Team:UESTC-China/Design
Overview
Compared with chemical reagent deinking, biological enzyme is more suitable in office due to its low health hazard and green environmetal protection characteristics. In order to find high efficient and stable enzymes, we screened a large number of the
latest literatures and finally selected four deinking enzymes. They are: EG1 and EGL7 cellulase from Trichoderma and Aspergillus fumigatus, xylanase from Aspergillus niger, laccase from Botrytis aclada, and lipase from Aspergillus
niger. Moreover, cellulosome is introduced to our bio-deinking system in order to get higher efficiency.
Figure1. The whole Bio-deinking system.
Design flow
1. Paper and tonner components
The realization of deinking can not only depend on the direct degradation of the ink, but also on degradation of the composition of the paper. We need to first check the composition of the ink and paper. And then, from a variety of components, we should
find out the most effective deinking components as the substrate of the enzyme. Thus, we can determine what types of enzyme should be selected according to the substrate of the enzyme.
2. Determination of enzyme species
Enzymes degradating the same substrate from different species, have different optimum temperatures, pH and other properties. Finding an enzyme with good performance is very helpful to achieve efficient deinking. We identified 5 criteria to help us screen
for suitable enzymes.
Table1. Five criteria help us choose good sources of enzymes.
| 1 | Choosing enzymes that work at room temperature to save energy on deinker hardware； |
|---|---|
| 2 | Choosing enzymes that are active in the moderate pH range (6-8) to avoid the corrosive effects of strong acids and bases； |
| 3 | Choosing enzymes that have high thermal stability and are not easy to deactivate； |
| 4 | Choosing enzymes that are insensitive to common inhibitors； |
| 5 | Selecting the enzyme from which the gene sequence can be obtained； |
3. Introduction of cellulosome
During the process of choosing the enzyme type and its species, we learned about cellulosome, which is a good method to enhance the deinking efficiency. Cellulosome is a kind of self-assembled of enzyme complexes. It can enhance the targeting effect of
cellulose and promote synergies between the enzymes. Thus, we introduce cellulosome to our bio-deinking system, hoping higer efficiency can be achieved.
4. Cellulosome and Plasmids design
Natural cellulsome complex is so complicated that it is hard to be used. The sequences of each parts of cellulosome were found in NCBI and GeneBank. Using these sequence information, the natural cellulosome system was simplified and we designed our own
cellulosome structure as well as its corresponding plasmids. The self-designed cellulosome can assemble cellulase and xylanase with the portion of 2:1.
Paper and ink toner component
Paper structure and composition
Main ingredient of all paper is plant material such as Cellulose, Hemi-cellulose and modified Lignin. Loading or filling material such as clay, CaCO3, Talc, TiO2 are used for higher brightness and better printability.
Rosin, alum or combination of other chemicals is used to make paper water resistant. Some kind of paper with special purpose, such as coffee filter paper containing wet strength polymer to withstand hot water soaking. Colored paper
may contain dye or pigment.
Table2. Main composition of office waste paper.
| Plant material | Cellulose |
|---|---|
| Hemi-cellulose | |
| Lignin | |
| Minerals | Coating Pigments |
| Fillers | |
| Chemical Additives | All Kinds |
Of all the components, plant materials on paper surface always connect with ink toner(see in Figure2.). So, degradation of cellulose, hemi-cellulose and lignin helps to release the ink attached to the surface of paper.
Figure2. Paper Structure.
Ink toner structure and composition
The basic components of toners are polymers, resins, pigments or dyes, iron oxide, amorphous silica, charge control agents, paraffin wax, surfactants and other inorganic/organic additives. The actual makeup of these toners differs from manufacturer to
manufacturer and is kept a secret with the purpose of bringing uniqueness to itsproperties.
However, certain polymers like polyester, polystyrene, or polyacrylate are commonly found in most toners. Certain other components, for instance, surfactants and paraffin wax, can enhance the adherence of toner particles to the surface of the paper. So,
degradation of these components can help weaken the binding force between ink and paper fibers. So, degradation of these components can help weaken the binding force between ink and paper fibers.
