1 Introduction

In our project, the wet lab experiments played a central role. Specifically, we conducted numerous experiments to develop the BIND-PETase module by creating various PETase variants and evaluating their functionality. We aimed to improve PETase using an in silico evolution pipeline. As a result, we identified a new enzyme called BIND-bearPETase, which demonstrated exceptional performance in PET degradation.

Additionally, we focused on the development of the ElectroCount Biosafety (ECB) module, allowing us to explore the potential for safer and more efficient applications in PET degradation.

2 BIP Overview

We have found BIND-bearPETase (BBa_K5436124) from the various BIND-PETases listed in Table. 9.2.1 by performing multiple assays in the flow shown in Fig. 9.2.1. A summary of the results is shown in Table. 9.2.1.
For the various mutant BIND-PETases designed at the beginning, Curli Fiber formation by CsgA was evaluated by performing the Congo Red Assay and the activity of the PETase was evaluated by performing the pNPB Assay.
Those that showed Curli Fiber formation and PETase activity were further subjected to Storage Activity Assay and Reusability Assay. Those that showed excellent storage activity and reusability in these assays were further validated for PET degradation activity in the Pet Pellet Degradation Assay and PET Powder Degradation Assay. Through these validations, we determined that “BIND-bearPETase (BBa_K5436124 ),” which we submitted as New Composite Parts, has excellent activity as a PETase, post-storage activity, and reusability.
The results of each assay are shown in Congo Red Assay~ PET powder Degradation Assay, and the flowchart to determine “BIND-bearPETase(BBa_K5436124)” from each mutant is shown in Discussion.

Experiment flow. png

Fig. 9.2.1. Experiment flow

Table. 9.2.1. BIND-PETase variants and an overview of their characteristics and results

BIND-PETase variant	description	Curli Fiber Formation(Congo Red Assay)	PETase activity(pNPB Assay)	Activity while Stored(Storage Activity Assay)	Activity while Reused(Reusability Assay)	PET Degradation Activity
WT	Wild type sequence from I. Sakaiensis	✓	✓	4℃：increase RT：decrease	a little decrease
E. coli optimized	WT codon optimized for E. coli	✓	✓	4℃：no significant change RT：decrease	a little decrease
5 mut.	5 amino acid substitution sequence of E. coli optimized	✓ (weak)	×	-	-
dura	10 mutants of WT, with superior activity and heat tolerance	✓	✓	4℃：increase RT：increase	increase	low
ID1	ESM_PETase	×	×	-	-
ID2	ESM_PETase_affinity_screenig	×	×	-	-
ID3	ESM_PETaseaffinity_REU_screening	✓ (weak)	✓	-	-
ID4	DuraPETase_EvoProGrad_EMS2_evocouplings_affinity_screening	×	×	-	-
ID5	DepoPETase_EvoProGrad_EMS2_evocouplings_affinity_screening	×	×	-	-
ID6	LCC_mut_IsPETase_M1M3	×	×	-	-
ID7	LCC_mut_IsPETase_M3	No validation due to failed plasmid construction
ID8	LCC_mut_IsPETase_M1M2M3	No validation due to failed plasmid construction
ID22	DepoPETase-M4_DuraPETase-M10	✓	✓	4℃：increase then decrease RT：increase then decrease	decrease
ID23	DepoPETase-M2-3_DuraPETase-M10	✓	✓	4℃：increase RT：increase	increase	high
ID24	DepoPETase-M2_DuraPETase-M10	✓	✓	4℃：increase RT：increase	increase	high
ID25	DepoPETase-M3_DuraPETase-M10	✓	✓	No verification due to time constraints
ID26	DepoPETase-M2-2_DuraPETase-M10	✓	✓	No verification due to time constraints
ID27	DepoPETase-M2-1_DuraPETase-M10	✓	✓	No verification due to time constraints

3 Confirmation of expression of BIND-PETase(WT), BIND-bearPETase

For BIND-PETase (WT), we performed SDS-PAGE and Western Blotting to evaluate whether our RBS was working properly. We also performed Western Blotting for BIND-bearPAGE. We also confirmed the expression of BIND-bearPETase in the New Composite Parts registry. For other mutants of BIND-PETase, we substituted the expression verification of BIND-PETase with the functional verification of BIND-PETase.

