Team:UESTC-China/Demonstrate

description

Overview

To achieve the purpose of using enzymes to replace traditional physical and chemical deinking methods, we have tried four different enzymes. And in the experiment, we found that cellulase, xylanase, and lipase could realise biodeinking by helping the ink detach from the waste paper and degrade harmful substance. And the results of multi-enzyme synergetic experiment has showed that the best deinking effect can occur when the ratio of cellulase and xylanase is 2:1. In our wet lab, through Dot-Blot, Western-Blot and enzyme activity definition, cellulase EGL7(BBa_K3819003), xylanase XynB(BBa_K3819062), and lipase Lipase(BBa_K3819128)'s expressions and functions have successfully been proved. To improve the function of enzymes, we designed the cellulosome system and we are continously on the way to verify it. Click on the figure below to learn more about demonstration.

Figure1. Results Flow

Recombination and Expression

1. Results

Enzyme Expression

A.Target genes insertion

Pichia pastoris is a kind of biological chassis which has high expression efficiency, low glycosylation level and great adaptation to exogenous gene. The insertion of Pichia pastoris is much more difficult than E.coli for the reason that more than 6ug linearized plasmids and freshly prepared competent yeast cells are needed in each electroporation. However, Pichia pastoris GS115 is much more frequently to lose the inserted plasmids than X33.

To insert plasmids into Pichia pastoris' genomes, UESTC-China digested plasmids by restriction enzymes at sites on promotor(Figure2. (b)), carrying out electroporation through homologous recombination(Figure2.(a)).

Figure2. Plasmids Linearization. (a)Schematics of PCR strategy for homologous recombination reaction. (b)pGAPZa-EGL7 digested by BglⅡ, produces a 4460bp band; (c)pGAPZa-EG1 digested by BglⅡ, produces a 4508bp band; (d)pGAPZa-XynB digested by BglⅡ, produces a 3974bp band; (e)pGAPZa-Scaf digested by AvrⅡ, produces a 5582bp band; (f)pGAPZa-SdbA digested by BspHⅠ, produces a 4700bp band; (g)pGAPZa-Liplac digested by AvrⅡ, produces a 6386bp band. Controls are the circular(not linearized)plasmids, which will give ≥2 bands: a DNA supercoil band (the shorter one), and a DNA helix band (the longer one). Markers are Trans 2K Plus Ⅱ and 5K marker from Transgen.

After electroporation and cultivation on MDHz plates for 2 days, colony PCR was conducted and visualized by 1.2% agrose gel electrophoresis. However, high false positive rate forced us to do PCR on a large scale and it wasted so much time. We used 96 wells plate to screen single colonies with YPDz(zeocin concentration is 100ug/ml) culture(Figure3.). After three continuous passages, the cell's concentration in each well would reach the same. Then UESTC-China inoculated them on MDHz plates agian. Their colonies' size and growth rate would relate with the number of plasmids' multicopy.

Figure3. 96 wells plate screening. Every well was filled with 200ul YPDz culture and yeast cells would proliferate in it. Take 2ul culture in wells to inoculate on MDHz plates after continuous passages.

We designed non-specific primers(pGAP forward and 3'AOX1) and six specific primers for six plasmids as our double insurance. Using non-specific primers, the six plasmids will produce a ~540bp PCR products as GS115's intrinsic band, while the inserted fragment will produce another band, which can be calculated by the length of inserted fragments plus 540bp.

Figure4. Colony PCR results of recombined GS115. In each photo the left arrow refers to specific primers' product, and the right one refers to non-specific primers' product.(a)pEGL7, produces 694bp and 2207bp bands respectively; (b)pEG1, produces 1072bp and 2254bp bands respectively; (c)pXynB, produces 662bp and 1448bp bands respectively; (d)pScaf, produces 1901bp and 3047bp bands respectively; (e)pSdbA produces 707bp and 2441bp bands respectively; (f)pLiplac, produces 861bp and 4124bp bands respectively.

