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One-Step Partially Purified Lipases (Sclipa and Sclipb)

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One-Step Partially Purified Lipases (Sclipa and Sclipb) molecules Article One-Step Partially Purified Lipases (ScLipA and ScLipB) from Schizophyllum commune UTARA1 Obtained via Solid State Fermentation and Their Applications Yew Chee Kam 1 ID , Kwan Kit Woo 2 and Lisa Gaik Ai Ong 1,* 1 Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Kampar 31900, Malaysia; [email protected] 2 Department of Chemical Engineering, Lee Kong Chian Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Kajang 43000, Malaysia; [email protected] * Correspondence: [email protected]; Tel.: +60-5-468-8888 Received: 9 October 2017; Accepted: 29 November 2017; Published: 8 December 2017 Abstract: Lipases with unique characteristics are of value in industrial applications, especially those targeting cost-effectiveness and less downstream processes. The aims of this research were to: (i) optimize the fermentation parameters via solid state fermentation (SSF); and (ii) study the performance in hydrolysis and esterification processes of the one-step partially purified Schizophyllum commune UTARA1 lipases. Lipase was produced by cultivating S. commune UTARA1 on sugarcane bagasse (SB) with used cooking oil (UCO) via SSF and its production was optimized using Design-Expert® 7.0.0. Fractions 30% (ScLipA) and 70% (ScLipB) which contained high lipase activity were obtained by stepwise (NH4)2SO4 precipitation. Crude fish oil, coconut oil and butter were used to investigate the lipase hydrolysis capabilities by a free glycerol assay. Results showed that ScLipA has affinities for long, medium and short chain triglycerides, as all the oils investigated were degraded, whereas ScLipB has affinities for long chain triglycerides as it only degrades crude fish oil. During esterification, ScLipA was able to synthesize trilaurin and triacetin. Conversely, ScLipB was specific towards the formation of 2-mono-olein and triacetin. From the results obtained, it was determined that ScLipA and ScLipB are sn-2 regioselective lipases. Hence, the one-step partial purification strategy proved to be feasible for partial purification of S. commune UTARA1 lipases that has potential use in industrial applications. Keywords: sugarcane bagasse; used cooking oil; hydrolysis; esterification 1. Introduction Filamentous fungi are suitable for solid state fermentation (SSF) due to their substrate colonizing mycelium, limited water tolerance and extracellular enzyme production. Schizophyllum commune is a commonly distributed split gill white-rot mushroom [1,2] found worldwide. It is cultivated in Malaysia, as it is popularly eaten by the Malay community [1]. However, it is also found to degrade wood and caused severe infections in humans [2]. Recently, Singh et al. [3] has purified the lipase produced by S. commune which was cultivated on Leucaena leucocephala seeds under solid state fermentation conditions. Due to these findings, S. commune was selected for this study. The usage of lipase (triacylglycerol acylhydrolase EC 3.1.1.3) to convert water insoluble substrates is gaining more attention [4]. Enzymes are the alternative option in the continual pursuit for non-pollutant processes, moving in the direction of greener technologies [5]. As compared to chemical synthesis, biocatalysts or enzymes, which are classified as eco-friendly, are able to reduce the Molecules 2017, 22, 2106; doi:10.3390/molecules22122106 www.mdpi.com/journal/molecules Molecules 2017, 22, 2106 2 of 13 thermodynamic barrier that separates products from substrates and significantly lowers the energy consumption [5]. Studies of the selective triacylglycerol hydrolysis capabilities of lipases and their manipulations using diverse approaches have been reported [4] for various applications. Enzymes are a preferred choice due to their ability to work under mild conditions, ease of use, their production of less toxic by-products, and the fact the reactions are solvent-free or performed in an aqueous phase [5]. Lipases are suitable for working with raw, defined, sophisticated or unstable substrates under mild conditions and produce more stable products [5]. It is appealing that under certain conditions, lipases offer a natural approach compared to chemical catalysts [5]. Thus, this speed up the reaction processes and product procurement. The seafood industry generates wastes mainly from the discarded parts of fish, i.e., heads, fins, guts, scales and skins, that have potential for fish oil extraction and subsequent use in the food or nutraceutical industries [6] as an alternative to decrease land filling and environment pollution. Coconut oil, from the kernel of Cocos nucifera L., a clear liquid with pleasant aroma, is edible and used in