Role of Enzymes in Biofuels

There are so many advantages for the production of biofuels from lignocellulosic waste/biomass from agriculture and food industries. They are not only cleaner alternatives but also offer several advantages such as:

  • • Reduces pollution
  • • Can deal with the shortage of food and feed
  • • Renewable sources of energy over fossil fuels
  • • Maintain the economy of countries around the globe

Biomass can be taken as an organic matter grown by photosynthetic conversion of solar energy.

Most of the lignocellulosic biomass comprises cellulose, hemicelluloses and lignin. These lignocellulosic wastes can be converted to simpler monomers by the action of different enzymes (Binod et al. 2019). These biofuels can be classified into first-generation and second-generation based upon the sources. The major drawback of using first-generation biofuels is the limited production of food as well as increasing the prices of food items. The biofuels derived from complex lignocellulosic waste serve as the second generation biofuel. For conversion of lignocellulosic waste into biofuel needs the action of various enzymes. But major technical barrier for adopting enzymes to convert biomass to biofuels is the high cost of the enzymes. An effective strategy is to use some immobilization methods for enzymes which leads to reusability of enzymes and thereby makes them economically viable. There is no single enzyme available which can convert complex lignocellulosic biomass. Various combinations using different enzymes can be used (see Figure 3).

Action of different enzymes on lignocellulosic wastes for biofuel production

Figure 3. Action of different enzymes on lignocellulosic wastes for biofuel production.


These are the enzymes responsible for the hydrolysis of cellulose. Conversion of cellulose to simpler glucose is done by three different enzymes:

  • • Eudoglucanases (EC involved in p 1 -4 glycosidic bonds
  • • Exocellulases (EC catalyzes the breakage of long chains to short chains
  • • p-glucosidases (EC hydrolyze the glycosidic bonds


Xylan is the major component present in hemicelluloses. Xylan degradation in simpler sugars is catalyzed by enzymes known as xylanases. In previous studies, xylanases have been derived from various microorganisms such as Trichoderma reesei, Humicola insolens or Bacillus (Binod et al. 2019). Different enzymes work singly or via combination with other enzymes for complete hydrolysis of xylan.

  • • Eudol-4P-xylanases (EC catalyze the glycosidic bonds and results in the production of oligomers.
  • • p-xylosidase (EC works on oligosaccharides and cellobioses. These enzymes act upon oligosaccharides and cellobioses.
  • • a-arabinofuranosidase (EC removes the arabinose and glucuronic acids from xylan.
  • • a-glucuronidase (EC 1,2 glycosidic bonds are hydrolyzed in xylose.
  • • Esterases (EC catalyzed the xylose units of xylan.

Lignin Peroxidises (EC

These are lignin-degrading enzymes which cleave lignin.

Laccases (EC

These are fungal enzymes that can act on lignin and convert them into simpler sugars.

4.5 Lipases (EC

These are enzymes involved in hydrolysis and esterification reactions. These have been studied extensively due to their high stability (Venna et al. 2008, Verma and Kanwar 2008). Lipases can catalyze transesterification reactions in different oils. Transesterification and esterification reactions can be catalyzed by lipases. Transesterification reactions of vegetable oils can convert the biomass into biofuels (Tan et al. 2010). Various recent studies have been earned out on using lipases for the production of biodiesel.

The major limitation of using enzymes for biofuel production is the high cost involved. However, various nanomaterials have been used for the immobilization of enzymes (Venna et al. 2013). It not only provides cost-cutting strategy by reusing the enzymes but also provides stability. Chitosan is more suitable support for the immobilization of enzymes due to low cost, biocompatibility and high strength (Foresti and Feneira 2007, Huang et al. 2007). In the following section, various immobilization strategies of different enzymes using chitosan nanomaterial for biofuel production have been discussed.

Other problems in the application of enzymes in biodiesel synthesis are enzyme deuaturation, high cost and scale-up at reactor level. Enzymes in biodiesel synthesis have inactivation problem due to some substrates/by-products, i.e., methanol and glycerol, respectively (Kumari et al. 2009). Methanol can decrease enzyme activity and biocatalyst efficiency (Ghamguia et al. 2004). Furthermore, a hydrophilic byproduct, glycerol can be absorbed on the surface that decreases the enzyme activity. Hence, the selection of an ideal acyl acceptor is an important step in biodiesel production. Several protective measures can be used including stepwise addition of methanol to the reaction mixture, use of methyl or ethyl acetate as acyl acceptors and use of longer alkyl chains alcohols such as t-butanol which can be used to overcome this problem (Royon et al. 2007, Kumari et al. 2009). The high cost involved in enzymatic processes can be reduced using nanomaterials due to high surface area and reusability. Magnetic nanomaterials are materials of choice for efficient separation of products as well as enhanced thermodynamic properties.

