Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose

BMC Biotechnology

Linguo Zhao²  Qian Pang²  Jingcong Xie¹  Jianjun Pei²  Fei Wang²  Song Fan¹ 

[1] College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China

[2] Jiangsu key Lab of Biomass Based Green Fuels and Chemicals, Nanjing, China

关键词: Immobilization;    Fusion protein;    Cellulose binding domain (CBD);    Glucose-tolerant β-glucosidase;   

Others  :  835087
DOI  :  10.1186/1472-6750-13-101

 received in 2013-09-04, accepted in 2013-11-11,  发布年份 2013

【 摘 要 】

Background

The complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation.

Results

The glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in V_max/K_m in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188.

Conclusions

The hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process.

【 授权许可】

   
2013 Zhao et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Aristidou A, Penttila M: Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechnol 2000, 11:187-198.
• [2]Bhat MK: Cellulases and related enzymes in biotechnology. Biotechnol Adv 2000, 18:355-383.
• [3]Brethauer S, Wyman CE: Review: continuous hydrolysis and fermentation for cellulosic ethanol production. Bioresour Technol 2010, 101:4862-4874.
• [4]Xiao ZZ, Zhang X, Gregg DJ, Saddler JN: Effects of sugar inhibition on cellulases and beta-glucosidase during enzymatic hydrolysis of softwood substrates. Appl Biochem Biotechnol 2004, 115:1115-1126.4.
• [5]Sternberg D: β-glucosidase of Tichoderma: its biosynthesis and role in saccharification of cellulose. Appl Environ Microbiol 1976, 31:164-174.
• [6]Chauve M, Mathis H, Huc D, Casanave D, Monot F, Ferreira NL: Comparative kinetic analysis of two fungal β-glucosidases. Biotechnol Biofuels 2010, 3:3. BioMed Central Full Text
• [7]Seidle HF, Marten I, Shoseyov O, Huber RE: Physical and kinetic properties of the family 3 beta-glucosidase from Aspergillus niger which is important for cellulose breakdown. Protein J 2004, 23:11-23.
• [8]Teeri TT, Reinikainen T, Ruohonen L, Jones TA, Knowles JKC: Domain function in Trichoderma reesei cellobiohydrolase. J Biotechnol 1992, 24:169-176.
• [9]Tomme P, Warren RAJ, Miller RC Jr, Kilburn DG, Gilkers NR: Cellulose-binding domains: classification and properties. In Enzymatic Degradation of Insoluble Carbohydrates. Edited by Saddler JN, Pennaer MH. Washington, DC: American Chemical Society; 1995:142-163.
• [10]Lymar ES, Li B, Renganathan V: Purification and characterization of a cellulose-binding (beta)-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Appl Environ Microbiol 1995, 61:2976-1980.
• [11]Gundllapalli SB, Pretorius IS, Cordero ORR: Effect of the cellulose-binding domain on the catalytic activity of a β-glucosidase from Saccharomycopsis fibuligera. J Ind Microbiol Biotechnol 2007, 34:413-421.
• [12]Pei JJ, Pang Q, Zhao LG, Fan S, Shi H: Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose. Biotechnol Biofuels 2012, 5:31. BioMed Central Full Text
• [13]Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratorymanual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
• [14]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685.
• [15]Chen M, Xia LM, Xue PJ: Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate. Int Biodeter Biodegr 2007, 59:85-89.
• [16]Hua YW, Chi MC, Lo HF, Hsu WH, Lin LL: Fusion of Bacillus stearothermophilus leucine aminopeptidase II with the raw-starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity. J Ind Microbiol Biotechnol 2004, 31:273-277.
• [17]Ouyang J, Dong ZW, Song XY, Lee X, Chen M, Yong Q: Improved enzymatic hydrolysis of microcrystalline cellulose (Avicel PH101) by polyethylene glycol addition. Bioresour Technol 2010, 101:6685-6691.
• [18]Zhou QZK, Xiao DC: Immobilization of beta-galactosidase on graphite surface by glutaraldehyde. J Food Eng 2001, 48:69-74.
• [19]Stahlberg J, Johansson G, Pettersson G: A new model for enzymatic hydrolysis of cellulose based on the two-domain structure of cellobiohydrolase I. Biotechnology 1991, 9:286-290.
• [20]Tormo J, Lamed R, Chirino AJ, Morag E, Bayer EA, Shoham Y, Steitz TA: Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. EMBO J 1996, 15:5739-5751.
• [21]Mamo G, Hatti-Kaul R, Mattiasson B: Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7. Extremophiles 2007, 11:169-177.