In the present work

In the present work, a lipase producing Entrobacter sp. MG40 was isolated from oil contaminated soils. Cross-link enzyme aggregated of this Lipase was prepared and covalently immobilized on the magnetic graphene oxide nanocomposites (mGO-CLEAs-lipase). FTIR spectra shown that the peak around 532-614 cm?1 could be evaluated to the stretching vibration of Fe–O in Fe3O4 nanoparticles. The maximum activity of free and immobilized lipase was obtained at pH 8.0 and 9.0, respectively. The immobilized lipase showed a broad range of temperature activity about 40-60 °C, compare to free enzyme. In the case of mGO-CLEAs-lipase, the observed lower Km value state a greater lipase affinity for the pNPP substrate, about 2.25 folds. Enzyme activity of mGO-CLEAs lipase was the highest up to 5 cycles, but it continuously decrease over 5 cycles. Results of storage stability displayed mGO-CLEAs-lipase retained about 85% of its original activity after 24 days of incubation, in which free enzyme lost its total activity at the same time. These results verified that mGO-CLEAs lipase had chief protection on the storage stability of lipase. Biodiesel production form Ricinus communis as a non-edible oil feed stock, was also achieved about 3.1 folds by mGO-CLEAs-lipase compare to free enzyme, making it promising as a good nanobiocatalyst for efficient production of biodiesel.

Key words: lipase, immobilization, CLEA, biodiesel, graphene oxide, nanobiocatalyst

1. Introduction
Recently, biodiesel has fascinated great consideration as a biodegradable, renewable, clean-burning and non-toxic fuel (Ranganathan et al., 2008; Yadav et al., 2017; Budžaki et al., 2018; Hama et al., 2018). The greenhouse gas (GHG) release of biodiesel (B100) are 4.5-fold lower than gasoline, and 3-times lower than petro-diesel (Schumacher et al., 1995; Ali et al., 1995; Mofijur et al., 2013; Yadav et al., 2017). All these factors mark it a perfect fuel for the future. But, the production of biodiesel, is about 30 % more expensive than that of petroleum-based diesel (Supple et al., 2002; Zhang et al., 2003). This is principally because of the using high quality, expensive and generally non-refined virgin oils, well-known as first group of biodiesel feedstock. The cost of feedstock oil production is the most important problem in biodiesel production which include about 70 % of the total biodiesel value. (Parawira W. 2009; Verma et al., 2016). Recently, non-edible oil feedstocks are attainment global consideration since they can be grown simply in different parts of the world particularly wastelands which are not suitable for refining food crops. Therefore, it make them more economical compared to edible oils because of more ecologically friendly, elimination for food competition, and production of useful by products (Kansedo et al., 2009; Sajjadi et al., 2016). Pongamia pinnata, Jatropha curcas, Azadirachta indica, Croton megalocarpus and Calophyllum inophyllum are some of the main non-edible resources for biodiesel production (Takase et al., 2015; Verma et al., 2016; Hasni et al., 2017). Hence, more prominence is nowadays assumed to non-edible seed oil resources as a favorable source for biodiesel production (Sajjadi et al., 2016; Mardhiah et al., 2017; Roschat et al., 2017; Teoa et al., 2018). In this study, we used Ricinus communis oil as an efficient non-edible oil feedstock for biodiesel construction.
The important catalysts used for biodiesel transesterification are alkali, acid and enzyme. The alkali-catalyzed procedure provides higher TG alteration at short time reaction. Conversely, biodiesel production with alkali based catalyst is considered by some intrinsic procedure disadvantages including the difficulties in glycerol separation, purification, catalyst recovery and recycling, saponification problems, the need to wash out biodiesel impurities, treat waste water and eliminate salts, and their energy-intensive nature (Reyero et al., 2015; Fjerbaek et al., 2009; Ranganathan et al., 2008). Due to low quality and yield of biodiesel by alkali transesterification from low-cost resources that have high water and FFA, this strategy does not deal the best manner for biodiesel production in terms of commercial and ecological issues (Meher et al., 2006; Lew et al., 2014). Although, the acidic catalysis trans-esterify both TGs and FFA to biodiesel, but, it is deliberated by some problems including the significantly lower transesterification rates, multi steps separation of biodiesel from glycerol and a requisite for higher reaction temperatures (Meher et al., 2006; Lew et al., 2014). To reduce the problems related with using acid and/or alkali for biodiesel transesterification, a lipase-catalyzed manner has been offered and extensively explored, recently (Fjerbaek et al., 2009; Xie and Huang, 2018; Bandikaria et al., 2018). Some benefits of the lipase-based biodiesel transesterification include biodegradability and environmentally-friendly, simplified production procedure, room-temperature reaction conditions, lower energy depletion, easier product separation and recovery, higher purity of glycerol by-product and minimized impurities, high substrate specificity, no soap formation in the system, lower alcohol to oil ratio, recycle of immobilized enzymes, single-step conversion of both FFA and TG to biodiesel (Lew et al., 2014; Hama et al., 2013; Tan et al., 2010). The main drawbacks mainly related with the lipases are the lower reaction rates, activity reduction by methanol and the higher enzyme prices (Brun et al., 2011). Lipase efficiency can be improved by screening of new, more effective lipase sources. Furthermore, the lipase can be recycled by covalent immobilization, which leads to enhanced of biodiesel productivity (Lew et al., 2014; Tan et al., 2010; Hama et al., 2013; Wang et al., 2017; Teoa et al., 2018).
In this project, we used the both mentioned strategies to find an excellent nano-biocatalyst for effective biodiesel making. At first, we screened a high potent lipase (inducible, high methanol-tolerant, short time rate) producer from local oil contaminated soils. Secondly, purified lipase was immobilized on coated graphene oxide in order to enhance enzyme stability and reusability. Finally, potential of this potent nano-biocatalyst (CLEA-lip-mGO) was considered for efficient biodiesel production from non-edible feedstock.