Chitosan Nanoparticles for Enzyme Immobilization

Nanofibers are solid particles with dimensions ranging from 1-100 mn (Zhao et al.

2011). They are lucrative support matrix for enzyme immobilization. The major advantages are high surface area and more enzyme loading per unit with low diffusion resistance (Wang et al. 2006, Nan et al. 2007). Moreover, the shear stress developed in the batch reactor would disrupt the enzyme carrier. This limits the reusability of immobilized enzymes for a large number of cycles. This limitation can be resolved using a packed bed reactor (PBR) (Wang et al. 2011). Nanomaterials have been successfiilly employed in biodiesel production. A high conversion (90%) has been achieved in biodiesel production using T. lamtginosus lipase (Xie and Ma 2009). Lipase immobilization using GA cross-linker exhibited a lower transesterification rate (40%) as compared to EDC cross-linker (60%). However, maximum biofuel production was achieved using immobilized Cepacia lipase (Wang et al. 2009). Butyl biodiesel production has been studied using electrospuu polyacrylonitrile nanofiber bound PCL (Sakai et al. 2010). Maximum biodiesel production (94%) has been obtained using immobilized PCL (Li et al. 2011a). During this process, it retained its activity even after 10 cycles.

Thus, the most significant method is immobilizing enzymes using nanocomposites. Magnetic nanocomposite bound lipase gave high conversion of biodiesel (> 90 %) within 30 hours in batch operation and further suggested the importance of novel matrix for immobilization. The immobilized lipase from Burkholderia sp. has high methanol tolerance and reusability (Tran et al. 2012). The biofuel production process has been affected by shear stress that has, in turn, created a negative impact on immobilized lipases. A packed bed bioreactor has been developed to minimize shear stress and biodiesel conversion (Wang et al. 2011). Maximum conversion (88%) has been achieved in 192 hours using the packed bed bioreactor. Moreover, this type of reactor showed enhanced stability of nano-immobilized biocatalysts. A nanobiocatalytic system has been developed for biodiesel production using the PBR system for effective and continuous biodiesel production based on soybean oil metlianolysis with nano-immobilized lipase using chitosan (Wang et al.

2009). Maximum activity and stability have been repotted with new biocomposite in the single-PBR at an optimal flow rate. The optimum conversion rate (75%) was recorded at 12 horns. Wang and coworkers (2011a) developed an effective nanobiocatalytic system for biodiesel production that made highly efficient use of lipase. Multiple PBR gave high conversion than using the single PBR. Thus, a packed bed bioreactor has shown great potential for biodiesel production using nanobiocatalytic systems.

High conversion rate and good stability in the four-packed bed reactor may be due to longer residence time of the reaction mixture in the reactor and elimination in the inhibition of the lipase-uanoparticle by-products. Effective stability and reusability of the immobilized enzyme system lower the cost of biodiesel production. The reactor studies provide the basis of technology for the scale-up of the biodiesel production process.

Chitosan Nanofibers

Chitosan nanofibers were prepared using an ultrasonic-assisted method with a diameter of 5 mil and length less than 3 pm (Wijeseua et al. 2015). These were analyzed by AFM and ТЕМ. These nanofibrils can be used as a precursor for further nanoparticles. Electrospiiming technology can be used for spinning synthetic polymers. Electrospiiming can be controlled by the suspension of the viscous solution. An electrospim fabric of chitosan was prepared by Ohkawa and coworkers (2004). They discovered that trifluoroacetic acid was the most effective solvent system because of amino groups of chitosan form interactions with triflouroacetic acid. A similar method of nanofibers has been developed by Schifmau and Schauer in 2007. hi this method, low molecular weight (70 KDa), medium (190-310 KDa) and high molecular weight with 500 KDa chitosans have been used along with trifluoroacetic acid. However, the high molecular weight of fibers had resulted hi more length of nanofibers. The length can be increased by cross-linking by using glutaraldehyde. Whereas, in another method, trifluoroacetic acid and dichloromethane have been used for developing chitosan nanofibers (Torres-Giner et al. 2008).

Chitosan Nanocomposites

Chitosan nanocomposites have been prepared using graphene oxide (Lau et al. 2014). X-ray diffraction studies have confirmed that graphene oxide is well embedded in chitosan which shows its appropriate immobilization. It was further confirmed by thermo-gravimetric analysis. Thus, chitosan lipase immobilized has better esterification with the enzyme activity of 64U. Esterification conversion was unproved from 78% to 98% with lipozyme. Carbon nauotubes have been employed for significant drug release using chitosan and nanocomposites (Shamieeu et al. 2018).