Determination of Enzymes
Cellulase
(1) Roles in deinking process
Cellulase is a complex mixture，including three different compounds: endoglucanase, cellobiohydrolase and β-glucosidase. Endoglucanase randomly cleaves the amorphous region of the cellulose chain(see in Figure3.), while other two enzymes begin cleavation
at chain ends, producing monosaccharide. Considering that most ink toners are not at the ends of cellulose fiber, we only choose endoglucanase to deink. For convenience, we will call endoglucanase cellulase.
Figure3. Endoglucanase deinking principle.
(2) Species identification
Aftering comparing lots of cellulases in the latest articles, we chosed EG1（GenBank EU935217）and EGL7（Gene ID: 3505017(AFUA_6G01800)）as nominates of cellulase, they can maintain enough activity at room temperature, and have high resistance to pH changing.
Figure4. Part of Cellulase comparison chart.
Xylanase
(1) Roles in deinking process
Xylanase belongs to the hemicellulase system. Xylan hydrolase is a family of xylan degradation enzymes, including β-1, 4-endoxylanase, β -xylo-sidase, α-L-arabinase, α-D-glucosidase, acetyl xylanase, and phenolic esterase. They can degrade xylan hemi-cellulose.
In the xylan hydrolase family, β-1, 4-endoxylanase plays a key role in cleaving hemi-cellulose. And by breaking the hemi-cellulose, ink particle is released from the hemi-cellulose on paper surface. Therefore, the removal of ink toner
is accompanied by the removal of hemi-cellulose.
Xylanase is often used in combination with cellulase in the traditional deinking process to assist and strengthen the deinking effect of cellulase. After adding xylanase, compared with cellulase alone, the deinking effect is better, the paper produced
by the whiteness is higher, deinking time is reduced, and the efficiency is higher.
Figure5. Xylanase deinking principle.
(2) Species identification
Like how we chose cellulase, we also listed a comparison chart when chose xylanase. At the end, the wild xylanaseB (XynB, GenBank: JX560731.1) from Aspergillus nigerIA-001 was selected. XynB has excellent pH and temperature
stability.
The XynB（JX560731.1） selected by us has wide pH range. The enzyme activity can be maintained at a higher pH range from 4 to 8. In addition, the modified XynB selected by us has the characteristics of tolerance to heavy metal
ions such as copper ions and certain protease resistance. Its enzyme activity is also high, making it can adapt to the complex deinking environment.
Figure6. Part of Cellulase comparison chart.
Laccase
(1) Roles in deinking process
Laccase, a versatile oxidoreductase enzyme, which can effectively degrade residual lignin. Laccase enables the lignin in the paper to degrade into monomers and can also directly interact with lignin to generate phenoxy free radicals on the fiber surface
which contributes to the strength of paper, especially the wet strength. Furthermore, laccase is able to degrade phenols in ink or toner or paper fillers which enables the deinking waste water to meet the discharge standard.
Figure7. Laccase deinking principle.
(2) Species identification
We chose Blac (GenBank: JN559771.1) as candidates laccase which has excellent PH stability and temperature stability.
Lipase
(1) Roles in deinking process
The natural substrate of lipase is triacylglyceride. The ester bond of triacylglyceride can be hydrolyzed by lipase. The function of lipase in deinking waste paper is to attack the ester bond of ink or ink binder. It can weaken the bond between ink and
paper fiber. After treated by lipase, ink particles can be completely removed by floating and washing.
Figure8. Lipase deinking principle.
(2)Species identification
The Aspergillus in the filamentous fungus is one of the important sources for obtaining high-performance lipase. We chose the lipase from Aspergillus niger F044 which NCBI accession number is DQ647700.1.
Introduction of Cellulosome
Why we introduced cellulosome？
Cellulosome is a self-assembled multi-enzyme complex.
The core of the cellulosome design is the scaffoldin protein, which contains a cellulose binding module (CBM) that binds cellulose substrates（see in Figure9.) to enhance the targeting effect of enzymes.
