To train for the future of manufacturing, companies cant rely on the teaching methods of the past. Applications of virtual reality (VR) and the metaverse are opening new frontiers for immersive learning experiences, and industries such as manufacturing are reaping the rewards.

Since travel is expensive and time-consuming, and classrooms can be limited in scope, virtual reality (VR) offers a promising alternative to facilitate rich, personal interactions using digital tools.

The goal is not to replicate real life, but to extend it, explains Gemba CEO, Nathan Robinson. Gemba delivers executive training across industry applications using a VR-based platform. With their masterclasses, the company aims to take advantage of the metaverse, as opposed to simply digitizing what would be done in an in-person classroom.

One of the companys most popular classes is Leading the Factory of the Future, co-led by Dr. Frank Piller. In the masterclass, Piller leads C-suite executives across industries through establishing and running manufacturing facilities in the era of Industry 4.0.

Piller is a leading expert in innovation management and the Chair of the Institute for Technology & Innovation Management at RWTH Aachen University. To date, he has taught the future factories masterclass more than 20 times, both in-person and in the metaverse. Now, we will never go back to traditional teaching, said Piller, describing the courses transition to VR.

Prior to being transferred over to the Gemba platform, Pillers class was delivered in-person, and saw participation from C-suite executives across the globe. When asked about the transition process, Piller explained that in many ways teaching in the metaverse is easier than over Zoom. For Piller, instruction is more natural and allows for more engaging personal interactions than a Zoom call. Where some participants and instructors might consider all virtual teaching options equal, Piller explained that was far from the case. The type of interactions and discussions he witnesses in the metaverse are simply not possible in a standard video call.

Piller also finds that many participants have little to no experience with the metaverse, so at least for Gemba, they rely on user-friendly Oculus hardware and a 30-minute orientation session to help executives get the most out of the masterclass. With this small upfront investment, Piller sees a high degree of engagement with participants who can focus on the content as opposed to the technology.

When transitioning the in-person class to the VR platform, the goal was to ask, what is meaningful in VR? So, instead of the class being about VR or the metaverse, it focuses on using the unique platform to deliver a one-of-a-kind teaching environment.

Piller says the goal is not to simply move their in-person class to a virtual platform, but to truly teach in a way that is not possible in traditional courses. With the Gemba platform, these types of classes can combine digital tools with live interactions to better instruct on leading large-scale companies of the future.

One interesting aspect of the Gemba platform is their approach to avatars in the metaverse. For the first phase of their masterclasses, the focus was on the course design and delivery, with significantly less emphasis put on the avatar design. As the VR system is wireless, the course relies on graphics limited by a smartphone, as opposed to a wired, computer system. So, avatars needed to be simple enough for wireless delivery to allow for more focus to be put on the VR setting. In their second phase, they adopted the Meta avatars for the platform, and soon they will be rolling out a third phase that allows additional customization options for participants. Piller explained his preference for simple avatar design, as it helps to eliminate bias and encourage participants to focus on the content and discussions. However, he also envisions that generative AI will continue to improve avatar technology over the coming years.

One of the most significant benefits of the metaverse over conventional virtual courses is the truly immersive learning environment. Unlike with a Zoom-based course, participants cannot access or be distracted by a second monitor or their smartphone. Participants are left with no choice but to provide their undivided attention to the course material, discussion, and tasks. Plus, activities can be more physical, like jumping into a box to select an answer or opinion to a question, as opposed to simply checking a survey box that pops up on a Zoom call. Or participants can walk along a line to illustrate the degree to which they agree or disagree with a statement. All of these interactions can be paired with 3D video and instruction that lends to an immersive training experience.

In many ways, the COVID-19 pandemic irreversibly altered our day-to-day lives. Robinson noted that the pandemic accelerated the adoption of certain technologies that previously received a great deal of pushback. Prior to the pandemic, many companies were insistent that employees were not capable of working from home. However, once it became a necessity for safety, a massive shift was rapidly adopted to transform the everyday working experience.

One excellent example of this is the adoption of the metaverse. Gembas platform existed before the pandemic, and many investors were skeptical in the early days that C-suite executives and large corporations would ever adopt such a training system. For Robinson, he believes the shift in 2020 saved at least five years in terms of the pushback to VR. But the Gemba masterclasses, including Pillers course, were already looking beyond 2020, to deliver a method of instruction which was simply not possible in an in-person setting. The goal was to provide a training system that would always be preferable to traditional, in-person courses.

Transformation is not about technology; its about people, explained Robinson. VR represents the opportunity to experience awe-inspiring learning experiences that still center human narratives. With VR, you can travel anywhere in the world, bring in a any global leader in a given field, and enforce an immersive environment.

Now, global clients such as Coca-Cola, Johnson and Johnson, and Caterpillar are using the Gemba platform and masterclasses to deliver safety training, leadership programs, and advanced manufacturing instruction for employees at all levels. One benefit is the ability to rapidly deliver new training programs at scale, with no travel required. For Pillers course, the metaverse makes it possible for participants to receive guest instruction from multiple leaders in next-generation manufacturing, including Porsche, Bosch, and Siemens. Porsche, for example, leads a session on data-driven decision-making in Industry 4.0.