Non-Magnetic Nanoparticles

Nanoparticles with a 1-100 nin uanoscale diameter have been used for biological applications (Verma et al. 2016). Matrices involving various nanoparticles as carrier results in enhanced activity and catalytic efficiencies of enzymes that in turn depend upon the stability, reusability and physical properties (Verma et al. 2013). Adsorption method has been employed to immobilize lipase derived from Candida antarctica В (CAL-В). Polystyrene nanoparticles have been used as a carrier and have been synthesized using the nano-precipitation technique. The major interactions involved in adsorption are hydrophobic interactions. However, immobilization efficiency has a greater effect on change in pH. The activity depends upon the ionization state that in turn has a deep impact on the conformation state of the enzyme. The activity of this enzyme was compared with commercial enzyme Novozyme 435 and the activity of immobilized lipase was 1.16-fold higher than commercial preparation and 1.81-fold higher than free enzyme.

Magnetic Nanoparticles

Reusability is one of the important properties of immobilized enzymes, and nonmagnetic particle requires a high speed centrifugation for separation (Chen et al. 2008). However, many workers have preferred using magnetic nanomaterials which can enhance the separation (Dyal et al. 2003. Huang et al. 2003, Lei et al. 2011, Wang et al. 2011, Thangaraj et al. 2016, Marta et al. 2017). Nanoscale magnetic particles possess a unique properties of superparamagnetism (Lu et al. 2007, Vaghari et al.

2016). These do not form aggregates and thus can form solutions (Lu et al. 2007). Most of the iron oxides have been used in the fabrication of magnetic nanoparticles as they have more biocompatibility and less toxicity. Dyal and coworkers (2003) evaluated the activity and stability of Candida rugosa lipase (CRL) by using a magnetic nanoparticle that has been immobilized using covalent binding. The main interaction involved in these nanoparticles is when the amine group of lipases reacts with acetyl/amine group of nanoparticles. These types of interactions lead to an improvement in the operational stability of Candida rugosa lipase. In another similar study, hydrophobic magnetic nauoparticles have been used to immobilize crude porcine pancreas lipase (Lee et al. 2009). Maximum enzyme activity was observed when lipases were immobilized on the hydrophobic surface of nanoparticles. Sodium dodecyl sulfate can be used as a ligand for hydrophobic magnetic nauoparticles. Immobilized lipases have exhibited more activity with a uniform size that has further enhanced the cthermal stability of the immobilized biocatalyst. Immobilized lipase showed 1.42-fold higher specific activity than the fr ee enzyme. The SDS ligand on the nanoparticles’ surface acted as a spacer between the nanoparticles and the enzymes, which resulted in a flexible enzyme structure form. CRL was also immobilized on functionalized superpararnagnetic nanoparticles (poly(GMA)-grafted Fe304/SiOx) by Lei et al. (2011). The diameter of the functionalized magnetic nanoparticles was 100 tun and showed higher saturation magnetization (8.3 kA/m).

Higher thermal stability and pH stability were observed in the case of immobilized lipase that has retained 83% of residual activity after six cycles of reusability. This type of magnetic nanoparticles showed 1.41-fold enhanced activity along with 31- fold high stability than that of free enzyme. The effect of various SiO, ratios for coating on Fe304 and further functionalization of Fe304/SiO, magnetic nanoparticles using organosilane compounds, 3-aminopropyltriethoxysilane (APTES) and 3-mercaptopropyltrimethoxysilane (MPTMS), for lipase immobilization was examined. When the Fe304:SiO., ratio was 1:0.25, the immobilization efficiency was the highest. Nano-immobilized lipases along with silica and magnetic material using APTES exhibited maximum catalytic activity. Moreover, APTES helped in improving surface properties for the characterization of nanoparticles.

