Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass

Biotechnology for Biofuels

Reinu E Abraham¹  Madan L Verma¹  Colin J Barrow¹  Munish Puri¹ 

[1] Centre for Chemistry and Biotechnology (CCB), Geelong Technology Precinct, Waurn Ponds, Deakin University, Geelong, Victoria 3217, Australia

关键词: Hydrolysis;    Enzyme;    Nanoparticle;    Immobilisation;    Cellulase;    Hemp hurd;   

Others  :  792195
DOI  :  10.1186/1754-6834-7-90

 received in 2013-12-03, accepted in 2014-05-21,  发布年份 2014

【 摘 要 】

Background

Previous research focused on pretreatment of biomass, production of fermentable sugars and their consumption to produce ethanol. The main goal of the work was to economise the production process cost of fermentable sugars. Therefore, the objective of the present work was to investigate enzyme hydrolysis of microcrystalline cellulose and hemp hurds (natural cellulosic substrate) using free and immobilised enzymes. Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability.

Results

Up to 94% protein binding was achieved during immobilisation of cellulase on nanoparticles. Successful binding was confirmed using Fourier transform infrared spectroscopy (FTIR). The free and immobilised enzymes exhibited identical pH optima (pH 4.0) and differing temperature optima at 50°C and 60°C, respectively. The K_M values obtained for the free and immobilised enzymes were 0.87 mg/mL and 2.6 mg/mL respectively. The immobilised enzyme retained 50% enzyme activity up to five cycles, with thermostability at 80°C superior to that of the free enzyme. Optimum hydrolysis of carboxymethyl cellulose (CMC) with free and immobilised enzymes was 88% and 81%, respectively. With pretreated hemp hurd biomass (HHB), the free and immobilised enzymes resulted in maximum hydrolysis in 48 h of 89% and 93%, respectively.

Conclusion

The current work demonstrated the advantages delivered by immobilised enzymes by minimising the consumption of cellulase during substrate hydrolysis and making the production process of fermentable sugars economical and feasible. The activity of cellulase improved as a result of the immobilisation, which provided a better stability at higher temperatures. The immobilised enzyme provided an advantage over the free enzyme through the reusability and longer storage stability properties that were gained as a result of the immobilisation.