3.1 Confirmation of expression of BIND-PETase(WT)

BIND-PETase (WT) was introduced into E. coli BL21 (DE3) and we tried to confirm the IPTG-induced expression by SDS-PAGE. E. coli were crushed and SDS-PAGE was performed (refer to Experiments). The results are shown in Fig. 9.3.1A. A very large number of bands were identified with and without IPTG; there was no clear difference in the bands of BIND-PETase(WT) with a molecular weight of approximately 45 kDa; the BIND-PETase(WT) band of E. coli BL21(DE3). It is possible that the BIND-PETase(WT) band overlapped with the endogenous protein, making it difficult to detect. Therefore, we conclude that it is difficult to confirm the expression of BIND-PETase by SDS-PAGE.
SDS-PAGE results for BIND-PETase (WT, *E. coli* optimized, 5 mut.). png

Fig. 9.3.1A. SDS-PAGE results for BIND-PETase (WT, E. coli optimized, 5 mut.)

Two colonies each of BIND-PETase (WT, E. coli optimized, 5mut) were cultured with and without IPTG induction for each colony. The E. coli were crushed, proteins were extracted, and the proteins were phased. The samples were treated with HFIP treatment, which depolymerizes CsgA. BIND-PETase (E. coli optimized, 5mut), which was being validated at the same time, was also treated in the same way and then phased.

Therefore, Western Blotting, which can specifically detect proteins, was performed(refer to Experiments). The reason why no difference was detected in the bands corresponding to IPTG+/- BIND-PETase by SDS-PAGE was considered to be a problem with HFIP treatment in sample preparation prior to SDS-PAGE, and sonication treatment, which is more commonly used as a cell disruption method, was also performed. The results of Western Blotting are shown in Fig. 9.3.1B. Western Blotting was not performed with BIND-PETase (E. coli optimized, 5mut).

Western Blotting results for BIND-PETase(WT).png

Fig. 9.3.1B. Western Blotting results for BIND-PETase(WT)

Three colonies of BIND-PETase (WT) were cultured with and without IPTG induction for each colony. E. coli were disrupted by HFIP or sonication, proteins were extracted, and the proteins were phased. E. coli BL21(DE3) were disrupted by sonication, proteins were extracted, and phased. his-tag antibody was used to detect BIND-PETase(WT).

From Fig. 9.3.1B, a band was detected around 45 kDa in the IPTG-induced sample, confirming the expression of BIND-PETase. the reason why it was difficult to confirm the expression of BIND-PETase (WT) by SDS-PAGE is that its expression level is low and it is likely to be hidden by the endogenous protein of E. coli BL21(DE3), which was likely masked by an endogenous protein in E. coli BL21(DE3). This low expression of BIND-PETase may be due to the fact that BIND-PETase is a protein that is displayed on the membrane^[1].

3.2 Confirmation of expression of BIND-bearPETase

Western blotting was also performed for BIND-bearPETase, which has a his-tag at the end of the PETase, so it was detected with a his-tag antibody in the same way as BIND-PETase (WT). The results are shown in Fig. 9.3.2.

Western Blotting results for BIND-bearPETase.png

Fig. 9.3.2. Western Blotting results for BIND-bearPETase

Three colonies of BIND-bearPETase were cultured and IPTG induced. E. coli BL21(DE3) were disrupted by sonication, proteins were extracted, and phased. his-tag antibody was used to detect BIND-bearPETase.