Figure9. Scaffoldin proteins bind to cellulose through CBM domain.
In addition, the most important structural domains of cellulosome are cohesin and dockerin domain. They are a pair of domains with specific interactions. Enzymes with dockerin domain can be anchored together on scaffoldin protein with cohesin domains,
which promotes the spatial proximity of various enzymes and play a synergistic role (see in Figure10.).
These advantages provide a good idea for developing a method to improve the effective degradation of cellulose substrate. We can achieve higher deinking efficiency through cellulosome.
Cellulosome and Plasmids design
Cellulosome Design
1. Assembly way
Cellulosome has two main assembly forms, random assembly or speicific assembly. The first thing when we designed what our cellulosome structure would be like is choosing one of this two assembly forms.
Generally, scaffoldin proteins have two types of cohesin. Type I cohesin binds enzymes with type I dockerin, and type II cohesin binds dockerin displaying on the cell surface, which causes scaffoldin proteins to be anchored on the cell surface. Between
different species, cohesin don't have species intersectionality i.e. and only the type I cohesin and type I dockerin from the same species can bind specifically.
For specific assembly approaches, the type I cohesins from different genotypes are installed on scaffoldin and the corresponding genotypes of type I dockerins are installed on the different enzymes. This allows us to specifically control the proportion
and location of the enzymes which bind with scaffoldin protein. For example, in Figure10, we control the enzyme 1 to 4 binding with scaffoldin protein from left to right in a 1:1:1:1 ratio.
Figure10. Specific Assembled Cellulosome.
Different with specific assembly, all the type I cohesin are from the same species in random assembly approach. As you can see in Figure11, the location and portion of the enzymes are both not fixable, and have randomness during every single assembly
process. Theoretically, if we have four kinds of enzymes with the same type I dockerin, and have four same type I cohesin in scaffoldin protein, we might get 4×4×4×4 kinds of cellulosome morphology in one reaction ( Figure11 shows
three of them).
Figure11. Some possibilities of random assembled cellulosome.
Both random assembly and specific assembly have its advantages and disadvantages. We liste them on Table3. Considering that we might finally let enzymes become enzyme preparation when using in the deinking hardware, it is important for us to know the
details of which enzymes' ratio has the best efficiency and make some improvement in the far future, so we finally decided to choose specific assembly method.
Table3. Pros and cons comparison of two kinds of cellulosome assembly approach.
| Pros | Cons | |
|---|---|---|
| Random Assembly | Advantageous for expanding the list of enzymes | It is impossible to control the enzyme proportion accurately, and the enzyme proportion of each constructed cellulosome may be different. |
| The realization is simple and the success rate of constructing cellulosome is higher | With such randomness and complex influencing factors, it may be difficult for us to analyze the results | |
| Specific Assembly | Provide highly controllable ordering of each enzyme | Increase the difficulty of the experiment |
| Facilitate subsequent improvement | Disadvantageous for expanding the list of enzymes to be incorporated into the cellulosome complex |
2. Enzymes anchored on scaffoldin
According to the deinkning principles of different enzymes which are stated in the determination of enzymes section, we know that cellulase and xylanase are more relevant to degrade the plant materials, which connected with ink particles on paper surface.
So it's helpful if we use CBM domain on scaffoldin protein to durg cellulase and xylanase on the paper surface and then work together.
Theoretically, all the enzymes can anchor on the scaffoldin protein. However, the substrate of lipase and laccase both contains ink itself. Anchoring them on scaffoldin protein might reduce their efficiency because its possibility of contacting with ink
freely is reduced. Thus, in the first edition of our cellulosome system, we decided only cellulase and xylanase be anchored on the scaffoldin protein. And we also need to determine whether we will change the structure based on the
enzymes deinking experimental results.
3.The proportion of the enzymes
Natural scaffoldin protein has 9 cohesin domains. By now, only 2 researches have successfully recombinated the whole length of natural scaffoldin protein because it's too large to expressed in other chassis organisms. Most of the researches built recombinant
scaffoldin with three cohesin domains. Thus, considering the probability, we decided to install no more than three cohesins on the first edition of our self-design scaffoldin protein.
