Elsevier

Process Biochemistry

Volume 101, February 2021, Pages 26-35
Process Biochemistry

Stabilization of recombinant d-Lactate dehydrogenase enzyme with trehalose: Response surface methodology and molecular dynamics simulation study

https://doi.org/10.1016/j.procbio.2020.11.001Get rights and content

Highlights

  • Trehalose has a protective effect on the d-Lactate dehydrogenase (DLDH) structure.

  • RSM design indicated the best conditions for DLDH stabilization with trehalose.

  • Enzyme random coil convert to β-sheet and α-helix in the presence of trehalose.

  • Obtained results confirm that the treated enzyme is more stable than the free one.

  • The enzymatic catalytic efficiency of the DLDH increases in the presence of trehalose.

Abstract

In this work, the effect of trehalose was investigated on the structural stability of the d-Lactate dehydrogenase (DLDH) enzyme. Initially, response surface methodology (RSM) was applied to optimize different process variables for the stabilization of DLDH using trehalose. The stabilization of DLDH with trehalose was examined under various experimental conditions, including trehalose concentration, pH, temperature, and incubation time. The effect of the processing parameters was tested using the RSM method and a central composite design (CCD) model. According to the ANOVA results, there was a close correlation between the predicted and experimental values of the response parameter. The optimum values of trehalose concentration, pH, temperature, and incubation time were found to be 1.25 M, 8, 30 °C, and 25 min, respectively, for achieving the maximum activity. Then, the kinetic and thermodynamic parameters of DLDH in the absence and presence of trehalose were examined. The DLDH half-life at 40 °C was 28.88 min, while, in the presence of trehalose, the enzyme had a longer half-life (33.01 min) at this temperature. Also, in the end, molecular dynamics (MD) simulations of the DLDH enzyme was performed at the optimum conditions to obtain fundamental insights into the conformational transitions of the DLDH enzyme in the absence and presence of trehalose.

Introduction

Lactate dehydrogenase enzymes offer a wide range of catalytic properties. NAD-dependent l-lactate dehydrogenase (l-nLDH) and NAD-dependent d-Lactate dehydrogenase (d-nLDH) catalyze the conversion of pyruvic acid to lactic acid [1]. Lactic acid fermentation is utilized in food processing and has different medicinal and chemical applications [1,2]. Nowadays, the main industrial application of lactic acid is producing polylactic acid, a biodegradable polymer [3,4]. Lactic acid can be produced through either microbial fermentation or chemical synthesis whereby microbial fermentation has recently received attention because of its purer lactate production [3]. d-Lactate dehydrogenase (DLDH) is not produced in the body and is applied as a marker of disease diagnosis such as acute appendicitis, bacterial infection [5], and kidney damage [6,7]. DLDH (EC 1.1.1.28) catalyzes the NADH-dependent conversion of pyruvate to D-lactic acid and the reverse reaction [8]. This enzyme is applied in diagnostic biosensors to determine the types of diseases associated with increased d-Lactate concentration in urine or serum [9]. So far, several biosensors have been used for d-Lactate detection [6,10,11]. For example, Satomura and co-workers developed a stable d-Lactate electrochemical sensing system using a dye-linked DLDH [12]. The low stability of enzymes limits their use in various industries while adding stabilizing agents is one of the simplest and preferred methods for increasing the enzyme thermo-stability [13]. Osmolytes are small organic molecules involved in maintaining osmotic pressure in cells, stabilizing the native conformation of proteins, and protecting their structure from different types of stress [14,15]. Osmolytes are usually natural compounds from different groups of polyols, sugars, methylamines, and amino acids. Compounds such as proline, glycine betaine, glycerol, sorbitol, trehalose, 4-phenyl butyric acid, trimethylamine-N-oxide, and 6-aminohexanoic acid are found in microorganisms, animals, and plants [14]. Osmolytes accumulate in the cells under adverse environmental conditions (high salt concentrations, high temperatures, or desiccation) and protect proteins from denaturation as well as their loss of function [[16], [17], [18]]. Protecting osmolytes such as polyols which include sucrose, trehalose, glycerol, and other sugars enhance the protein stability via preferential exclusion, while non-protecting osmolytes, such as urea, destabilize protein structures [[19], [20], [21]]. It was shown that the protective effects of various osmolytes are the result of increasing the stability of the protein structure [22]. Trehalose is a common molecule among the compatible polyols utilized by nature to protect organisms at high and low temperatures [23]. Also, trehalose is known to be the best stabilizer for the function and structure of numerous macromolecules. It can also enhance the stability of folded protein states against denaturation conditions [[24], [25], [26]]. Plants, insects, and certain organisms such as yeast and nematodes produce trehalose molecules as a protecting agent of biomolecules under non-physiological conditions [27,28]. One hypothesis that has been proposed to explain the stabilizing effect of trehalose on proteins at the molecular level is that the trehalose molecules are replaced by water molecules on the protein surface and form hydrogen bonds at specific sites on the protein surface [29,30]. Although the trehalose effect on biomolecules has been broadly investigated, the detailed mechanism responsible for its protective ability at the molecular level has remained unknown so far. Herein, the effect of trehalose on the structural stability of DLDH was examined. Initially, the process variables were optimized using response surface methodology (RSM) to stabilize the DLDH enzyme with trehalose in the best situation. The RSM is a set of statistical and mathematical techniques which is beneficial for analyzing the influence of the independent variables [31]. In RSM, the interaction of several variables can be examined simultaneously. In the end, the molecular dynamics (MD) simulations were utilized to examine the conformational changes caused by trehalose within the structure of the enzyme.

Section snippets

Materials

Escherichia coli BL21 strain was obtained from Iranian GenBank (Pasteur Institute of Iran), Phosphate buffered saline (PBS), trehalose, Tris/HCl, NADH, and sodium pyruvate were purchased from Sigma-Aldrich Corporation. Glycine, sodium acetate (CH3COONa), and Bradford solution for determining the protein concentration were obtained from Sinaclon Co. KH2PO4 and K2HPO4 were purchased from Merck company. All other chemicals were of analytical grade and obtained from Merck & Co., Inc (Kenilworth,

Experimental design

In conventional optimization methods, a variable would be applied for monitoring the operating penetration of parameters, which was an expensive and time-consuming technique. Nowadays, the multivariate statistical procedures are utilized for optimizing the effective parameters [58,59].

Conclusion

In this study, the central composite design (CCD) model was applied to design experiments for visualizing the effect of independent variables to stabilize the DLDH enzyme with trehalose. Four independent variables include the trehalose concentration, pH, temperature, and incubation time. The quadratic polynomial model was selected to predict the value of the trehalose treated-DLDH activity. The obtained results suggest that estimating the optimal conditions to obtain the maximum activity using

CRediT authorship contribution statement

Mahdiye Zaboli: Methodology, Writing - review & editing, Formal analysis. Faranak Saeidnia: Data curation, Conceptualization. Maryam Zaboli: Writing - review & editing, Formal analysis, Validation, Investigation, Software. Masoud Torkzadeh-Mahani: Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgment

We would like to thank the Graduate University of Advanced Technology for the support of this work.

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