B.Fermentation and expression

Then UESTC-China tested if the recombined colony could produce and express target proteins successfully in consideration of yeast's unpredictable expression level. By adding different tags on the C-terminal of target genes, which could combine with specific antibodies and show their existence by substrate chemiluminescence. Therefore, we could easily judge the presence of proteins in fermentation broth（Figure5.) except SdbA which is displayed on the surface of cells and could only be verified in sediment. Other 6 proteins were secreted into the supernatant directly.

Figure5. The Dot-Blot results of recombined GS115 fermentation broth. All samples were acquired after fermentation in YPD for 96h. EGL7(His-Tag), EG1(His-Tag), XynB(His-Tag), Scaffoldin(Myc-Tag), Laccase(His-Tag) and Lipase(Myc-Tag) were tested by adding 2ul fermentation supernatant on NC membrane and exposing 60s.

In experiment, recombined GS115 cells with high multicopy plasmids were more likely to have high expression levels as might have been expected.

C.Function test for four enzymes

Although Pichia pastoris GS115's protein glycosylation level is artificially declined and UESTC-China choose GS linker to form fusion protein, we were still unclear of their performance in our lab. After fermentation in YPD for 3~4 days, we tested four enzymes' activities.

Figure6. The DNS results of EGL7 and XynB reflects enzymes' activity. Both solutions were acquired after 96h of fermentation. (a)EGL7 reaction results. 2ml fermentation supernatant reacted with 2ml 0.8%(w/v) sodium carboxymethyl cellulose (CMC) for 30min, then added 5ml DNS to shut down the process. During boiling in water bath, DNS combined with reducing sugar and produced dark brown, while the control group just shown unobvious color change. (b)XynB reaction results. Except changing substrate from CMC to 1% Xylan solution and using ammonium sulfate deposition method to acquire concentrated protein, other procedures are the same as EGL7's activity defining procedure.

DNS method is widely used in polysaccharide hydrolase mediated reducing sugar systems, and the color of reaction system is proportional to the reducing sugar content. For cellulase and xylanase, which could digest cellulose and xylan respectively and produce reducing ends, the color of reaction system would be darker than the control group, in which DNS was added before reaction to kill enzyme's activity(Figure7.).

During the DNS experiment, a weird phenomenon surprised us: the control group of EGL7's enzyme activity test also changed color to brown, although we could still clarify the difference of color between the control group and the experiment group. Compared with the control group of XynB's results, which had concentrated and changed protein's medium before reaction, we concluded that YPD, the culture used for fermentation, would decompose and produce reducing sugar, which was an intervention considering DNS's principle.

The enzyme activity of Lipase could be measured by Alkali Titration. Olive oil emulsified with polyvinyl alcohol(PVA) by high-speed homogenizer was digested by Lipase and produced fatty acid. Then added two drops of phenolphthanlein incicator and used 100mM NaOH solution to titrate until reaction systems turned red and maintained more than 30s. Heat inactivated Lipase solution had been used in the control group.

Figure7. Alkali Titration results of Lipase. 1ml concentrated protein solution mixed with 5ml 50mM, PH=5.0 phosphate buffer and 4ml emulsion, then reacted at 40℃ for 15min. In the experiment system, produced fatty acid neutralizes 100mM NaOH solution. Therefore, the color of the experiment system was stable during the process of titration while the system turns red direclty in the control group.

For laccase, the change of ABTS solution's absorbance value could reflect its enzyme activity for laccase. The laccase could react with ABTS and produce ABTS free radicals, whose absorption coefficient at 420nm is much higher than ABTS. However, we failed to detect any changes between the control group and the experiment group due to low enzyme activity. It might be necessary for us to introduce modifications or change chassis to improve laccase's activity, which is impossible for us to accomplish in a limited time.

D.SDS-PAGE and Western-Blot analysis

Different from enzymes, Scaffoldin could only be tested by SDS-PAGE and Western-Blot. For insurance, we also conducted SDS-PAGE on the proteins in GS115(pEG1) and GS115(pXynB)'s fermentation broth. Considering that constitutive expression was also weak in unconcentrated fermentation broth, it was hard to distinguish the bands we expected for low protein content(Figure8. (a)). But the bands turned clear after ammonium sulfate deposition(Figure8. (b)).