And because cellulose is the main component of waste paper, cellulase plays a more important role in deinking process compared with xylanase. We designed the scaffoldin protein that can let the portion of cellulase: xylanase to be 2:1.
Plasmids design
1. pEGL7 and pEG1 plasmid
pEGL7 and pEG1 express EGL7 and EG1 cellulase with CcsDockerin domain respectively. The functional domain of cellulase and dockerin domain (CcsDockerin, which can combines with CcsCohesin on scaffoldin) are linked via GSlinker, yielding a new fusion protein
able to bind cellulosome. Moreover, we added a His-tag on the C-terminal of this fusion protein, which can help us complete protein purification, western blot and fluorescence microscope analysis.
Figure12. pEGL7 and pEG1 plasmids and their expressed proteins.
2. pXynB plasmid
pXynB plasmid expresses xylanase with CtDockerin domain. The C-terminal of the catalytic domain is separated from CtDockerin by a short GSlinker sequence. His-tag was added on the C-terminal of the whole fusion protein.
Figure13. pXynB plasmids and it expressed fusion protein.
3. pLiplac plasmid
We put the laccase gene on a plasmid together with the lipase gene for the reason that these enzymes both work in the form of free enzyme instead of binding to the dockerin domain. The genes of lipase and laccase are constructed on the same plasmid, and
they are connected using 2A peptides. For 2A peptide, we chose E2A, which can be cut after the laccase and lipase are expressed, so that the two enzymes can perform their respective functions.
Otherwise, In order to ensure that the lipase can be smoothly secreted, we introduced SMT3(Gene ID: 852122),the SUMO fusion sequence is used to increase the expression of lipase, and the SMT3 and lipase sequences are connected by GS linker (GGGGS).
Figure14. pLiplac plasmids and it expressed fusion proteins.
4. pScaf plasmid
pScaf plasmid expresses scaffoldin, and there are two kinds of cohesin in the scaffoldin we designed: CcsCohesin and CtCohesin, whose proportion is 2:1. Scaffoldin also has a CBM domain and a Type II dockerin domain. Pro/Thr linker was used to connect
and seperate the different domain. Comparing to Scaffoldin （BBa_K2155012 ） protein of Team iGEM16_NWPU, we put CBM domain in the middle of two CcsCohesin so
as to reduce the possibility of steric hindrance. Myc-tag was added on the C-terminal of the scaffoldin protein.
Figure15. pLiplac plasmids and it expressed fusion proteins.
5. pSbdA plasmid
pSdbA expresses SdbA, an anchor protein, with flag tag in C-terminal. SdbA protein makes scaffoldin and its assembled enzymes anchors together on cell wall. SdbA protein can be used as a tool to detect whether the cellulosome is successfully assembled
or not. For example, SdbA makes scaffoldin and enzymes on the scaffoldin anchor on the surface of the chassis organism. If we treat the protein of cellulosome system with its antibody which has fluorescence, corresponding fluorescence
can occur on the surface of Pichia pastoris cells, which indicates that cellulosome complex is successfully assembled on the surface（see in Figure16.）.
Figure16. Chassis organism treated with different antibodies under a fluorescence microscope.
Chassis Organism
We choose eukaryotes this year. For one reason that we choose cellulase and xylanase as the main deinking enzymes, we consider eukaryotes as the chassis organisms to make the enzymes work better, because eukaryotes expression system can post-translationally
process proteins. Also, cellulosome is mostly studied on eukaryotes.
We found that Saccharomyces cerevisiae and Pichia pastoris were usually used as chassis organisms in eukaryotic expression system. Both of them belong to iGEM risk level 1, so there are no safety concerns.
However, as S. cerevisiae could not overcome the problem of glycosylation, we finally chose Pichia pastoris. Pichia pastoris can secrete and express exogenous proteins at a high level, for the reason that there are few proteins secreted by Pichia pastoris
itself into the culture medium, so it is convenient to purify and produce soluble recombinant proteins with correct folding.
