| In recent years, although the study of enzyme promiscuity has been widelyconcerned, there is no a simple and rapid method to exploring it, and the number of natureenzymes is limited. The activities of some natural enzymes for some substrate arerelatively low. Therefore, researching on the enzyme promiscuity mechanism, andestablishing effective enzyme promiscuity evolution strategy, Is not only a challengingjob for protein engineering and the biological engineering, but also a problem need tosolve for today’s industrial production. Especially for the enzymes which have beenwidely used in the industry. Compared to the other lipase, CalB has many excellentproperties, so it attracted more and more people to pay attention to. Candida Antarcticalipase B (CalB) is extracted from Candida Antarctica. Because of its special structure,CALB have very strong catalytic activity not only for a non-water soluble substances, butalso for water soluble matter, it also have a high degree of stereoselectivity in thehydrolysis and in organic synthesis reactions. But up to now, the expression of CalB indeveloped mature high efficiency expression system is very low, and the product has poorstability, wild type of CalB has a low catalytic efficiency for longer carbon chainssubstrate compounds, all those make the CalB large-scale application in industry isrestricted. By using the method of bio engineering for directional modification of theCalB, expecting to get a new lipase which have high activity not only for short but alsofor long chain substrate compounds, and the new lipase still keep its stability, has becomeone of the the current hot spot.In this paper, we take the Candida Antarctica lipase B (Candida Antarctica lipase B,CalB) as the object of study, First of all, we optimized the induction conditions of therecombinant strains of pET-22b-CalB-Rosetta (DE3) we got. We get a best inducibleexpression of conditions, which make us got about improved approximately10foldsamount of protein expression compared to the amount of the same period. By using themethod of computer simulation, we make the wild type CalB and different types ofsubstrates docking. According to the reaction mechanism of enzyme and substrate, weobtain ideal conformation docking products, Analysis of the main interaction amino acidsof the enzyme and substrate. At the same time, we measured the distance between theseamino acids and the activity center. Then we identified the amino acids mutation sites. Wethen according to the CASTing method combine the mutation sites, group A(Thr138, Val139), B (Ile189, Val190), C (Leu278), D (Ala281, Ala282), E (Ile285), We designedNDT (double site mutation) and NNK (single site mutation) degenerate primers, Usingthe method of iterative saturation mutagenesis to mutate the mutation group, After threerounds of mutation we obtained a activity increased about10fold mutant strain281F-282R/189I/138V-139C compared to the wild type. The mutants are homologymodeling, then docking with the substrate p-nitrophenyl octanoate. We analyzed the causeof increased activity of the mutants. We speculate that the increased activity may beassociated with the mutant protein intramolecular hydrophobic interaction, at the sametime for the docking results analysis, we found that, to improve the hydrophobicinteraction between amino acid of the activity center and the substrate, to reduce thedistance of the hydrophobic amino acid and substrate, regional pockets becomeappropriate larger, are likely to increase the activity of enzyme to substrate.In this paper, we explore the effects of the active center region evolution on theprotease activity, and from the perspective of computational biology, we analyze thepossible causes of the effects that the active center region evolution for enzyme activity.And research strategies which may change enzyme catalytic promiscuity. This study isexpected to provide a new source of enzyme of high activity and stability for thebiological enzyme catalysis. |
PDF Full Text Request