4 Congo Red Assay

BIND-PETase requires the formation of Curli Fiber by CsgA, that PETase is displayed on the membrane surface. We evaluated this by using Congo Red, which stains Curli Fiber(refer to Experiments). If the pellet formed after the addition of Congo Red is red and the supernatant is light in color, it can be confirmed that Curli Fiber has formed normally. The results of absorbance measurement are shown in Fig. 9.4.1, and detailed results for each mutant are shown in Table. 9.4.1 of the 16 BIND-PETase variants tested in the experiment. The formation of Curli Fiber was confirmed in 12 of them. In particular, a lot of Curli Fiber was formed in BIND-PETase (WT, ID22, 25). The difference in the ability of each mutant to form Curli Fiber may be due to the different expression levels of CsgA in each mutant and the different structure of PETase, which may affect the tendency of CsgA polymerization.

Intensity of Curli Fiber Formation For Each Variant

Fig. 9.4.1. Intensity of Curli Fiber Formation in each mutant

The horizontal axis represents the mutant type, and the vertical axis represents the level of Curli Fiber formation. IPTG(+/-) samples stained with Congo Red were pelletized and the absorbance (wavelength 490 nm) indicated by Congo Red in the supernatant was measured. Curli Fiber formation was evaluated by comparing the difference in absorbance between IPTG(-) and IPTG(+) samples. The absorbance was corrected by the value of OD 600. The higher the value, the greater the amount of Congo Red bound to the Curli Fiber, indicating the formation of more Curli Fiber.

Table. 9.4.1. Result and analysis of the formation of Curli Fiber in each mutant

Sample name	formation of Curli Fiber	Figure (left: IPTG+, right:IPTG-)	Analysis
WT	✓	[click this link]	The IPTG(+) pellet was stained red, and the result of the supernatant indicates that Curli Fiber has formed.
E. coli optimized	✓	[click this link]	The IPTG(+) pellet was stained red, and the result of the supernatant indicates that Curli Fiber has formed.
5 mut.	✓ (weak)	[click this link]	The IPTG(+) pellet was a little stained red, and the result of the supernatant indicates that a little Curli Fiber has formed.
dura	✓(*)	[click this link]	*The supernatant of the IPTG+ pellet is clear and colorless. This indicates the formation of a very large number of Curli Fiber. On the other hand, it is likely that the IPTG- sample also formed Curli Fiber for some reasons. Therefore, although the absorbance value of the supernatant is low, it is likely that more Curli Fiber has formed.
ID 1	×	[click this link]	Both IPTG(+) and IPTG(-) sample pellets were not stained, and the absorbance results show that little or no number of Curli Fiber was formed.
ID 2	×	[click this link]	Both IPTG(+) and IPTG(-) sample pellets were not stained, and the absorbance results show that little or no number of Curli Fiber was formed.
ID 3	✓ (weak)	[click this link]	IPTG(+) pellets are slightly more stained than IPTG(-) pellets. The absorbance results indicate that a small amount of Curli Fiber was formed.
ID 4	×	[click this link]	Both IPTG(+) and IPTG(-) sample pellets were not stained, and the absorbance results show that little or no number of Curli Fiber was formed.
ID 5	×	[click this link]	Both IPTG(+) and IPTG(-) sample pellets were not stained, and the absorbance results show that little or no number of Curli Fiber was formed.
ID 6	×	[click this link]	Both IPTG(+) and IPTG(-) sample pellets were not stained, and the absorbance results show that little or no number of Curli Fiber was formed.
ID 22	✓	[click this link]	IPTG(+) pellets were stained dark red and the supernatant was nearly colorless. The absorbance results also indicate that a lot of Curli Fiber was formed.
ID 23	✓	[click this link]	IPTG(+) pellets were stained dark red and the supernatant was nearly colorless. The absorbance results also indicate that a lot of Curli Fiber was formed.
ID 24	✓	[click this link]	IPTG(+) pellets were stained dark red and the supernatant was nearly colorless. The absorbance results also indicate that a lot of Curli Fiber was formed.
ID 25	✓	[click this link]	IPTG(+) pellets were stained dark red and the supernatant was nearly colorless. The absorbance results also indicate that a lot of Curli Fiber was formed.
ID 26	✓	[click this link]	IPTG(+) pellets were stained dark red and the supernatant was nearly colorless. The absorbance results also indicate that a lot of Curli Fiber was formed.
ID 27	✓	[click this link]	IPTG(+) pellets were stained dark red and the supernatant was nearly colorless. The absorbance results also indicate that a lot of Curli Fiber was formed.

