Abstract
Objective: Energy and environmental issues are one of the most severe challenges for human survival and development in the 21st century. Therefore, the global interest in developing lignocellulosic biomass resources is growing, and cellobiohydrolase is one of the key enzymes for converting cellulose into sugars. Based on this, we want to breed strains with high cellobiohydrolase production. Methods: The strains with high production of cellobiohydrolase were screened by Congo red medium firstly. Then, strains producing transparent circle were screened again by enzyme activity assay, so as to screen out the strain with high production of cellobiohydrolase (with Trichoderma reesei as control). Observed the growth state of the strain and identified the strain. Atmospheric and Room Temperature Plasma mutagenesis was used to mutagenize the strain, and selected dominant mutants in order to further improve the enzyme activity. Finally, the enzymatic property analysis was carried out to evaluate the stability of cellobiohydrolase. Results: 12 strains were obtained through the primary screening by Congo red staining. After fermentation and re-screening, a strain with high enzyme activity was obtained, which was H9, and the maximum enzyme activity was 32.4 IU/mL. The colony of the strain nearly round, thick and fluffy, with a slight bulge in the middle, it was light gray flocculent in the initial stage of culture, and turns light green in the later stage; It was identified as Cladosporium. In this study, 36 positive mutants were screened, of which H9-18 mutant strain had the highest enzyme activity of 43.2 IU/mL, which was 33.33% higher than that before mutation. The results of the enzymatic property analysis showed that the cellobiohydrolase produced by the H9-18 strain selected in this study showed highest enzyme activity of 43.2 IU/mL when the temperature was 50 ℃. And the highest enzyme activity was 44.1 IU/mL when the pH was 4.8. Fe2+ had the strongest promoting effect on the enzyme activity of 38.67%, while Na+ had the most obvious inhibitory effect of 34.03%. Conclusion: This experiment finally obtained a strain H9-18 with high cellobiohydrolase production efficiency and stable enzyme activity, which further laid a theoretical and experimental foundation for the efficient utilization of lignocellulosic biomass resources.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wenli, Z., et al.: A comprehensive green utilization strategy of lignocellulose from rice husk for the fabrication of high-rate electrochemical zinc ion capacitors. J. Clean. Prod. 327, 129522 (2021)
Dawei, C., et al.: Sustainable utilization of lignocellulose: preparation of furan derivatives from carbohydrate biomass by bifunctional lignosulfonate-based catalysts. Catal. Commun. 84, 159–162 (2016)
Xiaoxue, Z., et al.: A structure-activity understanding of the interaction between lignin and various cellulase domains. Biores. Technol. 351, 127042 (2022)
Zhenying, P., Yijing, L., Bo, W., Fubao, S., Feng, X., Xueming, Z.: Mild fractionation of poplar into reactive lignin via lignin-first strategy and its enhancement on cellulose saccharification. Biores. Technol. 343, 126122 (2022)
Xie, X., Liu, B.: An effective approach to improve ethanol productivity by simultaneous saccharification and fermentation of rice straw. Int. J. Green Energy 15(10), 545–549 (2018)
Baig, K.S., Turcotte, G., Doan, H.: Adsorption of cellulose enzymes on lignocellulosic materials and influencing factors: a review. Int. J. Waste Resour. 6, 3 (2016)
Buchanan, C.J., Mitchell, W.J.: Two beta-glucosidase activities in Fibrobacter succinogenes S85. J. Appl. Bacteriol. 73(3), 243–250 (1992)
Jianlong, H., et al.: A novel strategy for producing cellulase from Trichoderma reesei with ultrasound ssisted fermentation using spent mushroom substrate. Process Biochem. 104(1), 110–116 (2021)
Puspawati, S., Wagiman, Ainuri, M., Nugraha, D.A., Haslianti: The production of bioethanol fermentation substrate from Eucheuma cottonii seaweed through hydrolysis by cellulose enzyme. Agric. Agric. Sci. Procedia 3, 200–205 (2015)
