shake flasks for screening. To verify the mutation that had been introduced into gene, each mutant was sequenced by Genscript (Nanjing, China). | Get A Quote |
A sensitive and reliable high-throughput approach to screen lipoxygenase (LOX) mutants was developed to permit a directed evolution study in an attempt to increase the activity and thermostability of LOX from Anabaena sp. PCC 7120 using error-prone polymerase chain reaction (PCR) and DNA shuffling. The best mutant, named D22, contained one key amino acid change (N305D) and its half-life of inactivation at 50 °C was increased by approximately twofold in comparison to the wild-type enzyme, while specific activity 1.87-fold higher than the wild-type enzyme. The N305D substitution was confirmed to be critical for the thermostability and catalytic activity of LOX by site-saturation mutagenesis. Indeed, the react... More