Figure8. The SDS-PAGE analysis of recombined GS115(pEG1) and GS115(pXynB) fermentation broths, showing target protein existence in substrate. (a)EG1, Line1, 2, 3, 4 are GS115(pEG1) supernate, GS115(pEG1 ) lysate, GS115 supernate, and GS115 lysate without concentration, and EG1 with Ccsdockerin is about 60.3KDa; (b)XynB, Line 1 and 2 are GS115(pXynB) supernate and GS115 supernate respectively after 75-fold concentration, and XynB with Ctdockerin is about 43.4KDa; (c)Scaffoldin, Line1, 2, 3, 4, 5, 6 are concentrated GS115(pEGL7) supernate, GS115(pEGL7) supernate, GS115(pEGl7 ) lysate, concentrated GS115 supernate, GS115 supernate, and GS115 lysate respectively. Scaffoldin is 98.5KDa in theory but 130-180KDa in practice because of glycosylation.

Some bands were not clear enough to be found by SDS-PAGE, so we chose Western-Blot to identify target proteins' expression.

Figure9. The western blot analysis of recombined GS115 fermentation broth. Control is GS115’s concentrated fermentation supernatant. (a)XynB, which is 43.4KDa. Line1, 2, 3 refer to sediment, supernatant and concentrated supernatant; (b)Scaffoldin, which is 98.5KDa. Line1, 2, 3 refer to sediment, supernatant and concentrated supernatant; (c)Laccase, which is 56.7KDa.

We were surprised by Scaffoldin's size shown in its WB result, which is about 130KDa to 180KDa and much bigger than the theoretical result: 98.5KDa. So we thought the Scaffoldin might be glycosylated but its influence on cellulosome assembly was unknown.

2. Conclusion

In conclusion, UESTC-China succesfully accomplished the basic design: recombination and expression. For enzymes, they all showed great activities except laccase. For scaffoldin, western blot helped us find its existence and enlarged protein size, which offered possible reasons to the cellolusome assembly in future as well.

Deinking Efficiency Validation

While the expression experiments were ptomoting smoothly, we began using business enzymes to detect the deinking efficiency. We divided a A4 paper into many cells, and every cell had 15 ink dots originally (see In-situ deinking experiment method below). Papers were soaked in enzyme solutions for some time, and then we used sticky cylinder rolling through the papers, which provided physical force to peel the ink off the papers. We can easily detect how much residual ink points were still on the paper after deinking treatment by counting the number of ink points per cell. After counting, we used T test checking the significant difference between different treatments. Our results are listed below:

1. Results

（1）Cellulase

Cellulase can cut cellulose fibers on which ink toners attach, so that the ink can be removed from the surface of the paper by the slight force provided by the sticky cylinder. We can see in Figure10. that residual ink significantly decreases（p＜0.01）when treated time is more than 20min and less than 40min.

Figure10. Deinking Efficiency of 1mg/ml business cellulase at different times. Inactive business cellulase (Cel-) or active cellulase (Cel+) .

Because the least residual ink is on 40min treatment, It was shown that residual ink distribution of papers which were treated with active and inactive cellulase respectively in Figure11. We can see in Figure11. that the mean of residual ink shift left which means the residual ink decreases.

Figure11. Residual ink distribution when treated with 1mg/ml cellulase in 40min. Inactive business cellulase (Cel-) or active cellulase(Cel-).

（2）Xylanase

Xylanase can cut hemi-cellulose fibers (including lignin) on which ink toners attach, so that the ink can be removed from the surface of the paper by the slight force provided by the sticky cylinder. We can see in Figure12. that residual ink significantly decreases（p＜0.01）when treated time is more than 1min and less than 40min.

Figure12. Deinking Efficiency of business 0.5mg/ml xylanase at different times. Inactive business xylanase (Xyn-) or active cellulase (Xyn+) .