5 pNPB Assay

In the pNPB assay, the activity of PETase was evaluated by observing the degradation activity of BIND-PETase towards pNPB. When pNPB is hydrolyzed by PETase, butyric acid and the fluorescent yellow pNP are produced. Since pNP exhibits an absorption peak at 405 nm, this was measured(refer to Experiments). The results of the absorbance measurements are shown in Fig. 9.5.1, and detailed results for each variant are provided in Table. 9.4.1.
Among the 16 types of BIND-PETase tested in the experiment, PETase activity was confirmed in 11 of them. Particularly high activity was observed in BIND-PETase variants (ID23~27). DuraPETase, which has been shown in previous studies to have higher activity than PETase (WT), also demonstrated similarly high activity in the form of BIND-duraPETase. Moreover, the PETase variants we designed (ID23~27) exhibited higher activity than duraPETase in the pNPB assay, indicating that the activity of PETase was improved. Among them, BIND-PETase (ID23, 27) exhibited more than double the activity of BIND-PETase (WT).
On the other hand, the variants that did not show activity either have inherently low PETase activity, or the fusion with CsgA may have structurally hindered the binding of the substrate to the PETase enzyme.

Fig. 9.5.1. Degradation Activity of pNPB for Each Variant

The horizontal axis represents the types of variants, and the vertical axis represents pNPB degradation activity. The IPTG (+/-) samples were reacted with pNPB, and after the pNPB assay, the absorbance at 405 nm (indicative of pNP production) was measured, and the difference between IPTG (+) and IPTG (-) was calculated. The absorbance values were corrected with OD 600, and higher values indicate higher PETase activity towards pNPB.

Table. 9.5.1. The results and analysis of Reusability assay in each mutant

Variant	PETase Activity	Image (Left: IPTG+, Right: IPTG-)	Discussion
WT	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results also confirmed hydrolytic activity.
E. coli optimized	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results confirmed hydrolytic activity, though lower than WT.
5mut.	×	[click this link]	Both IPTG (+/-) samples remained clear, indicating no pNP production. Absorbance results confirmed the absence of hydrolytic activity.
dura	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results confirmed hydrolytic activity, which was higher than WT.
ID1	×	[click this link]	Both IPTG (+/-) samples remained clear, indicating no pNP production. Absorbance results confirmed the absence of hydrolytic activity.
ID2	×	[click this link]	The IPTG(+) sample appeared slightly yellow, but since IPTG(-) also showed similar coloration, it is unlikely that the hydrolysis of pNP by BIND-PETase (ID2) occurred.
ID3	✓ (weak)	[click this link]	Both IPTG (+/-) samples remained clear with no significant difference, but a small amount of pNP was detected by absorbance. ID3 has weak activity.
ID4	×	[click this link]	Both IPTG (+/-) samples remained clear, indicating no pNP production. Absorbance results confirmed the absence of hydrolytic activity.
ID5	×	[click this link]	Both IPTG (+/-) samples remained clear, indicating no pNP production. Absorbance results confirmed the absence of hydrolytic activity.
ID6	✓	[click this link]	Both IPTG (+/-) samples remained clear, but absorbance confirmed a small amount of pNP production. ID6 has weak activity, lower than ID3.
ID22	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results confirmed hydrolytic activity, which was lower than WT and similar to the E. coli optimized version.
ID23	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results confirmed hydrolytic activity, which was the second highest among the tested variants.
ID24	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results confirmed hydrolytic activity, which was the third highest among the tested variants.
ID25	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results confirmed hydrolytic activity, which was higher than WT and duraPETase.
ID26	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results confirmed hydrolytic activity, which was higher than WT and duraPETase.
ID27	✓	[click this link]	IPTG(+) samples turned yellow due to pNP production. Absorbance results confirmed hydrolytic activity, which was the highest among the tested variants.