bakery, confectionary, cooking, pharmaceutical and cosmetics [7]. On the other hand, butter has been in our daily lives since the early ages, and is a common item in the kitchen. In this study three raw triacylglycerols (TAGs), namely crude fish oil (long chain), coconut oil (medium chain) and butter (short chain), were selected for lipase hydrolysis studies. TAGs are rich sources for the production of monoacylglycerols (MAGs) and diacylglycerols (DAGs). MAGs and DAGs are utilized as emulsifiers in cosmetics, drugs and food industry [8]. MAGs and DAGs can be chemically synthesized by glycerolysis of vegetable and animals lipids at high temperatures ranging from 200 to 290 ◦C, producing unwanted by-products [4,8]. Therefore, lipase is an alternative for lipids hydrolysis under mild conditions in the presence of excess water, generating lower wastes [8] and faster products production. Besides hydrolysis, lipases also have the ability to esterify glycerol and free fatty acids to form TAGs [9]. As hydrolysis and esterification are reversible, water contents must be monitored in order to drive the reaction towards the desired products. Since lipases have specific affinity towards different substrates, thus acetic, lauric and oleic acids, which represent short, medium and long fatty acids, were chosen in this study for this purpose. Thin layer chromatography (TLC) is a low cost reaction monitoring and an alternative method mainly used to detect chemical compounds, and it has been used for commercial characterization of oils for several decades [8,10,11], as it is rapid and sensitive. Junior et al. [8] recently studied the hydrolysis of triolein using Lipozyme RM IM from Rhizomucor miehei and the results were evaluated using TLC analysis. According to Bayoumi et al. [12], high-purity of lipases is not required in the detergent industry, but rather the crude or partially purified version can be used as it is more cost effective. The industry is more concerned with the functionality and performance of the enzyme under certain conditions, rather than the enzyme purity. Ammonium sulfate was selected to precipitate and partial purify the crude lipase extract due to its inert effect of protein structure, solubility in water and being cheaply available [12]. Thus, the aim of this study is to evaluate the actions of one-step partial purified lipases on substrates of different chain lengths. 2. Results and Discussion 2.1. Optimization of Fermentation Parameters In this study, eight variables i.e., inoculum density, moisture ratio, urea, incubation temperature, sugarcane bagasse (SB) solids, glucose, SB particle size and used cooking oil (UCO) ratio that affect lipase productivity were screened. The analysis of variance (ANOVA) was applied to test the interaction effects of the variables (Table1). The p-value less than 0.05 indicated that the model terms are significant. Among the selected parameters, only moisture (p-value: 0.0021) and UCO (p-value: 0.0122) ratios were the significant variables. From these results, the correlations between the two factors and their effects on lipase production were evaluated using a 3-Level Factorial Design. Molecules 2017, 22, 2106 3 of 13 Table 1. Statistical analysis of the model (ANOVA) for 2-Level Fractional Factorial Design. MoleculesSource 2017, 22 Sum, 2106 of Squares Degrees of Freedom Mean Square F Value p-Value Prob.3 of 13 > F Model 0.078 7 0.011 4.22 0.0014 Table 1. Statistical analysis of the model (ANOVA) for 2-Level Fractional Factorial Design. A 1 9.663 × 10−3 1 9.663 × 10−3 3.64 0.0637 B 2Source Sum0.029 of Squares Degrees 1 of Freedom Mean 0.029 Square F Value 10.77 p-Value Prob. 0.0021 > F 3 −3 −3 C Model 4.760 ×0.07810 1 7 4.7600.011× 10 4.221.80 0.0014 0.1875 4 H A 1 0.0189.663 × 10−3 11 9.663 0.018 × 10−3 3.64 6.89 0.0637 0.0122 AB 7.701 × 10−3 1 7.701 × 10−3 2.91 0.0959 B 2 0.029 1 0.029 10.77 0.0021 AC 4.641 × 10−3 1 4.641 × 10−3 1.75 0.1930 C 3 4.760 × 10−3 1 4.760 × 10−3 1.80 0.1875 AH 4.681 × 10−3 1 4.681 × 10−3 1.77 0.1912 H 4 0.018 1 0.018 6.89 0.0122 Lack of fit 0.030 8 3.772 × 10−3 1.59 0.1658 AB 7.701 × 10−3 1 7.701 × 10−3 2.91 0.0959 1 2 3 4 AC 4.641 × 10Inoculum−3 density; 1moisture ratio; 4.641urea; × 10UCO−3 ratio.1.75 0.1930 AH 4.681 × 10−3 1 4.681 × 10−3 1.77 0.1912 TheLack model of fit F-value0.030 of 14.24 (p < 0.0001)8 was obtained3.772 × from 10−3 the1.59 ANOVA analysis0.1658 (Table2), which implies the model is significant.1 Inoculum density; There 2 ismoisture only a ratio; 0.01% 3 urea; chance 4 UCO that ratio. such a “Model F-value” could occur due to noise. From the results, there is no correlation between the moisture and UCO ratios The model F-value of 14.24 (p < 0.0001) was obtained from the ANOVA analysis (Table 2), (p-value > 0.05). which implies the model is significant.
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