Then, we selected GS115 as the candidate specie according to foreign and domestic studies on the heterologous expression of cellulase. GS115 strain is the most mature strain in Pichia pastoris. It is histidine deficient type and can only live with added
additional histidine, which ensures biosafety and is convenient for screening. However the other choice —— X33 is wild type without nutritional deficiency (After repeated comparisons, we summarized the following advantages and disadvantages
of the two kinds of yeast in Table4). Considering the effective screening in subsequent experiments, we finally selected GS115 as our chassis organism.
Table 4. GS115 versus X33.
| Saccharomyces cerevisiae | Pichia pastoris | |||
|---|---|---|---|---|
| Adventages | Eukaryotes can post-translationally process proteins. | |||
| 1. Saccharomyces cerevisiae is safe and easy to cultivate in laboratory, and has been widely used in the food industry for a long time. It does not produce toxins, and has been identified as a safe organism by the FDA of the United States, and the expression products do not need to undergo a lot of host safety experiments. | 1. The function of expression and secretion is strong. | Its secreted protein is less, which is conducive to the purification of the target protein. | ||
| The signal peptide suitable for target protein can be quickly obtained through existing research. | ||||
| Good folding ability. | ||||
| 2. Its product is not easy to produce inclusion body and the secretion of it is stable. | 2. Defective and phenotypic strains have been studied extensively and can be constructed on demand. | |||
| 3. It can absorb heavy metal ions, which is beneficial to expand the influence of the project. | 3. High degree of commercialization. | |||
| 4. The genetic background is clear, the transformation is more simple with many ways. | 4. It has been proved that glycerol and glucose can be used as carbon sources, and rhamnose can also be induced after modification. | |||
| 5. The substrate is simple and easy to operate. | ||||
| Disadvantages | 1. Low yield, difficult to carry out high density fermentation (difficult to secrete, protein above 30K Dalton is difficult to secrete, and low expression efficiency). | 1. The fermentation period is long, and the protease produced in the process is easy to decompose the target protein. There is also evidence that exogenous proteins are easy to stay in the endoplasmic reticulum and cannot be secreted during pichia pastoris expression under certain conditions. | ||
| 2. The promoter is unstable (only TATAbox and its location is unstable, and the spacing between the sequence used to enhance expression and the gene is required not too far away). | 2. The AOX12 promoter must be induced with methanol, but after modification, rhamnose can be used as induction. | |||
| 3. Target proteins are susceptible to superglycosylation. | 3. It is complicated and difficult to carry out renovation. | |||
| 4. The process of cell division is not stable (plasmid transmission is not stable) with many mutations. | 4. Methanol induction involves product safety issues (if we are going to use bacteria directly to deink). | |||
| 5. Inducible promoters require inducers, which are expensive and inhibit yeast growth. | ||||
Expression vector
After reviewing the literature, we found that Pichia pastoris GS115 expression vector was mostly pIC9K in studies, but methanol was needed for induction during the expression process and it is not safe. So, we chose another pGAPZαA without methanol induction,
which is a constitutive expression vector and can shuttle freely. It can be stored, replicated and amplified in Escherichia coli at first, and then linearized and introduced into yeast cells to undergo homologous recombination with
its chromosomes. After the gene expression framework of the target protein integrated into the chromosome, the expression of foreign genes can be realized.
References
[1] Ataeefard, Maryam, and Mohammad Reza Saeb. “A Multiple Process Optimization Strategy for Manufacturing Environmentally Friendly Printing Toners.” Journal of Cleaner Production, vol. 108, Elsevier Ltd, 2015, pp. 121–30, doi:10.1016/j.jclepro.2015.07.016.
[2] Biedermann, A., et al. “Implementing Statistical Learning Methods through Bayesian Networks (Part 2): Bayesian Evaluations for Results of Black Toner Analyses in Forensic Document Examination.” Forensic Science International, vol. 204, no. 1, Elsevier
Ireland Ltd, 2010, pp. 58–66, doi:10.1016/j.forsciint.2010.05.001.