Ghazi, A.R., Münch, P.C., Chen, D., Jensen, J., Huttenhower, C.: Strain identification and quantitative analysis in microbial communities. J. Mol. Biol. (2022). https://doi.org/10.1016/j.jmb.2022.167582
Yuan, L., et al.: Characteristics of hydrogen production of an Enterobacter aerogenes mutant generated by a new atmospheric and room temperature plasma (ARTP). Biochem. Eng. J. 55(1), 17–22 (2011)
Peshin, A., Mathur, J.M.S.: Purification and characterization of glucosidase from Aspergillus niger strain 322. Lett. Appl. Microbiol. 28(5), 401–404 (2011)
Velmurugan, K.C.: Comparative studies using various substrates for enhancing yield of Pleurotus spp. Int. J. Curr. Microbiol. App. Sci. 9(11), 2831–2852 (2020)
Xiaobei, W., et al.: The atmospheric and room-temperature plasma (ARTP) method on the dextranase activity and structure. Int. J. Biol. Macromol. 70(8), 284–291 (2014)
Ren, F., Chen, L., Tong, Q.: Highly improved acarbose production of Actinomyces through the combination of ARTP and penicillin susceptible mutant screening. World J. Microb. Biot. 33(1), 16 (2017)
Rongming, L., et al.: An engineering Escherichia coli mutant with high succinic acid production in the defined medium obtained by the atmospheric and room temperature plasma. Process. Biochem. 48(11), 1603–1609 (2013)
Azizi, A., Sharifi, A., Fazaeli, H., Azarfar, A., Jonker, A., Kiani, A.: Effect of transferring lignocellulose-degrading bacteria from termite to rumen fluid of sheep on in vitro gas production, fermentation parameters, microbial populations and enzyme activity. J. Integr. Agric. 5, 1323–1331 (2020)
Acknowledgement
This work is supported by Ph.D. Startup Fund NO. [2020]18 of Guizhou University of Traditional Chinese Medicine.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Appendix
Appendix
Support Material 1
The genomic DNA of the strain was extracted, selected the universal primers of ITS sequence of fungus, amplified and sequenced. The sequencing results were compared with the GenBank database by the BLAST program and combined with the result of morphological observations, the strain was identified as Cladosporium.
Description | Max score | Total score | Query cover | E value | Per. ident | Accession |
|---|---|---|---|---|---|---|
Cladosporium cladosporioides strain BFMY-2 small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MT573533.1 |
Cladosporium caricinum strain CCPc-3 small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MZ198356.1 |
Cladosporium caricinum strain CCPc-2 small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MZ198355.1 |
Cladosporium caricinum strain CCPc-1 small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MZ198354.1 |
Cladosporium colombiae isolate MT19_ITS_12_SIPPE_ZMJ small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MW922818.1 |
Cladosporium cladosporioides internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MW793722.1 |
Cladosporium sp. Isolate SG small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MW602954.1 |
Cladosporium tenuissimum strain 3-12F small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MW077084.1 |
Cladosporium cladosporioides isolate Cc18_01 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MT854328.1 |
Cladosporium cladosporioides isolate LY-A2 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence | 972 | 972 | 100% | 0.0 | 100.00% | MT732011.1 |
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Nature Switzerland AG
About this paper
Cite this paper
Zhao, W., Ju, Z., Zheng, Y., Mei, S., Shi, H. (2023). Breeding and Efficiency Evaluation of a High-Yielding Cellobiohydrolase Strain. In: Wen, S., Yang, C. (eds) Biomedical and Computational Biology. BECB 2022. Lecture Notes in Computer Science(), vol 13637. Springer, Cham. https://doi.org/10.1007/978-3-031-25191-7_10
Download citation
DOI: https://doi.org/10.1007/978-3-031-25191-7_10
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-031-25190-0
Online ISBN: 978-3-031-25191-7
eBook Packages: Computer ScienceComputer Science (R0)