Aspergillus oryzae lipase-catalyzed synthesis of glucose laurate with excellent productivity
© Lin et al. 2016
Received: 8 November 2015
Accepted: 20 December 2015
Published: 5 January 2016
As nonionic surfactants derived from naturally renewable resources, sugar fatty acid esters (SFAEs) have been widely utilized in food, cosmetic, and pharmaceutical industries.
In this study, six enzymes were screened as catalyst for synthesis of glucose laurate. Aspergillus oryzae lipase (AOL) and Aspergillus niger lipase (ANL) yielded conversions comparable to the results obtained by commercial enzymes such as Novozyme 435 and Lipozyme TLIM. The productivity obtained by AOL catalysis in anhydrous 2–methyl–2–butanol (2M2B) (38.7 mmol/L/h and 461.0 μmol/h/g) was much higher than the other literature results. Factors affecting the synthetic reaction were investigated, including water content, enzyme amount, substrate concentrations and reaction temperature. The process was greatly improved by applying the Box-Behnken design of response surface methodology (RSM). Solubilities of glucose in 14 different organic solvents were determined, which were found to be closely associated with the polarity of the solvents.
Aspergillus oryzae lipase is a promising enzyme capable of efficiently catalyzing the synthesis of sugar fatty acid esters with excellent productivity.
Sugar fatty acid esters (SFAEs) are nonionic surfactants which can be produced from naturally renewable resources: carbohydrates (e.g., glucose and sucrose) and fatty acids (e.g., lauric and palmitic acids). SFAEs are tasteless, odorless, nontoxic, nonirritant, and biodegradable; their functional properties such as critical micelle concentration (CMC) and hydrophilic–lipophilic balance (HLB) can be altered over a broad range by tuning the constitutive sugar and fatty acid moieties of the sugar esters, and some of them also possess insecticidal (Puterka et al. 2003) and antimicrobial (Ferrer et al. 2005) activities. All these advantageous features have made SFAEs attractive for use in food, cosmetic, and pharmaceutical industries (Ye and Hayes 2014; Gumel et al. 2011; Kobayashi 2011; Chen et al. 2007).
Enzymatic preparation of sugar esters in organic media has been proved to be superior to the currently dominating chemical synthesis in terms of mild reaction conditions, simple operational procedures, high productivity, excellent regioselectivity, and easy product separation (Ye and Hayes 2014; Gumel et al. 2011; Kobayashi 2011; Chen et al. 2007). The synthetic reaction is normally performed in an organic solvent such as tert-butanol and 2-methyl-2-butanol (2M2B) (Ferrer et al. 2000, 2005; Ye and Hayes 2014; Gumel et al. 2011; Kobayashi 2011; Ikeda and Klibanov 1993; Flores et al. 2002; Pöhnlein et al. 2014; Walsh et al. 2009; Cao et al. 1996).
In terms of enzyme selection, among different lipases (EC 184.108.40.206) that have been tested, Novozym 435 from Novozyme (Candida antarctica lipase B immobilized on acrylic resin) is the most popular one for catalyzing sugar ester synthesis (Ferrer et al. 2005; Ye and Hayes 2014; Gumel et al. 2011; Kobayashi 2011; Flores et al. 2002; Pöhnlein et al. 2014; Cao et al. 1996; Lin et al. 2015). It is necessary to search for some other lipases that are cheap and catalytically efficient for this application.
Factors affecting the synthetic process include substrate concentrations, enzyme amount, reaction temperature, etc. The traditional one-variable-at-a-time optimization approach is not sufficient to maximize the conversion because it is not only laborious and time-consuming, but also cannot guarantee determination of optimal conditions due to the fact that possible interactions among various operational factors have not been considered. The use of the response surface methodology (RSM) as a tool for experimental design and optimization has been applied to a variety of processes because it can enable the building of models and the evaluation of the significance of the different factors considered as well as their interactions (Bezerra et al. 2008). With the aid of experimental design via RSM, optimal conditions can be obtained and optimization achieved by running only a small number of experimental trials.
In this current study, synthesis of glucose laurate by transesterification in 2M2B between d-glucose and vinyl laurate was taken as a model reaction to demonstrate that Aspergillus oryzae lipase (AOL) is a promising catalyst for SFAE synthesis, and Box-Behnken design (BBD) of response surface methodology (RSM) was successfully applied to improve the conversion and productivity.
Materials and methods
Novozym 435 and Lipozyme TLIM were purchased from Novozymes (China) Investment Co., Ltd. Lipases from Penicillium expansum (PEL), Rhizopus chinensis (RCL), A. oryzae (AOL) and A. niger (ANL), all produced by spraying the concentrated supernatant from fermentation with addition of a certain amount of starch as a thickening agent, were kindly donated by Shenzhen Leveking Bioengineering Co. Ltd., Shenzhen, China. Vinyl laurate was purchased from Sigma-Aldrich China Inc. α-d-Glucose, lauric acid, molecular sieves (4 Å), and all other reagents used were of analytical grade from local manufacturers in China.
Dissolution and solubility of glucose
Different methods of dissolving glucose in organic solvents have been tested, which include agitation, vortex mixing, sonication, microwave, and their combinations, and the one that yielded the fastest dissolution turned out to be a combination of vortex mixing and incubating/shaking. Glucose (100 mg) was added to a test tube containing 2.0 ml of a given solvent (dried with molecular sieves), followed by vortex mixing for 5 min and then shaken in an incubator/shaker at 30 °C and 220 rpm for 12 h. After settling at room temperature (25 °C) for 30 min and then centrifugation at 6000 rpm for 5 min, the supernatant was obtained for determining the glucose solubility by using the dinitrosalicylic acid (DNS) method (Miller 1959). All solubility tests were performed at least three times subjected to less than 5 % error.
