Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase.

Rosa-María Oliart-Ros,Alfonso Alexander-Aguilera,Giselle-Lilian Badillo-Zeferino,Jorge-Guillermo Domínguez-Chávez,Dinh Duc Nguyen,Ashok Kumar Nadda,Irving-Israel Ruíz-López,María-Guadalupe Sánchez-Otero,Azmat Ali Khan,Rodolfo Quintana-Castro

doi:10.3390/molecules26247569

Abstract

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.

Highlights

Lipases (E.C. 3.1.1.3) are ubiquitous enzymes, as they have been found to occur in most living organisms; in vivo, they catalyze the hydrolysis of triacylglycerides to glycerol and fatty acids [1]
For LipMatCCR11-cross-linked enzyme aggregates (CLEAs) preparation, glutaraldehyde was selected as the reticulant agent since LipMatCCR11 has 11 lysine residues in its structure, none of which is directly involved in the active site or the domains of interaction with Ca+2 and Zn+2 (Figure 1A)
The immobilization of the recombinant thermoalkaliphilic lipase LipMatCCR11 in the form of cross-linked aggregates (CLEAs) allowed us to obtain a robust biocatalyst in solid form, free of support, with greater thermostability, and reusable and resistant to storage

Summary

Introduction

Lipases (E.C. 3.1.1.3) are ubiquitous enzymes, as they have been found to occur in most living organisms; in vivo, they catalyze the hydrolysis of triacylglycerides to glycerol and fatty acids [1]. Some of them possess a flexible alphahelix (called “lid”) covering their active site, which in its “closed conformation” makes it inaccessible to substrates; this form exists in equilibrium with an “open form”. In the presence of hydrophobic interfaces, the equilibrium between the open and closed forms shifts to the “open form” through important conformational changes, where the active site becomes accessible and hydrophobic surfaces are exposed, increasing enzyme activity, a phenomenon called interfacial activation. This phenomenon is very important for all applications of lipases [8,9]. Thermoalkaliphilic lipases are interesting due to their high stability towards high temperatures and pH, chaotropic agents, detergents, and some protease activity [1,6,10]

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