A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: Characterization and application to enzymatic biodiesel production
Introduction
Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) make up a versatile group of enzymes that have the ability to hydrolyze triglycerides at the lipid-water interface. Lipases show wide substrate specificity and they are often used in chemo-, enantio- and stereo-selective reactions of biotechnological importance [1], [2].
Although immobilization of lipases may produce some improvements in enzyme features, like stability, activity, selectivity or specificity [3], [4], this process has a cost that in certain cases may promote some difficulties to the implementation of the processes. To overcome this drawback, we focused on a whole-cell biocatalyst, which enables the direct use of lipase-producing microorganisms. Among several microorganisms, Aspergillus oryzae is a promising host because it has high protein productivity in the expression of heterologous genes using improved promoters [5], [6] and can easily be immobilized on porous biomass support particles (BSPs [7]). Thus far, A. oryzae strains that have been genetically engineered to express several microbial lipases have been developed for use as whole-cell biocatalysts in biodiesel production and enantioselective transesterification [8], [9], [10], [11]. Since practical conditions that promote the reaction include a high temperature and a high concentration of organic solvents, there is a necessity to develop a robust whole-cell biocatalyst. Therefore, introducing robust lipases into A. oryzae would further expand the application of this technology.
Lipases from thermophiles often show their extreme stability at elevated temperature and in organic solvents [12]. Thus, they have become objects of special interest for structural investigations and also for industrial applications [13]. The lipase from Geobacillus thermocatenulatus (BTL2), isolated by Schmidt-Dannert et al. [14], is an interesting lipase possessing unique structural characteristics. BTL2 comprises 389-amino-acid residues with a molecular mass of 43 kDa, which is relatively larger than those of other microbial lipases [15]. In contrast to most lipases, BTL2 has two lids and a zinc-binding domain that is typically observed in the same family [15]. Moreover, BTL2 shows high stability toward moderately high temperature (40 °C) and organic solvents [14]. Such excellent characteristics of BTL2 have led to extensive studies such as high level expression in Escherichia coli [16] and Pichia pastoris [17], immobilization on various kinds of supports [18], [19], [20], [21], and applications including the kinetic resolution of chiral substrates [22] and aliphatic ester synthesis [20].
In the present study, a recombinant A. oryzae whole-cell biocatalyst expressing BTL2 (r-BTL) was developed. The characteristics of r-BTL were evaluated and compared with those of a conventional A. oryzae whole-cell biocatalyst expressing Fusarium heterosporum lipase (r-FHL), which provides high alkyl ester contents of more than 90% during alcoholysis [23]. In addition, r-BTL was used for biodiesel production from palm oil, which is one of the most abundant, cheapest and available vegetable oils and would be a more sustainable biodiesel-feedstock than rapeseed oil, as shown in life cycle assessments [24], [25]. However, because of the low fluidity of palm oil at room temperature, a moderately high temperature is desirable for processing reaction mixtures in transesterification. Given the high thermostability of BTL2, r-BTL was applied to the methanolysis of palm oil at a moderately high temperature.
Section snippets
Strains and chemicals
A. oryzae niaD300, which is a niaD mutant derived from the wild type strain RIB40, was used as a recipient strain for transformation. The E. coli strain used for gene manipulation was Nova Blue {endA1 hsdR17 (rK12− mK12+) supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB+ lacIqZΔM15::Tn10 (Tetr)]} (Novagen, Madison, WI, USA). Reticulated polyurethane foam BSPs (Bridgestone Co Ltd., Osaka, Japan) with 6 mm × 6 mm × 3 mm cuboids were used to immobilize A. oryzae. Palm oil was obtained from Wako Pure
Evaluation of the catalytic performance of r-BTL
The gene sequence of BTL2, cloned into pNAN8142 completely corresponded to the GenBank reference sequence (X95309). Among several transformants carrying pNAN8142BTL2, the transformant that provided the highest reaction rate in methanolysis was selected and employed in the subsequent experiments.
To evaluate the catalytic performance of r-BTL, the lipase-hydrolytic activities at high temperature and organic solvent tolerance of r-BTL were investigated and compared with those of r-FHL. The optimum
Discussion
In the present study, to develop a robust whole-cell biocatalyst that would tolerate high temperature and organic solvents, BTL2 was cloned and introduced into a high-lipase-expression system in A. oryzae. The characteristics of r-BTL were investigated via a lipase activity assay using pNPB and were compared with those of r-FHL. As shown in Fig. 1, Fig. 2, r-BTL showed significantly high lipase activity and thermostability at elevated temperature that ranged from 40 to 70 °C. In addition, r-BTL
Acknowledgements
This work was partially supported by Regional Innovation Creation R&D Programs, the Ministry of Economy, Trade and Industry (METI) and Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan.
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