Because the least residual ink is on 30min treatment, residual ink distribution of papers that were treated with active and inactive cellulase respectively was drawn in Figure13. We can see in Figure13. that the mean of residual ink shift left which means the residual ink decreases.

Figure13. Residual ink distribution when treated with 0.5mg/ml xylanase in 30min. Inactive business xylanase (Xyn-) or active xylanase (Xyn+).

We also detected the lignin content before and after treatment with xylanase, the results showed that lignin content dramatically decreased after treating with xylanase in 30min as expected (see in Figure14.).

Figure14. Ligin content after treated with 0.5mg/ml xylanase in 30min. Inactive business xylanase (Xyn-) or active xylanase (Xyn+).

（3）Laccase

Theoretically, laccase can cut lignin on which ink toners attach, so that the ink can be removed from the surface of the paper by the slight force provided by the sticky cylinder. However, according to our experiment data, residual ink with active laccase treatment is significantly higher than（p<0.01）with inactive laccase treatment no matter the mediator is ABTS or HBT (See in Figure15. and Figure16.).

Figure15. Residual ink distribution when treated with 1mg/ml laccase with 1mM ABTS. Inactive business laccase（Lac-) or active laccase (Lac+)

Figure16. Residual ink distribution when treated with 1mg/ml laccase with 10mM HBT. Inactive business laccase（Lac-) or active laccase (Lac+) .

Moreover，paper that is treated with laccase will turn yellow in a short time（see in Figure17.).

Figure17. Paper's color after treating with laccase for 40 min. (a) Buffer; (b) 1mg/ml laccase + 10mM HBT; (c) 1mg/ml laccase+1mM ABTS

（4）Lipase

Lipase can degrade ester content in ink toner, so that the ink can dissolve from the surface of the paper. We can see in Figure18. that residual ink significantly decreases（p<0.01）when treated time is more than 30min and less than 40min.

Figure18. Deinking Efficiency of business lipase at different times. Inactive business lipase (Lip-) or active cellulase (Lip+).

（5）Multi-enzymes

In addition to the single-enzyme experiment, we also conducted a multi-enzyme synergy experiment. In this experiment, we tested the proportion of synergistic enzymes based on the possibility of the cellosome we designed. The results showed that the proportion of enzymes with the least residual ink dots when the ratio of cellulase and xylanase is 2:1.

Figure19. Deinking efficiency of multiple business enzymes in 30min or 40min. Cel means we used 1mg/ml cellulase solution to treat the papers. Cel-Xyn means we used 1mg/ml cellulase and 0.5mg/ml xylanase solution to treat the papers. Cel-Xyn-Lac means we used 1mg/ml cellulase, 0.5mg/ml xylanase, 1mg/ml laccase solution to treat the papers. Cel-Xyn-Lac-Lip means we used 1mg/ml cellulase, 0.5mg/ml xylanase, 1mg/ml laccase and 1mg/ml lipase solution to treat the papers.

2. Discussion

In the deinking efficiency validation experiment, we conducted a single-enzyme experiment, and the results showed that the residual ink spots on paper were significantly reduced after the action of active cellulase, xylanase, and lipase. This part of the experiment shows that biological enzymes such as cellulase and xylanase have significant effects on deinking.

However, the laccase experiment showed the opposite result. The effect of inactivated laccase is more effective than that of active laccase, all the results of our repeated experiments show this situation. We guessed that it might be because we chose the thermal inactivation method to inactivate the laccase. Some substances in the heated water are oxidized, and the oxidized substances further oxidize ABTS and HBT, which promotes the generation of ABTS and HBT free radicals. Because the principle of the action of laccase is that the oxidized laccase transfers oxygen to ABTS, so that ABTS free radicals are generated, and ABTS free radicals act again. Not only that, the chromophore groups on paper treated with laccase are oxidized, making the paper yellow overnight, which will affect the use sence of the paper.

Although lipase has the effect of deinking, the results of enzyme synergistic experiments show that the synergistic effect of cellulase and xylanase is the best. Therefore, we believe that free laccase and lipase can be removed from our system, which makes our originally designed cellulosome system simple, leaving only the complex of Scaffoldin, cellulase and xylanase. Click here for more information in Engineering.