A typical reaction was carried out by adding 0.27 g glucose (corresponding to 0.3 mol−1 of the reaction system, but only partially dissolved) to 5.0 ml 2M2B (dried over molecular sieves for over a week prior to use) containing 0.3 M vinyl laurate (totally dissolved) and 1.0 g 4Å molecular sieves. After vortex mixing for 5 min to maximize the glucose dissolution in the solvent, 0.5 g of the enzyme was added, and the flask was placed in an incubator/shaker with agitation of 300 rpm at 40 °C to start the reaction. Periodically, a 100 µl sample was taken for HPLC analysis (Lin et al. 2015). The conversion was calculated based on the total amount of glucose added to the reaction system. In order to optimize the reaction conditions, the effect of each affecting factor (water content, enzyme amount, reaction temperature and substrate concentrations) was studied independently, i.e., to obtain the optimum for one factor at a time while keeping others constant. The product is 6-O-lauroyl-d-glucopyranose, which has been verified by using HPLC and structural analyses with NMR, IR, and MS (Lin et al. unpublished results). All conversion results were subject to less than 10 % error.
RSM experimental design
Three factors, namely reaction temperature, enzyme amount, and molar ratio of the substrates (VL/Glc), were selected for optimization. The appropriate range for each variable was selected based on the single-factor experiments (see “Enzymatic reaction” section). Then a 3–factor–3–level–Behnken design (BBD) of response surface methodology (RSM) was carried out using Design-Expert 8.0.6, a DOE software developed by Stat-Ease, Inc. Experimental results were analyzed by applying ANOVA (analysis of variance) technique implemented in the Design-Expert software.
Results and discussion
Solubility of glucose in different organic solvents
For our reaction involving two substrates of opposite polarity (i.e., the hydrophilic sugar and the hydrophobic fatty acid), an appropriate solvent has to be selected based on a compromise between the two solubilities, while meanwhile the enzyme compatibility and environmental and health risks have also to be considered. Tertiary alcohols such as tert-butanol and 2M2B have been considered as good candidates for this application (Ferrer et al. 2005; Ye and Hayes 2014; Gumel et al. 2011; Kobayashi 2011; Ikeda and Klibanov 1993; Flores et al. 2002; Pöhnlein et al. 2014; Walsh et al. 2009). 2M2B was used throughout this study.
AOL-catalyzed synthetic reaction and its affecting factors
The impact of enzyme amount on the glucose laurate synthesis has also been examined (Fig. 4b). The conversion was raised first and then declined as the enzyme amount increased with an optimum obtained at 0.4 g. Part of the reasons for the later decline may be related to the hydrolysis of the product as mentioned above. Additionally, too high a concentration of the immobilized enzyme makes the mixing of the reaction slurry difficult. Taking the enzyme cost into consideration, using excessive enzyme is counterproductive. In this study, use of 0.4 g of the enzyme in the 5 ml reaction system is recommended.
Other factors affecting the enzymatic reaction include the reaction temperature and substrate concentrations. A range of temperatures (40–80 °C) has been tested, and the optimal reaction temperature was found to be 60 °C (Fig. 4c). In terms of substrate concentrations, when the concentration of vinyl laurate was fixed at 0.3 M (totally dissolved) while the amount of glucose varied in a range of 0–0.4 mol−1 of the reaction system (most of it was initially undissolved and unreacted), the formation of glucose laurate increased gradually, accompanied by a decrease in the conversion (Fig. 4d). The reduction in conversion with an increase in the applied glucose amount is not surprising, for the reported conversion here was calculated based on the amount of glucose that was applied, while most of this added glucose was initially undissolved and unreacted. Applying the sugar in solid state is favorable for the reaction because its suspended particles continuously replenish the dissolved pool of the substrate as it is consumed by the reaction, thus pushing the reaction forward. On the other hand, when the glucose input was fixed at 0.3 mol−1 of the reaction system while vinyl laurate was added varying in the range of 0–1.2 M, an optimal conversion was obtained at a VL concentration of 0.6 M (Fig. 4e).
The above single-factor experiments suggested the optimum for each reaction condition: reaction temperature at 60 °C, 400 mg enzyme, 2:1 M ratio for VL/Glc (with 0.3 mol of Glc per liter of the reaction system) in 5.0 ml dried 2M2B. After a reaction was conducted under these conditions for 5 h, a conversion of 50.9 % was obtained, which is obviously improved as compared to those obtained previously.
Optimization through RSM
Variables and levels used for the Box-Behnken design
Enzyme amount (mg)
VL/Glc molar ratio
Reaction temperature (°C)
Conversion and productivity obtained in this study as compared to those reported in literature
(Ikeda and Klibanov 1993)
(Yan et al. 1999)
(Ferrer et al. 2000)
(Ferrer et al. 2005)
(Pöhnlein et al. 2014)
Palmitic acid methyl ester
Methyl ethyl ketone
Pseudomonas sp. lipoprotein lipase
Helvina lanuginosa lipase
Tidestromia. lanuginosus lipase
Sugar input (M)
Enzyme amount (mg/ml)
Reaction time (h)
Reaction temperature (°C)
Specific productivity (μmol/h/g)
XL conceived of the study and carried out the RSM design. KZ assisted in data analysis and supervised the whole experiment. QZ and KX assisted in experimentation. PJH assisted in result discussion and draft revision. ZY was responsible for draft preparation and submission. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (Grant number 21276159).
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