A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: Characterization and application to enzymatic biodiesel production

Highlights

• Aspergillus oryzae (r-BTL) harboring lipase gene (BTL2) was constructed.

• r-BTL showed higher thermo- and solvent-tolerance lipase activity.

• Enzymatic production of biodiesel from palm oil was performed.

• r-BTL efficiently catalyzed methanolysis at elevated temperature (40–50 °C).

• r-BTL efficiently catalyzed methanolysis at a high methanol concentration

Abstract

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40–50 °C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50 °C and was maintained even after an incubation of 24-h at 60 °C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50 °C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including 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.