Carbon Nanotubes-Basecl Lipase Immobilization

Carbon nanotubes are a unique and promising material for enzyme immobilization. Carbon tubes with graphite can be easily rolled into cylinders that can help in easy and stable enzyme immobilization (Yang et al. 2015). Carbon nanombes can be classified into single-walled carbon nanombes (SWNTs) and multi-walled carbon nanombes (MWNTs). Both of these can be used for immobilization (Lee et al. 2010b). Two different solvent systems have been used hi the case of pancreatic lipase immobilized onto SWNTs. Both buffer and ionic liquids can be used for immobilization. Ionic liquid enhances the immobilization efficiency with better dispersion of carbon nanombes than using a buffer solution. Lipases have been immobilized using nanotube-silica composites in multiwalled nanombes (Lee et al. 2010b) MWNTs were used as additives to prevent lipase inactivation during the sol-gel process. Three different lipase activities from Candida rngosa, Candida antarctica type В and Thermomycs lanuginous immobilized on MWNT increased 10-fold to perform transesterification reaction which may be due to enhanced stability of the catalyst using multiwalled nanombes. Immobilization involved physical adsorption and displayed high activity retention up to 97%. Transesterification rates increased using both aqueous and organic solvents by 2.2 and 14-fold, respectively (Shah et al. 2015). Mohammad et al. (2017) reported a simple adsorption method to immobilize CRL onto acid- fimctionalized MWNTs (F-MWNTs). Carboxyl groups as polar groups were induced in MWNT by stirring with an acid mixture containing sulfuric and nitric acids. The charged carboxyl moieties on the MWNTs’ surfaces could be connected with other polar moieties (NH, and OH) on the CRL. The immobilized CRL on acid F-MWNTs had improved structural integrity and mechanical strength. The activity and thermal stability of the immobilized CRL on MWNTs were twofold enhanced compared to those of the free enzyme.

Electrospun Nanofibers

The major problem associated with carbon nanombes for immobilization is mass transfer limitation and difficulty in recycling due to good dispersion (Wang et al.

2009). Electrospun nanofibers have more potential to avoid these above-mentioned problems (Nakane et al. 2007). Lipases were attached to the surface of electrospun nanofibers as the carrier or entrapped in the uanofibers. Nanofiber membranes with carboxyl groups were made from poly(acrylonitrile-co-maleic acid) (PANCMA) via au electrospinning process (Pavlidis et al. 2010). Immobilization has impacted the increase in enzyme activity from 33.9% to 37.6% mg/g with enhanced enzyme loading that reached up to 21.2 mg/g from 2.36 mg/g as compared to the hollow fiber membrane. The efficiency of the biocatalysis increased because the Km value of the immobilized lipase decreased. Huang and coworkers (2011) reported the development of immobilized CRL on an electrospun cellulose nanofiber membrane via covalent binding. The nanofiber was made by electrospun cellulose acetate. These have been stimulated to produce aldehyde group that can enhance the covalent bonding with enzyme molecules. The activity of immobilized CRL was 29.6 U/g under an optimal condition, and the thermal stability was higher than that of free enzyme. Immobilized Burkholderia cepacia (BCL) lipase has been immobilized using polycaprolacone nanofibers, and their overall effectiveness was further checked using an aqueous and organic medium (Song et al. 2012). The specific hydrolysis activity was higher than the transesterification activity. The immobilized BCL maintained 50% of its initial activities up to the 10th recycle in a non-aqueous media.

Biofuel production using immobilized lipases onto nanoparticles have been studied (Babaki et al. 2016). Raita et al. (2015) studied biodiesel production from palm oil using immobilized Thermomyces lamiginosus lipase (TLL) on magnetic nanoparticles. Optimization of biodiesel production has been performed and a 97.2% increase in yield was obtained with 23.2 mg/g enzyme loading. Moreover, these nano-immobilized catalysts showed stability at 50°C for 24 hours. These enzyme preparations could be reused for five cycles with 80% retained catalytic activity. Waste cooking oil was also converted to biodiesel via transesterification using nano- immobilized lipase (Mehrasbi et al. 2017). Karimi and coworkers (2016) immobilized BCL on superparamagnetic iron-oxide nauoparticles (SIONs) for biodiesel production. Maximum conversion (91%) of waste cooking oil to biodiesel has been observed in 35 hours. Immobilized Rhizomucor miehei lipase has been used, and a high diesel yield of 94% from waste cooking oil has been reported. The RML was immobilized onto polyamidoamiue (РАМАМ) grafted with magnetic MWCNTs (m-MWCN- PAMAM). The immobilized catalysts showed reusability up to 10 cycles along with a 27-fold higher transesterification activity. These results showed great potential for biodiesel production. Tran et al. (2012) developed alkyl-functionalized Fe304-Si0, nanocomposites for lipase immobilization. The immobilized lipase was used for biodiesel production. Maximum biodiesel production (90%) has been observed in batch operation for 30 hours. One-step extraction and transesterification process of biodiesel production from wet microalgae biomass using alkyl-grafted Fe304-Si0,- immobilized lipase have been studied. The biodiesel conversion was over 90% under optimal conditions, hi another similar study, transesterification of soyabean oil to biofuel has been observed using lipase immobilized on polyacrylonitrile nanofiber. The biodiesel conversion was 90%. The immobilized lipase on the nanofibers maintained 91 % of its initial activity for 10 cycles. All these results have strengthened that nanomaterials can be used as best carriers for immobilization of lipase and they have tremendous potential in biodiesel production.

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