Cellulosome Assembly

After determining the best enzymes combination, the final edition of cellulosome was designed accordingly. Based on our experimental results, the ratio of divergent ccscohesion and ctcohesion on scaffoldin is set as 2:1, to obtained the best result of the co-working of cellulase and xylanase. When validate assembly, we mixed Scaffoldin and XynB that have been successfully expressed by Pichia pastoris and verified in a 1:1 ratio as reaction system, incubated them at 37 ℃ for 3 hours so they could combine. We also mixed Scaffoldin protein and extracellular protein secreted by Pichia pastoris in a 1:1 ratio as our control group. Then we used the protein gel immunoprecipitation kit, mixed the treated protein mixture with a gel containing Myc-antibody and incubated it overnight at 4 ℃. Finally, we performed Dot-Blot experiments after centrifugation, washing and purification of the targeted protein.

1. Results

First, we have successfully expressed Scaffoldin, the results are shown below:

Figure20. Scaffoldin expression. (a)Plasmid Linearization. (b)Colony PCR. (3)Dot Blot. (4)Western Blot.

We proved the feasibility of assembly theoretically. The schematic Figure21. is as follows.

Figure21. Theoretical feasibility of EGL7, EG1 and XynB connected to Scaffoldin

The experiment of cellulosome assembly is still undergoing, and our preliminary results are as follows.

In theory, if Scaffoldin could combine with XynB by coh-doc domain, which have been fused with Scaffoldin and XynB respectively, the gel containing Myc-antibody would drag Scaffoldin and Scaffoldin-Cohesion-Dockerin-XynB down together. If we use Dot-Blot to test the existence of protein with 6×his tag, Scaffoldin-Cohesion-Dockerin-XynB could be detected uniquely. Of course, nothing would be detected in control.

Figure22. Schematics of defining docking between Scaffoldin and XynB by immunoprecipitation.

Figure23. The Dot-blot results of Scaffoldin and XynB docking. Positive control is the Scaffoldin-XynB docking product after 3h incubation at 37℃; Negative control is the (GS115 supernatant protein)-XynB docking and post-purifying product; Experiment is the Scaffoldin-XynB docking and post-purifying product. Every sample is dotted on two blots and gives 3 repeats. Line 1 is the Dot-blot result of sample disposed by anti-Myc-tag antibody, while Line 2 is disposed by anti-6×His-tag antibody.

Unfortunately, we didn't acquire the expected results of the experiment. In theory, positive control would be detected by both anti-6×His-tag antibody and anti-Myc-tag antibody and negative control would result nothing. At the same time, the experimental group would also be detected by both anti-6×His-tag and anti-Myc-tag antibody if Scaffoldin and XynB could dock, while nothing would appear if Scaffoldin and XynB couldn't dock. However, a weird result came out. The dots of the experiment sample could only be detected by anti-Myc-tag antibody, which means only XynB was left after immunoprecipitation.

2. Discussion

Due to limited time, Immunoprecipitation is hard to do perfectly even though we have tried our best. However, considering that coh-doc domain has been used for years, we are confident of its function. After several attempts, we finally ruled out some possibilities and focused on three factors:

(1) Low expression quantity

Low expression quantity is the main factor currently. In the early development phases of Pichia pastoris, it is common for the intracellular protein production of secreted proteins to be less than 1% of total protein, and protease degradation may begin before most products can be visualized. Therefore, after purification or ammonium sulfate precipitation concentration, there are not enough proteins to verify the assembly of cellulosome through our experimental methods.

(2) Our method

There might be something we are still unknown that makes XynB nonspecifically attach to gel in Immunoprecipitation Kit, so free XynB can't be washed out and finally precipitated, which could explain why XynB appears on the control groups and the experimental groups. And the anti-Myc-tag antibody attached to gel might have lost its function after a period of time so Scaffoldin was not dragged by it successfully. In addition, after more than 10 hours incubation, Scaffoldin and XynB's structure might have changed. All possibilities above need a new method to define the docking of Scaffoldin and XynB.