Two additional validations were conducted on BIND-PETase (WT, E. coli optimized, ID 22~24), which had demonstrated sufficient basic functionality as BIND-PETase through the Congo Red Assay and the pNPB Assay. These validations were the storage activity assay, which investigated the activity after storage, and the Reusability Assay, which examined the reusability of BIND-PETase.
BIND-PETase (ID3) was excluded due to the low activity of PETase. Additionally, while BIND-PETase (ID25~27) had also demonstrated sufficient basic functionality, further validation could not be performed due to time constraints.
On the other hand, in cases where neither Curli fiber formation nor PETase activity was confirmed in BIND-PETase, it is possible that either the expression of CsgA and PETase did not occur, or even if expressed, the fusion of CsgA and PETase structurally hindered polymerization or activity of one another.

6 Storage Activity Assay

In this assay, the activity of BIND-PETase was evaluated on the 5th and 11th days after storage at either 4°C or room temperature (refer to Experiments). The activity fluctuates depending on the storage temperature and type of PETase, and detailed results are shown in Table. 9.6.1.
The highest increase in activity during the storage period was observed in BIND-PETase (ID23) at 4°C and in BIND-PETase (ID24) at room temperature. Compared to BIND-PETase (WT, E. coli optimized), BIND-duraPETase and its designed variants (ID23, 24) exhibited a greater increase in activity over time.
Conversely, BIND-PETase (WT, E. coli optimized) showed little to no increase in activity, or even a decrease, indicating lower stability. These results suggest that BIND-duraPETase improved upon BIND-PETase (WT, E. coli optimized) in terms of storage stability. Furthermore, BIND-PETase (ID23, 24) may have further improved upon BIND-duraPETase.
The changes in activity after storage could be attributed to factors such as a decrease in stability, denaturation, or degradation of BIND-PETase. Alternatively, the increase in activity may be due to the continued expression or folding of BIND-PETase over time. The overall post-storage activity likely reflects a combination of these factors.

Fig. 9.6.1. Storage Activity in the condition of room temperature

Fig. 9.6.2. Storage Activity in the condition of 4°C

Table. 9.6.1. Results and Discussion of the Storage Stability Assay for Each BIND-PETase Variant

Variant	Activity	Discussion
WT	4°C: Increase Room temp: Decrease [click this link]	At 4°C, activity increased to 120% on day 5 and 136% on day 11. At room temperature, it decreased to 81% on day 5 and 55% on day 11, indicating instability at room temperature.
E. coli optimized	4°C: No significant change Room temp: Decrease [click this link]	At 4°C, no significant changes were observed, indicating relative stability.
dura	4°C: Increase Room temp: Increase [click this link]	At 4°C, activity increased to 183% on day 5 and 237% on day 11. At room temperature, activity increased to 224% on day 5 and 327% on day 11, suggesting stable expression and folding under both conditions.
ID 22	4°C: Increase, then decrease Room temp: Increase, then decrease [click this link]	At 4°C, activity increased to 177% on day 5 but dropped to 63% on day 11. At room temperature, it increased to 172% on day 5 but decreased to 108% by day 11.
ID 23	4°C: Increase Room temp: Increase [click this link]	At 4°C, activity increased to 294% on day 5 and 310% on day 11. At room temperature, it increased to 126% on day 5 and 394% on day 11.
ID 24	4°C: Increase Room temp: Increase [click this link]	At 4°C, activity increased to 198% on day 5 and 313% on day 11. At room temperature, it increased to 379% on day 5 and maintained at 391% by day 11.

7 Reusability Assay

In this Assay, the activity of BIND-PETase was evaluated by the pNPB assay when it was used 3 times in a row (cycles 1~3) (refer to Experiments). The increase or decrease in activity depended on the type of mutant, and the details of each mutant are shown in Table. 9.7.1.

Activity of BIND-PETase (WT, E. coli optimized, ID22) showed a slight decrease while reuse. On the other hand, activity of BIND-duraPETase and BIND-PETase (ID23 and ID24) showed an increase while reuse. This suggests that BIND-duraPETase and BIND-PETase (ID23, 24) are superior to BIND-PETase (WT, E. coli optimized) in terms of reusability of BIND-PETase.

