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More details about current works on enzymes conducted at PNNL will be updated soon!
The Relationship between Enhanced Enzyme Activity and Structural Dynamics of Lipase, Candida antarctica Lipase B
The enzyme is a biological catalyst that has been widely used for many industrial applications because it is selective for specific substrates and capable of catalyzing only desired chemical reactions under the mild conditions. Specifically, production of ester and amide derivatives using enzymes is the most effective process for a plethora of high-value products including biofuels, cosmetics, personal care, and pharmaceutical products. Unlike most other enzymes, lipase B from Candida antarctica (CALB) is distinguished by effectively catalyzing both hydrolysis and synthesis reactions with high enzyme activity, structural and thermal stability. However, despite many advantages of using CALB as an enzyme catalyst, it has been reported that the activity of CALB can be significantly reduced when bulky or non-linear carboxyl acids are used as the substrates, such as acids with big alcohol moiety (e.g. benzyl group). Since these branched or sterically demanding substrates are often required as sources for the commercial products in various markets from benzoate ester plasticizers for PVC products to benzoate ester emollients and stabilizers in personal care products, it is imperative to overcome these drawbacks and find the way to specifically increase the ability to react with bulky acids. However, due to the fact that experimental approach that performs iterative process evolving genes in vitro or selecting reaction solvents solely relies on the entirely random event, it is obvious to require a great deal of time and effort to obtain desired properties. Furthermore, an experimental approach cannot fully explain the reason why specific mutations or solvents lead to functional changes of the enzymes.
MD simulations have been used as a versatile tool for design or guidance of many experimental procedures. During this research period, I utilized MD simulations to find the optimal solvents and potential mutation sites for better enzyme activity of CALB.
Effect of Solvents
03. 2010 – 08. 2012 supported by the Ministry of Knowledge Economy, Korea through the Cleaner Production Technology Development Project.
This research was collaborated with Dr. Yoon-Mo Koo (Inha Unversity, Korea), Dr. Sung Ho Ha (Hannam University, Korea)
First, by performing computation modeling, I proposed rules for the selection of solvents that can stabilize CALB resulting in increased enzyme activity. Specifically, my simulation results discovered the effect of cation and anion on structure and dynamics of CALB. Based on the observations and conclusions from MD simulation, I suggested the favorable and unfavorable solvents for enzyme reactions, which are composed of specific cation - anion combinations, such as [Bmim][TfO] as the best ionic liquids and [Bmim][Cl] as the worst one. When I was a graduate student and research assistant at Inha University in 2010 to 2012, I experimentally verified that this prediction led to a higher enzyme activity for ester and amide products. After I conducted research at North Carolina State University, Dr. Koo group at Inha University and Dr. Sung Ho Ha of Hannam University in South Korea carried out experiments to support my simulation results. I truly expect that this research can provide insight into choosing optimal cation and anion combinations for enzyme reaction solvents without a great deal of time and effort that often requires experimental approach. As a successful result, I have published three research papers (see [1] – [3] in the Journal Paper sections).
Enzyme Mutations
10. 2013 – 12. 2015, Supported by Eastman Chemical Company
This research was collaborated with Dr. Stephanie K. Clendennen at Eastman Chemical Company (Kingsport, TN)
Second, I developed a new strategy for enzyme engineering using MD simulation techniques and found the critical mutation sites of amino acids for a better enzyme activity. Specifically, from this new modeling-based strategy, I was able to propose a design for the CALB mutants with specific amino acid changes adjacent to a catalytic cavity; these amino acid sites are heavily related to lipase activity and serve as gates for substrate ingress and egress. My predictions were experimentally verified by our collaborator, Dr. Stephanie Clendennen of Eastman Chemical Company (Kingsport, TN) and we have obtained a CALB lipase mutant with 12-fold higher productivity than the wild type. We expect that our findings with new leading mutant can result in the significant impact on reducing process cost of the aforementioned high-value product. This combined experiment and computation study resulted was filed for a U.S. patent which includes sequences of CALB mutants as well as the new computation approach that I developed (see [1] in a Patent section below). Moreover, Eastman Chemical company plans to use the mutants that we found in their process lines after all the patent processes are finalized.
Journal Papers
#: Brief explanation about the paper[1] Ho Shin Kim, Doyoung Eom, Yoon-Mo Koo, and Yaroslava G. Yingling. “Role of imidazolium cation on structure and activity of Candida antarctica lipase B enzyme in ionic liquids”, Physical Chemistry Chemical Physics 18, 32 (2016): 22062-22069, #Effect of solvent (cation) on CALB# Featured on journal cover
[2] Ho Shin Kim, Sung Ho Ha, Latsavongsakda Sethaphong, Yoon-Mo Koo, and Yaroslava G. Yingling. "The relationship between enhanced enzyme activity and structural dynamics in ionic liquids: a combined computational and experimental study." Physical Chemistry Chemical Physics 16, 7 (2014): 2944-2953 #Effect of solvent (anion) on CALB#
[3] Ho Shin Kim, and Yoon-Mo Koo. "Effects of physicochemical properties of ionic liquids on butyl acetate synthesis using Candida antarctica lipase B." Korean Journal of Chemical Engineering 29, 11 (2012): 1610-1614 #Effect of solvent: experiment#
Patents
#: Brief explanation about the paper[1] Stephanie K. Clendennen, Yaroslava G. Yingling, and Ho Shin Kim, “CALB variants”, US Patent App., 14/961,237 (2015)
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