[3] Shah F, Ranawat B, Mishra S. An Approach Toward Cellulase Production, Bioconversion, and Utilization[M]//Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts. Woodhead Publishing, 2019: 207-223.
[4] Shu, Zhengyu, et al. “Aspergillus Niger Lipase: Heterologous Expression in Pichia Pastoris, Molecular Modeling Prediction and the Importance of the Hinge Domains at Both Sides of the Lid Domain to Interfacial Activation.” Biotechnology Progress, vol.
25, no. 2, Wiley Subscription Services, Inc., A Wiley Company, 2009, pp. 409–16, doi:10.1002/btpr.147.
[5] Kittl, Roman, et al. “A Chloride Tolerant Laccase from the Plant Pathogen Ascomycete Botrytis Aclada Expressed at High Levels in Pichia Pastoris.” Journal of Biotechnology, vol. 157, no. 2, Elsevier B.V, 2012, pp. 304–14, doi:10.1016/j.jbiotec.2011.11.021.
[6] Fang, Wei, et al. “Cloning and Expression of a Xylanase xynB from Aspergillus Niger IA-001 in Pichia Pastoris.” Journal of Basic Microbiology, vol. 54, no. S1, Blackwell Publishing Ltd, 2014, pp. S190–S199, doi:10.1002/jobm.201300078.
[7] Budinova, Geisa A. L. .., et al. “Casein-Based Scaffold for Artificial Cellulosome Design.” Process Biochemistry (1991), vol. 66, no. C, Elsevier Ltd, 2018, pp. 140–45, doi:10.1016/j.procbio.2017.12.013.
[8] Bule, Pedro, et al. “Cellulosome Assembly: Paradigms Are Meant to Be Broken.” Current Opinion in Structural Biology, vol. 49, Elsevier Ltd, 2018, pp. 154–61, doi:10.1016/j.sbi.2018.03.012.
[9] Chockalingam, Karuppiah, et al. “Golden Gate Assembly with a Bi-Directional Promoter (GBid): A Simple, Scalable Method for Phage Display Fab Library Creation.” Scientific Reports, vol. 10, no. 1, NATURE PUBLISHING GROUP, 2020, pp. 2888–2888, doi:10.1038/s41598-020-59745-2.
[10] Deng, Lan, et al. “Cellulose Hydrolysis Ability of a Clostridium Thermocellum Cellulosome Containing Small-Size Scaffolding Protein CipA.” Journal of Biotechnology, vol. 212, Elsevier B.V, 2015, pp. 144–52, doi:10.1016/j.jbiotec.2015.08.016.
[11] Galera-Prat, Albert, et al. “Solution Conformation of a Cohesin Module and Its Scaffoldin Linker from a Prototypical Cellulosome.” Archives of Biochemistry and Biophysics, vol. 644, Elsevier Inc, 2018, pp. 1–7, doi:10.1016/j.abb.2018.02.016.
[12] Getzlaff, M., et al. “Magnetic Nanoparticles in Toner Material.” Nano-Structures & Nano-Objects, vol. 22, 2020, p. 100462, doi:10.1016/j.nanoso.2020.100462.
[13] Gilmore, Sean P., et al. “Designing Chimeric Enzymes Inspired by Fungal Cellulosomes.” Synthetic and Systems Biotechnology, vol. 5, no. 1, Elsevier B.V, 2020, pp. 23–32, doi:10.1016/j.synbio.2020.01.003.
[14] Hirano, Katsuaki, et al. “In Vitro Assembly and Cellulolytic Activity of a β-Glucosidase-Integrated Cellulosome Complex.” FEMS Microbiology Letters, vol. 366, no. 17, 2019, doi:10.1093/femsle/fnz209.
[15] Hyeon, Jeong-eun, et al. “Production of Minicellulosomes from Clostridium Cellulovorans for the Fermentation of Cellulosic Ethanol Using Engineered Recombinant Saccharomyces Cerevisiae.” FEMS Microbiology Letters, vol. 310, no. 1, Blackwell Publishing
Ltd, 2010, pp. 39–47, doi:10.1111/j.1574-6968.2010.02035.x.