The following factors may contribute to the change in activity while reuse. The decrease in activity may be due to the destabilization of the structure because of usage in the reaction.

Also, the structural change because of the environment after the reaction or collection by centrifugation may be the factors either. On the other hand, the increase in activity may be due to the reaction of BIND-PETase with pNPB, or the promotion of folding of BIND-PETase.
In addition, as a problem of experimental manipulation, it is possible that pNP, a degradation product of pNPB, was mixed into the cycle. However, the effect on the cycle should be small because it should be diluted enough when reused. However, there were some results that suggested that pNP were not removed enough from the samples of some mutants. For more accurate measurement, it is important to increase the times of washing BIND-PETase pellets and remove pNP as much as possible.

Fig. 9.7.1 Reusability of BIND-PETase variants

Table. 9.7.1. The results and analysis of Reusability assay in each mutant

Variant	Activity of BIND-PETase while reuse	Discussion
WT	a little decrease [click this link]	Activity was 83% in Cycle 2 and 88% in Cycle 3. Activity decreased slightly while reusing, but not significantly decreased. This BIND-PETase is considered to be relatively stable and reusable.
E. coli optimized	a little decrease [click this link]	Activity was 70% in Cycle 2 and 86% in Cycle 3. Activity decreased slightly while reusing, but not significantly decreased. This BIND-PETase is considered to be relatively stable and reusable.
dura	increase [click this link]	The activity was 91% in cycle2 and 274% in cycle3. The variation between samples in cycle1 and cycle2 was small and this suggests that activity of BIND-PETase didn’t decrease by reusing. On the other hand, the activity increased significantly between cycle2 and cycle3, and the variation among the samples was wide. So, it is possible that the activity of BIND-PETase in cycle3 was estimated to be higher due to the contamination of pNP generated in the previous cycles, but at least the activity was not lost.
ID22	decrease [click this link]	Activity decreased to 59% in cycle 2 and 52% in cycle 3. It is thought that the activity decreases with reuse. The more it is used, the lower the activity.
ID23	increase [click this link]	The activity was 193% in cycle2 and 144% in cycle 3. Although there is a possibility of pNP contamination because there is large variation between sample in cycle2 and sample in cycle3.
ID24	increase [click this link]	The activity was 168% in cycle2 and 128% in cycle3. As with ID23, there is a possibility of pNP contamination because there is large variation among samples. However, an increase in activity was observed.

8 Pet Pellet Degradation Assay

Using the PET single pellets and the composite pellets containing PET, polypropylene (PP), and polyethylene (PE) obtained from esa inc, interviewed by iGEM-Waseda as part of the iHP(refer to iHP). The degradation activity of BIND-PETase on these was evaluated by measuring the pellet. The objective of this experiment was to determine the qualitative degradation activity of BIND-PETase on the pellets. Since the purpose of this experiment was to qualitatively confirm the degradation to the pellets, only BIND-PETase (ID 23, 24), which was highly active in the pNPB Assay, was used. In this assay, reactions were performed for 5 days under two different conditions: pH 7 or pH 9 (refer to Experiments). The percentage weight loss of the pellets on day 5 of the reaction is shown in Fig. 9.8.2 and Fig. 9.8.3, and the qualitative trends regarding the degradation activity are summarized in Table 9.8.1 and Table 9.8.2.

Fig. 9.8.1. PET Pellets
(1. PET single pellets, 2. PETPEPP composite pellets)

Fig. 9.8.2. Mass Reduction Rate of PET Single Pellets

Table. 9.8.1. Degradation Activity for PET Single Pellets

Variant	Weight change of pellets (pH 7)	Weight change of pellets (pH 9)	Discussion
ID23	decrease	decrease	The weight of the pellets decreased at both pH 7 and pH 9, indicating that the PET in the pellets was degraded. The rate of decrease at pH 9 was particularly large.
ID24	increase	decrease	Although the pellet weight increased at pH 7, it is unlikely that the pellet weight increased during the reaction, suggesting that the BIND-PETase solution was not sufficiently wiped off from the pellet during the measurement. On the other hand, the weight decreased at pH 9, and the rate of decrease was greater than that of BIND~PETase (ID23), suggesting that the active PET degradation activity was higher.