(3) Glycosylation

We think that this failure may be related to the high glycosylation of the expressed proteins by Pichia pastoris. Our above experimental results(Western-Blot of Scaffoldin) also confirm our conjecture to a certain extent. Glycosylation is the reaction in which a carbohydrate is attached to a hydroxyl or other functional group of another molecule in order to form glycoconjugate. According to relevant literature, glycosylation caused by heteroexpression of proteins in Pichia pastoris will have impact on the structure and function of proteins.

3. Future plans

In the future, we will explore the severity of glycation in order to minimize the effects of it. Based on this situation, we will continue to investigate the assembly of Scaffoldin and enzymes to demonstrate if cellulosome could improve deinking efficiency. Although it is still on the way, we also make a design for it. We can use Co-immunoprecipitation to verify the interaction between scaffoldin proteins and enzymes.

Co-Immunoprecipitation (Co-Immunoprecipitation) is a classical method used to study protein interaction based on the specific interaction between antibody and antigen. It is an effective method to determine the rational interaction between two proteins in intact cells.

The idea is that when cells are cleaved under non-denatured conditions, many of the protein-protein interactions present in intact cells are retained. If X is immunoprecipitated with antibodies to protein X, the protein Y bound to X in vivo can also precipitate.

If this experiment is successful, we can not only avoid human influence to verify the interaction between scaffoldin and enzymes but also acquire interaction protein complexes in their natural state.

We can also use immunoprecipitation magnetic beads to verify the interaction between scaffoldin proteins and enzymes. The reproducibility and purity of magnetic beads are generally higher than that of agarose and magnetic beads generally do not require pre-purification steps.

Prospection

1. Improvement of expression level

According to our results, the concentration of target proteins in fermentation supernatant is not as high as we expected. To concentrate these target proteins, we need to use a huge amount of ammonium sulfate or adopt other expensive and tedious methods, which is impossible to apply on a large-scale. So it is essential to improve the expression level of our target proteins. In the future, we will try to improve strain by increasing promoter strength and gene copy number, to ensure a sufficient level of transcription of the heterologous gene.

2. Adjustment of enzymes' activity

Obviously, low enzyme activity makes Deinker unable to deink the waste paper. However, "higher is better" is also incorrect in our project. In fact, the quality of deinked paper might decrease. Imagine that a kind of cellulase with high activity degrades much more fiber than those combining ink particles, resulting in thinner deinked product and higher possibility of paper tearing. So adjusting enzyme activity to a suitable level plays an important role in our future plan, enabling Deinker to behave both stably and effectively.
In the future, we decide to adjust the activity of cellulase and xylanase by introducing mutations at their active sites or change to other enzymes with the same functions.

3. Exploring better enzymes' proportion

By exploiting the modular nature of each population(of different recombinant GS115s) to provide a unique building block for our cellulosome structure, the overall cellulosome assembly, cellulose hydrolysis, and deinking effect were easily fine-tuned by adjusting the proportion of different populations in the consortium, instead of using commercial enzymes to simulate.

There is an overall design of experiments:

Figure24. An overall design of experiments.

4. Other enzymes

Although we have tried our best to choose the most suitable enzymes for our project, there must be better choices waiting to be found.
Considering that we can appropriately expand the application scope of Deinker, from point to surface, we can really solve the problem of paper waste. For the reason that the compositions of different types of paper may be different as well, we need to find new enzymes to degrade other components of these papers, to assist cellulase and xylanase. We will continue to look for other enzymes and explore the role of these enzymes in paper deinking. We will carry out a series of experiments to investigate the synergistifrc effects of these enzymes with cellulase and xylanase and evaluate their deinking effects.

Methods

1. Gene, plasmid, reagent

The accession of Cellulase AF-EGL7 gene is 3505017 (AFUA_6G01800). The GenBank accession number of Endoglucanase I (EG1) in SSL of Trichoderma sessoniana is EU935217.