[16] Ichikawa, Shunsuke, et al. “Cellulosomes Localise on the Surface of Membrane Vesicles from the Cellulolytic Bacterium Clostridium Thermocellum.” FEMS Microbiology Letters, vol. 366, no. 12, 2019, doi:10.1093/femsle/fnz145.
[17] Kumar, Raj, et al. “On the Spectroscopic Cum Chemometric Approach for Differentiation and Classification of Inkjet, Laser and Photocopier Printed Documents.” Science & Justice, vol. 60, no. 4, Elsevier B.V, 2020, pp. 347–57, doi:10.1016/j.scijus.2020.01.004.
[18] Li, Xiaoyi, et al. “The Spatial Proximity Effect of Beta-Glucosidase and Cellulosomes on Cellulose Degradation.” Enzyme and Microbial Technology, vol. 115, Elsevier Inc, 2018, pp. 52–61, doi:10.1016/j.enzmictec.2018.04.009.
[19] Mori, Yutaka. “Purification and Characterization of an Endoglucanase from the Cellulosomes (Multi-Component Cellulase Complexes) of Clostridium Thermocellum.” Bioscience, Biotechnology, and Biochemistry, vol. 56, no. 8, Taylor & Francis, 1992, pp.
1198–203, doi:10.1271/bbb.56.1198.
[20] Nikolaev, A. .. “Flotation Recovery of Toner Containing Iron Oxide from Water Suspension.” Minerals Engineering, vol. 144, Elsevier Ltd, 2019, p. 106027, doi:10.1016/j.mineng.2019.106027.
[21] Ozturk, Sibel, et al. “Double Promoter Expression Systems for Recombinant Protein Production by Industrial Microorganisms.” Applied Microbiology and Biotechnology, vol. 101, no. 20, Springer Berlin Heidelberg, 2017, pp. 7459–75, doi:10.1007/s00253-017-8487-y.
[22] Peer, Ayelet, et al. “Noncellulosomal Cohesin- and Dockerin-Like Modules in the Three Domains of Life.” FEMS Microbiology Letters, vol. 291, no. 1, Blackwell Publishing Ltd, 2009, pp. 1–16, doi:10.1111/j.1574-6968.2008.01420.x.
[23] Sakka, Kazutaka, et al. “Unusual Binding Properties of the Dockerin Module of Clostridium Thermocellum Endoglucanase CelJ (Cel9D-Cel44A).” FEMS Microbiology Letters, vol. 300, no. 2, Blackwell Publishing Ltd, 2009, pp. 249–55, doi:10.1111/j.1574-6968.2009.01788.x.
[24] Sakka, Kazutaka, et al. “Analysis of Cohesin-Dockerin Interactions Using Mutant Dockerin Proteins.” FEMS Microbiology Letters, vol. 314, no. 1, Blackwell Publishing Ltd, pp. 75–80.
[25] Smith, Steven P., et al. “Continually Emerging Mechanistic Complexity of the Multi-Enzyme Cellulosome Complex.” Current Opinion in Structural Biology, vol. 44, Elsevier Ltd, 2017, pp. 151–60, doi:10.1016/j.sbi.2017.03.009.
[26] Tsatsis, D. .., et al. “Assessment of the Impact of Toner Composition, Printing Processes and Pulping Conditions on the Deinking of Office Waste Paper.” Journal of Environmental Chemical Engineering, vol. 7, no. 4, Elsevier Ltd, 2019, p. 103258,
doi:10.1016/j.jece.2019.103258.
[27] Yu, Yuan, et al. “Enhancing the Expression of Recombinant κ-Carrageenase in Pichia Pastoris Using Dual Promoters, Co-Expressing Chaperones and Transcription Factors.” Biocatalysis and Biotransformation, vol. 38, no. 2, Taylor & Francis, 2020, pp.
104–13, doi:10.1080/10242422.2019.1655001.