Fig. 9.8.3. Mass Reduction Rate of PET Composite Pellets

Table. 9.8.2. Degradation Activity for PET Composite Pellets

Variants	Weight change of pellets (pH 7)	Weight change of pellets (pH 9)	Discussion
ID23	decrease	decrease	The weight of the pellets decreased at both pH 7 and 9, indicating that the PET in the pellets was degraded. The rate of decrease was particularly large at pH 7. In addition, the activity of BIND-PETase (ID24) was higher than that of BIND-PETase (ID24) at both pH 7 and 9, as described below.
ID24	decrease	decrease	The weight of the pellets decreased at both pH 7 and 9, indicating that the PET in the pellets was degraded. The rate of decrease was particularly large at pH 7.

The degradation activity of BIND-PETase (ID23, 24) against PET pellets has been confirmed. For PET pellets, BIND-PETase (ID24) exhibited the highest degradation under pH 9 conditions. For composite pellets, BIND-PETase (ID23) demonstrated the most degradation at pH 7. However, since the size of each pellet varies, leading to different surface areas, the results can only provide a rough evaluation. Therefore, to measure the activity more quantitatively, we conducted a 9. PET Powder Degradation Assay.

9 PET Powder Degradation Assay

In this assay, BIND-PETase suspensions were reacted with PET powder to evaluate its degradation activity. This experiment was conducted to quantitatively compare BIND-PETase (ID23, 24) with high activity confirmed through the pNPB Assay and the foundational BIND-duraPETase from which they were constructed.

BIND-PETase catalyzes the reaction where PET is degraded into TPA, MHET, and BHET, as shown in Fig. 9.9.1. By quantifying these products using HPLC (High-Performance Liquid Chromatography), we assessed the PET degradation activity of each BIND-PETase. The reaction was carried out for three days under two conditions: pH 7 and pH 9 (refer to Experiments). The results are presented in Fig. 9.9.3 and Fig. 9.9.4, respectively.

Fig. 9.9.1. Scheme of PET degradation by PETase

Fig. 9.9.2. PET powder

Fig. 9.9.3. PET Powder Degradation Activity (pH 7)

Fig. 9.9.4. PET Powder Degradation Activity (pH 9)

Among each variant, the order of activity was confirmed as BIND-PETase(ID24) > BIND-PETase(ID23) > BIND-PETase(dura). For BIND-duraPETase, only a small amount of degradation products was detected regardless of the pH condition, and this amount did not significantly change between Day 1 and Day 3. In contrast, for ID23 and ID24, there was a notable increase in the amounts of MHET and BHET from Day 1 to Day 3, suggesting that these variants possess high PET degradation activity. The quantities of MHET and BHET were greater for ID24 than for ID23 under all conditions.

Additionally, the activity of BIND-PETase was confirmed to be greater at pH 9 than at pH 7. For all variants, the amount of the degradation products was higher at pH 9 compared to pH 7. From this, it can be considered that the optimal pH for BIND-PETase is closer to 9 than to 7.

Upon careful observation, it was found that more TPA was produced at pH 7 than at pH 9, however it should be considered that the amount of PET powder in this assay was likely excessive relative to the E. coli, which may have influenced these trends.

Overall, we were able to clarify that BIND-PETase(ID24) exhibits particularly exceptional PET degradation activity and to elucidate the impact of pH on the degradation reaction of BIND-PETase.