【 授权许可】

   
2014 Abraham et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Puri M, Abraham RE, Barrow CJ: Biofuel production: prospects, challenges and feedstock in Australia. Renewable and Sustainable Energy Rev 2012, 16:6022-6031.
• [2]Nakatani Y, Yamada R, Ogino C, Kondo A: Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose. Microb Cell Fact 2013, 12:66. BioMed Central Full Text
• [3]Kubicek CP: Systems biological approaches towards understanding cellulase production by Trichoderma reesei. J Biotechnol 2013, 163:133-142.
• [4]Ueda M, Goto T, Nakazawa M, Miyatake K, Sakaguchi M, Inouye K: A novel cold-adapted cellulase complex from Eisenia foetida: characterization of a multienzyme complex with carboxymethylcellulase, β-glucosidase, β-1,3 glucanase, and β-xylosidase. Comp Biochem Physiol Part B: Biochem Mol Biol 2010, 157:26-32.
• [5]Lan TQ, Wei D, Yang ST, Liu X: Enhanced cellulase production by Trichoderma viride in a rotating fibrous bed bioreactor. Bioresource Technol 2013, 133:175-182.
• [6]Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A: Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technol 2013, 127:500-507.
• [7]Frombo F, Minciardi R, Robba M, Rosso F, Sacile R: Planning woody biomass logistics for energy production: a strategic decision model. Biomass Bioenergy 2009, 33:372-383.
• [8]Sipos B, Kreuger E, Svensson S-E, Réczey K, Björnsson L, Zacchi G: Steam pretreatment of dry and ensiled industrial hemp for ethanol production. Biomass Bioenergy 2010, 34:1721-1731.
• [9]Rehman MSU, Rashid N, Saif A, Mahmood T, Han J-I: Potential of bioenergy production from industrial hemp (Cannabis sativa): Pakistan perspective. Renewable Sustainable Energy Rev 2013, 18:154-164.
• [10]Abraham RE, Barrow CJ, Puri M: Relationship to reducing sugar production and scanning electron microscope structure to pretreated hemp hurd biomass (Cannabis sativa). Biomass Bioenergy 2013, 58:180-187.
• [11]Banerjee S, Mudliar S, Sen R, Giri B, Satpute D, Chakrabarti T, Pandey RA: Commercializing lignocellulosic bioethanol: technology bottlenecks and possible remedies. Biofuels Bioprod Biorefin 2010, 4:77-93.
• [12]Verma ML, Barrow CJ, Puri M: Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production. Appl Microbiol Biotechnol 2013, 97:23-39.
• [13]Miletic N, Nastasovic A, Loos K: Immobilization of biocatalysts for enzymatic polymerizations: possibilities, advantages, applications. Bioresource Technol 2012, 115:126-135.
• [14]Puri M, Barrow CJ, Verma ML: Enzyme immobilization on nanomaterials for biofuel production. Trends Biotechnol 2013, 31:215-216.
• [15]Ansari SA, Husain Q: Potential applications of enzymes immobilized on/in nano materials: a review. Biotechnology Adv 2012, 30:512-523.
• [16]Das S, Berke-Schlessel D, Ji H-F, McDonough J, Wei Y: Enzymatic hydrolysis of biomass with recyclable use of cellobiase enzyme immobilized in sol-gel routed mesoporous silica. J Mol Catal B Enzym 2011, 70:49-54.
• [17]Zheng P, Wang J, Lu C, Xu Y, Sun Z: Immobilized β-glucosidase on magnetic chitosan microspheres for hydrolysis of straw cellulose. Process Biochem 2013, 48:683-687.
• [18]Verma ML, Chaudhary R, Tsuzuki T, Barrow CJ, Puri M: Immobilization of β-glucosidase on a magnetic nanoparticle improves thermostability: application in cellobiose hydrolysis. Bioresource Technol 2013, 135:2-6.
• [19]Ghose TK: Measurement of cellulase activities. Pure Appl Chem 1987, 59:257-268.
• [20]Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254.
• [21]Correia JAC, Júnior JEM, Gonçalves LRB, Rocha MVP: Alkaline hydrogen peroxide pretreatment of cashew apple bagasse for ethanol production: study of parameters. Bioresource Technol 2013, 139:249-256.
• [22]Kanwar R, Chaudhary R, Tsuzuki T, Kanwar JR: Emerging engineered magnetic nanparticles probes for molecular MRI of artherosclerotic plaque macrophages. Nanomedicine 2013, 7:735-749.
• [23]Khoshnevisan K, Bordbar A-K, Zare D, Davoodi D, Noruzi M, Barkhi M, Tabatabaei M: Immobilization of cellulase enzyme on superparamagnetic nanoparticles and determination of its activity and stability. Chem Eng J 2011, 171:669-673.
• [24]Hung T-C, Fu C-C, Su C-H, Chen J-Y, Wu W-T, Lin Y-S: Immobilization of cellulase onto electrospun polyacrylonitrile (PAN) nanofibrous membranes and its application to the reducing sugar production from microalgae. Enzyme Microb Technol 2011, 49:30-37.
• [25]Jordan J, Kumar CSSR, Theegala C: Preparation and characterization of cellulase-bound magnetite nanoparticles. J Mol Catal B Enzym 2011, 68:139-146.
• [26]Yuan X-Y, Shen N-X, Sheng J, Wei X: Immobilization of cellulase using acrylamide grafted acrylonitrile copolymer membranes. J Membr Sci 1999, 155:101-106.
• [27]Hegedüs I, Hancsók J, Nagy E: Stabilization of the cellulase enzyme complex as enzyme nanoparticle. Appl Biochem Biotechnol 2012, 168:1372-1383.
• [28]Cheng Z, Wu C, Yang W, Xu T: Preparation of bromomethylated poly(2,6-dimethyl-1,4-phenylene oxide) hollow fiber cation-exchange membranes and immobilization of cellulase thereon. J Membr Sci 2010, 358:93-100.
• [29]Liang W, Cao X: Preparation of a pH-sensitive polyacrylate amphiphilic copolymer and its application in cellulase immobilization. Bioresource Technol 2012, 116:140-146.
• [30]Zhang Y, Xu J-L, Li D, Yuan Z-H: Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100. Biocatal Biotransform 2010, 28:313-319.
• [31]Georgelin T, Maurice V, Malezieux B, Siaugue J-M, Cabuil V: Design of multifunctionalized γ-Fe2O3@SiO2 core-shell nanoparticles for enzymes immobilization. J Nanopart Res 2010, 12:675-680.
• [32]Singh R, Zhang Y-W, Nguyen NPT, Jeya M, Lee JK: Covalent immobilization of β-1,4-glucosidase from Agaricus arvensis onto functionalized silicon oxide nanoparticles. Appl Microbiol Biotechnol 2011, 89:337-344.
• [33]Blanchette C, Lacayo CI, Fischer NO, Hwang M, Thelen MP: Enhanced cellulose degradation using cellulase-nanosphere complexes. PLoS One 2012, 7:e42116.
• [34]Wang S, Su P, Ding F, Yang Y: Immobilization of cellulase on polyamidoamine dendrimer-grafted silica. J Mol Catal B Enzym 2013, 89:35-40.
• [35]Chang RH-Y, Jang J, Wu KCW: Cellulase immobilized mesoporous silica nanocatalysts for efficient cellulose-to-glucose conversion. Green Chem 2011, 13:2844-2850.
• [36]Ungurean M, Paul C, Peter F: Cellulase immobilized by sol-gel entrapment for efficient hydrolysis of cellulose. Bioprocess Biosys Eng 2013, 36:1327-1328.
• [37]Jones P, Vasudevan P: Cellulose hydrolysis by immobilized Trichoderma reesei cellulase. Biotechnol Lett 2010, 32:103-106.
• [38]Wang Y, Radosevich M, Hayes D, Labbé N: Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass. Biotechnol Bioeng 2011, 108:1042-1048.
• [39]Xu J, Huo S, Yuan Z, Zhang Y, Xu H, Guo Y, Liang C, Zhuang X: Characterization of direct cellulase immobilization with superparamagnetic nanoparticles. Biocatal Biotransform 2011, 29:71-76.
• [40]Sharma A, Khare SK, Gupta MN: Hydrolysis of rice hull by crosslinked Aspergillus niger cellulase. Bioresour Technol 2001, 78:281-284.
• [41]Cheng C, Chang K-C: Development of immobilized cellulase through functionalized gold nano-particles for glucose production by continuous hydrolysis of waste bamboo chopsticks. Enzyme Microb Technol 2013, 53:444-451.