[28] Clarkson; Kathleen A, et al. Trichoderma Longibrachiatum EGIII Cellulase. 6th ed., 1998.
[29] Pathak, Puneet, et al. “Optimization of Chemical and Enzymatic Deinking of Photocopier Waste Paper.” Bioresources, vol. 6, no. 1, North Carolina State University, 2010, pp. 447–63, doi:10.15376/biores.6.1.447-463.
[30] Vianna Bernardi, Aline, et al. “A Thermostable Aspergillus Fumigatus GH7 Endoglucanase Over-Expressed in Pichia Pastoris Stimulates Lignocellulosic Biomass Hydrolysis.” International Journal of Molecular Sciences, vol. 20, no. 9, MDPI, 2019, p. 2261,
doi:10.3390/ijms20092261.
[31] Jain, Kavish Kumar, et al. “Functional Expression of a Thermostable Endoglucanase from Thermoascus Aurantiacus RCKK in Pichia Pastoris X-33 and Its Characterization.” Molecular Biotechnology, vol. 60, no. 10, Springer US, 2018, pp. 736–48, doi:10.1007/s12033-018-0106-3.
[32] mKhan, Maria Tariq, et al. “Evaluation of Factors Affecting Saccharification of Sugarcane Bagasse Using Cellulase Preparation from a Thermophilic Strain of Brevibacillus Sp.” Current Microbiology, vol. 77, no. 9, SPRINGER, 2020, pp. 2422–29, doi:10.1007/s00284-020-02059-3.
[33] Jain, Kavish Kumar, et al. “Functional Expression of a Thermostable Endoglucanase from Thermoascus Aurantiacus RCKK in Pichia Pastoris X-33 and Its Characterization.” Molecular Biotechnology, vol. 60, no. 10, Springer US, 2018, pp. 736–48, doi:10.1007/s12033-018-0106-3.
[34] Chutani, Preeti, and Krishna Kant Sharma. “Concomitant Production of Xylanases and Cellulases from Trichoderma Longibrachiatum MDU-6 Selected for the Deinking of Paper Waste.” Bioprocess and Biosystems Engineering, vol. 39, no. 5, Springer Berlin
Heidelberg, 2016, pp. 747–58, doi:10.1007/s00449-016-1555-3.
[35] Xu, Yang, et al. “A Xylanase from Streptomyces Sp. FA1: Heterologous Expression, Characterization, and Its Application in Chinese Steamed Bread.” Journal of Industrial Microbiology & Biotechnology, vol. 43, no. 5, Springer, 2016, pp. 663–70, doi:10.1007/s10295-016-1736-8.
[36] Zhang, Hong-Lian, et al. “Expression of xylanase gene xynA from Streptomyces olivaceoviridis A1 in Escherichia coli and Pichia pastoris.” Shengwu gongcheng xuebao, vol. 19, no. 1, 2003, pp. 41–45.
[37] Gao, He, et al. “Expression of Aspergillus Niger IA-001 Endo-β-1,4-Xylanase in Pichia Pastoris and Analysis of the Enzymic Characterization.” Applied Biochemistry and Biotechnology, vol. 173, no. 8, Springer US, 2014, pp. 2028–41, doi:10.1007/s12010-014-1000-5.
[38] Han, Sung Ok, et al. “Isolation and Expression of the xynB Gene and Its Product, XynB, a Consistent Component of the Clostridium Cellulovorans Cellulosome.” Journal of Bacteriology, vol. 186, no. 24, American Society for Microbiology, 2004, pp. 8347–55,
doi:10.1128/JB.186.24.8347-8355.2004.
[39] Kittl, Roman, et al. “A Chloride Tolerant Laccase from the Plant Pathogen Ascomycete Botrytis Aclada Expressed at High Levels in Pichia Pastoris.” Journal of Biotechnology, vol. 157, no. 2, Elsevier B.V, 2012, pp. 304–14, doi:10.1016/j.jbiotec.2011.11.021.
[40] Zhang. “Study on high expression, enzymatic properties and application of acid lipase.” [D]. Jiangnan University,2018. (in Chinese)