GenBank accession number of Xylanase is: JX560731.1, and He Gao et al. optimized the gene sequence twice. GenBank Accession Number of Lipase is DQ647700.1; the GenBank accession number of Laccase is JN559771.1. We used the plasmid extraction kit and gel recovery kit (Axygen). PGAPZ αA plasmid (Feng Hui, China); Escherichia coli Trelief 5α (Tsingke, China); Zeocin Selection Reagent (Solarbio and InvivoGen); Restriction endonuclease (Thermo Scientific); Ligase (NEB); Western Blot Kit (TransGen Biotech, China); His-tag, MYC-TAG, Flag-Tag primary antibody and fluorescent labeled secondary antibody (CST); Lignin content detection Kit (Solarbio); Commercial enzymes (cellulase, xylanase and laccase are from ShanghaiyuanyeBio; lipase is from Macklin); other chemical reagents.

2. Equipments

Centrifuge(Eppendorf 5424), shaker(Shanghai ZhiCheng ZWY-100H and ZWYR-2102C), electrophoresis apparatus(Beijing Junyi JY300E, JY-SPFT, JY-SCZ2+), purification apparatus(AKTA), electrogyration apparatus(Bio-Rad), etc.

3. Construction of recombinant plasimds

We design the plasmids and send them to the company for synthesis. The cellulase plasmid expresses cellulase with Type1 dockerin (derived from clostridium fibrogiformis) that can be secreted out of the cell. The cellulase and dockerin are connected by flexible linker (GGGGS)3. Type1 Dockerin (derived from Clostridium fastigium) could combine with Type1 cohesin (derived from Clostridium fastigium) on the scaffoldin to anchor cellulase on the scaffoldin. And there is a tag, and the short peptide expressed by the tag can bind with the first antibody and the second antibody with fluorescent dye, so as to verify the binding of cellulase to the scaffoldin in the future.

Xylanase plasmids are very similar to cellulase plasmids, except that the C-terminal of xylanase is linked to Ctdockerin (from Clostridium thermophilus) instead of Ccsdockerin (from Clostridium fibrogivum).

The scaffoldin plasmid expresses the scaffoldin of the cellulosome, and the expressed scaffoldin contains: (1)Two Type1 Cohesin (Ccscohesin came from iGEM Part) that could interact with cellulase corresponding Type1 Dockerin. (2) A Type1 cohesin that could interact with the corresponding dockerin xylanase (CtCohesin/CipCoh4). (3) One CBM domain, which has a high affinity with paper cellulose, can improve the interaction efficiency of cellulosome system and paper as a whole. (4) A Type2 Dockerin could bond with Type2 cohesin (located on small scaffoldin responsible for cell anchoring), so that the whole scaffoldin was anchored on the cell surface. (5) A Myc-tag, and the scaffoldin expressed with the myc peptide. The assembly between the skeleton and the cell, and between the skeleton and the enzyme, can be observed by immunofluorescence through the primary antibody and the secondary antibody with fluorescent dye. The components are connected by rigid Linker (Pro/ THR-rich).

Anchor plasmids expressed anchored small scaffolds, which had three SLH cell wall anchor structural domains and a type2 cohesin over them, which could bond with type2 Dockerin on the cellulosome scaffoldin, thus anchoring the scaffoldin on the cell surface. Hungateiclostridium thermocellum naturally expressed the entire anchor scaffoldin (SdbA), whose GenBank accession number was U49980.1. An immunofluorescence tag, Flag-Tag(n-DykDDDDK-C), should also be added to the C-terminal of SdbA gene, consisting of 8 amino acid residues.

The gene sequence from 5' to 3 ' of Laccase and lipase plasmid is α-factor---SMT3（SUMO）---GS linker（GGGGS）2----ANL---Myctag ----E2A----α-factor---Lac---Histag. Among them, α-factor-- SMT3-- GS Linker (GGGGS)2 ----ANL belongs to the lipase part. SMT3 is the SUMO fusion sequence in yeast, which can help improve the expression of lipase. The connection between SMT3 and lipase is (GGGGS) 2Linker. Lac belongs to the laccase component, which is linked to the lipase component by a 2A peptide. Myc-tag and Histag can facilitate subsequent purification and determination of enzyme expression.