10 Discussion

10.1 Discussion about the wet lab experiments

A total of 16 BIND-PETase variants, excluding ID7 and ID8 due to unsuccessful plasmid construction, were created from existing PETase mutants and custom-designed PETase variants. Among these, BIND-PETase (WT, E. coli optimized, dura, ID22-27) confirmed the ability to form Curli Fiber and the hydrolytic activity of PETase through Congo Red Assays and pNPB Assays. With time constraints preventing further experimentation on BIND-PETase (ID25-27), the remaining variants underwent Storage Activity Assays and 7. Reusability Assays, revealing improved Storage Activity and Reusability for BIND-PETase (ID23,24). Additionally, the PET degradation capabilities were validated through 7 Pet Pellet Degradation Assays and 8 PET Powder Degradation Assays, showing that BIND-PETase (ID24) exhibits strong PET degradation activity.

BIND-PETase (ID23) demonstrated properties similar to BIND-PETase (ID24) and outperformed ID24 in some conditions of the pNPB Assay and Pet Pellet Degradation Assay. However, considering the low quantitativeness inherent in these assays due to the structure of pNPB and the weight measurement methods, as well as the significantly superior score of BIND-PETase (ID24) in the higher-resolution PET Powder Degradation Assay, it is concluded that BIND-PETase (ID24) is superior. Through this consideration, it was determined that BIND-PETase incorporating PETase (ID24) is particularly outstanding, resulting in the designation of BIND-bearPETase (BBa_K5436124) as a variant with exceptional functionality.

10.2 Discussion about the BIND-bearPETase(ID24)

The part that exhibited high activity in our wet lab experiments and that we registered as a New Composite Part is BIND-bearPETase (BBa_K5436124). BearPETase (BBa_K5436015) is a combination of the mutations found in DepoPETase^[2] and DepoPETase^[3]. These variants show enhanced enzymatic activity due to different factors, and by combining them, we anticipated even further improvements in activity. Therefore, based on previous research on DepoPETase and DuraPETase, we combined intermediate variants whose enzymatic activity had been evaluated through wet lab experiments. Excluding the overlapping mutations at Q119Y and Q119R, we ultimately created 81 combinations.

The mutations M1-M5, which were created during the screening process of DepoPETase, are as follows^[3].

(M1) N246D
(M2) N246D, Q119R
(M2-1) N246D, Q119R, R260Y
(M2-2) N246D, Q119R, T88I
(M2-3) N246D, Q119R, D220N
(M2-4) N246D, Q119R, S290
(M3) N246D, Q119R, T88I, D220N, S290P
(M4) N246D, Q119R, T88I, D220N, S290P, R260Y
(M5) T88I, D186H, D220N, N233K, N246D, R260Y, S290P

The PETase variants (ID23, ID24, and ID27), which showed improved functionality in various assays, including the PET Powder Degradation Assay, contained the following mutations.

BIND-bearPETase(ID24) : (M2) N246D, Q119R
BIND-PETase(ID23) : (M1) N246D, Q119R, R260Y
BIND-PETase(ID27) : (M2-3) N246D, Q119R, D220N

From this, it was determined that the mutations N246D and Q119R play a crucial role in enhancing the functionality of BIND-PETase variants. Additionally, according to Shi, who developed DepoPETase^[3], these mutations are known to stabilize key hydrogen bonds in PETase, which suggests that they also play an important role in DuraPETase.

However, it was concluded that the reason ID24 exhibited the highest activity among the variants was due to the large number of mutations. While adding beneficial mutations can enhance functionality, an excessive number may lead to a loss of stability. Therefore, the number of mutations that can be added to duraPETase from depoPETase is limited to two, as adding more could have a detrimental effect for the reasons mentioned above.

References

Yunpu J. et al. (2022), Hydrophobic cell surface display system of PETase as a sustainable biocatalyst for PET degradation, Microbiotechnology,Vol.13, https://doi.org/10.3389/fmicb.2022.1005480 ↩︎
Y Cui et al.(2021) Computational Redesign of a PETase for Plastic Biodegradation under Ambient Condition by the GRAPE Strategy. ACS Catal. 11(3), 1340–1350. doi: 10.1021/acscatal.0c05126 ↩︎
L Shi et al.(2023) Complete Depolymerization of PET Wastes by an Evolved PET Hydrolase from Directed Evolution. Angewandte Chemie International Edition 62(14) doi: 10.1002/anie.202218390 ↩︎ ↩︎ ↩︎