4. Amplification and linearization of recombinant plasmids

In order to obtain a large number of plasmid copies, the plasmids were first transformed into E. coli Trelief 5α and cultured in low-salt LB medium at 37 ℃ and 180 rpm for 18h. Then extracted the plasmids. The plasimds then linearized with restriction endonuclease for subsequent electroshock conversion.

5. Construction and expression of pichia pastoris engineering strain

The linearized plasmids were precipitated by ethanol, and then underwent electric shock transformation with pichia pastoris which had prepared the receptive state. After incubation at 30℃ for 1 h, MD and MDH plates were coated for screening, and cultured at 30℃ for 36-48 h, single colonies were selected in 96-well plates. Each well was screened with 180 μl YPD medium and 20 μl Zeocin, and cultured at 30℃ for 48h, and the 96-well plate screening was repeated twice. The positive clones were screened and fermented in YPD medium at 30℃ and 250rpm for 3-4 days fermentation.

6. Definition and determination of enzyme activity

Xylanase activity definition: the amount of enzyme required for conversion of xylan to 1 μmol xylose at a given pH and temperature is one unit of enzyme activity. The enzyme activity was determined by DNS method based on the amount of reducing sugar released after the enzyme reaction.

DNS method was also used for cellulase, and its enzyme activity was defined as: under certain pH and temperature conditions, the amount of enzyme required to transform 1 μmol product was an enzyme activity unit.

The enzyme activity unit of laccase is defined as: the enzyme activity unit of a laccase is the change of absorbance value of 1μmol ABTS transformed within 1min.

Lipase activity was measured by alkali titration. Unit lipase activity was defined as the enzyme amount of 1μmol fatty acid per minute under certain reaction conditions.

7. Fermentation enzyme production experiment

Positive colonies were tested for small-scale expression of recombinant xylanase in 30mL of YPD medium. The positive clones with the highest-level expression were used for large-scale expression and further studies.

The highest-expressing xylanase was grown in 400 mL of YPD medium with constant shaking (250 rpm) at 30◦C for 72 h. Samples were taken every 24 hours for microscopic examination of biomass. The protein expression level of the samples was observed by sdS-PAGE protein glue or Dot Blot analysis, and the protein content of the samples was determined by enzyme-plate analyzer, and the enzyme activity of the samples was determined.

8. In-situ deinking experiment

Due to time limits, we first used commercial cellulase, xylanase, laccase and lipase to conduct in-situ deinking experiments, first explored the method of in-situ deinking, and promoted the hardware design of the project.

In order to explore the effect of enzymes on paper deinking, a piece of A4 paper was divided into small cells of the same size, each containing the same number of points. Control experiments were designed. The bevel device is used to process the paper after soaking, and the adhesive cylinder falls free under the flat paper to take the ink. It is known that when the cylinder rolls around, it just completely covers 6×7 small cells, so 42 small cells can be obtained by each operation of a piece of paper, and the number of residual ink spots in it is counted. The advantage of designing experiments in this way is that the results of deinking can be measured quantitatively and the data statistics and processing are convenient.

9. Protein interaction handbook:

Two plans: If we successfully verify protein interaction, we will directly show our successful results.

If we don't, we will show our results for failure and the handbook.

In the future, we can use Co-immunoprecipitation to verify the interaction between scaffoldin proteins and enzymes.

Co-Immunoprecipitation (Co-Immunoprecipitation) is a classical method used to study protein interaction based on the specific interaction between antibody and antigen. It is an effective method to determine the rational interaction between two proteins in intact cells.

The idea is that when cells are cleaved under non-denatured conditions, many of the protein-protein interactions present in intact cells are retained. If X is immunoprecipitated with antibodies to protein X, the protein Y bound to X in vivo can also precipitate. If this experiment is successful, we can not only avoid human influence to verify the interaction between scaffoldin and enzymes but also acquire interaction protein complexes in their natural state.

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