One final benefit of teaching in the metaverse is the reduced environmental impact. Where traditional courses would see participants fly from all over the world to attend a three-day event, now people can join virtually from their own home. Robinson noted that on average, Gemba courses saved one ton of carbon dioxide per person, per masterclass, over the course of one year. So, not only can training be rapidly deployed, but it can assist with meeting sustainability metrics.

Although it seems the COVID-19 pandemic may have sparked a shift to virtual training offerings, Gemba seems to be standing out from the crowd by simply using the metaverse as a tool to deliver their training at scale. With recent successes, it will be interesting to see where the next phase of the platform takes the company and how the metaverse will continue to shape training at all levels of manufacturing.

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Teaching Industry 4.0 in the Metaverse - ENGINEERING.com

By Cassidy Morrison Senior Health Reporter For Dailymail.Com Updated: 01:19 01 Aug 2023

A central California city is reeling after a nondescript warehouse turned out to be an illegal lab with deadly pathogens, including coronavirus, HIV, and malaria.

If it were not for an errant hose sticking out of the back of the warehouse last spring, city officials would not have known that a shady biotech company with links to China had set up shop there, filling it with industrial freezers, hundreds of vials of viruses, and about 1,000 dead and dying lab mice.

Government investigators also found Covid diagnostic and pregnancy tests at the underground testing facility that they believed were being developed there, in addition to at least 20 stored infectious agents, including coronavirus, HIV, hepatitis, and herpes.

The lab was run under a company called Prestige Biotech unlicensed for business in California, whose president Xiuquin Yao said was a successor to the now-defunct company Universal Meditech Inc.However, officials dispatched to addresses linked to the companies turned up at empty office buildings or addresses in China that could not be verified.

The months-long investigation resulted in early July in the proper disposal of all dangerous chemicals and substances, labeled and unlabeled, and while officials there note that a probe into the origins of the lab is ongoing, they claim people in the surrounding area are safe.

While officials say the immediate danger has been resolved and power has been cut to the building, there will be lingering concerns about possible contamination in the area from improper waste disposal and leakage of dangerous bacteria and viruses that could pose health dangers to Reedley residents.

The black-market type lab operating in the sleepy town of Reedley, California, was brought to official attention in early March when a code enforcement officer, driving down the street noticed a garden hose sticking out of a building where it should not have been.

This lucky catch thrust into motion a combined state, local, and federal probe, one that Reedley City Manager Nicole Zieba had never seen before.

Ms Zieba said: This is an unusual situation. I've been in government for 25 years. I've never seen anything like this.

A warrant issued soon after the official happened across the code violation allowed those in the government to search the nondescript building, where they were shocked at what they found.

In one room were about 1,000 inhumanely stored white lab mice, roughly 200 of which were already dead. According to Assistant Director of the Fresno County Department of Public Health Joe Prado, the lab was conducting tests on the mice that would help develop Covid test kits found on site.

Mr Prado said: They were utilizing laboratory mice to see whether or not the Covid test kits were actually testing for Covid. So that was the purpose for the laboratory mice on-site.

Mr Prado did not add whether any of those Covid tests had been given or sold to the public.

They also found a wide array of vials containing biomaterials including blood and tissue, as well as many other unlabeled chemicals, some of which were found to contain the coronavirus, as well as bacterial and virus pathogens including HIV, chlamydia, E. Coli, streptococcus pneumonia, hepatitis B and C, herpes 1 and 5, rubella and malaria.

Mr Prado added: Here at the public health department we operate our own lab so were very well versed in the legal requirements and how to maintain and control an infectious agent. And there was just a complete absence of those controls in place at the warehouse.

More than 40 facilities certified as biosafety level 3 (BSL-3) or BSL-4 have either been built or have gone into construction since 2020, predominantly across Asia.

In addition to finding nearly a thousand lab mice either dead or in distress, court documents revealed that investigators also found refrigerators and freezers with blood and containers labeled as serum or plasma.

The officials were tasked with determining the provenance of the mysterious lab, which was found to be run by Prestige BioTech registered in Las Vegas.

City officials identified Xiuquin Yao as the companys president, who said Prestige BioTech shifted operations to the Reedley warehouse that was previously run by a now-defunct company called Universal Meditech Inc. Prestige was identified as UMIs successor, according to court documents.

But when officials were tasked with searching locations tied to either company, they turned up at abandoned offices or found linked addresses back in China that they could not verify.

City officials maintain that those operating under the name Prestige BioTech have not been forthcoming with information, though the investigation is ongoing and may turn up more answers down the line.

Ms Zieba said: There are no more biologicals. There are no more mice, but they still will see us abating 30 freezers and fridges, medical equipment, and all sorts of furniture in there. Theyll still see some activity nothing hazardous at this point.

Some of our federal partners still have active investigations going I can only speak to the building side of it.

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Unassuming warehouse in California turns out to be illegal Chinese-run virus laboratory that was genetically e - Daily Mail

In-cell engineering can be a powerful tool for synthesizing functional protein crystals with promising catalytic properties, show researchers at Tokyo Tech. Using genetically modified bacteria as an environmentally friendly synthesis platform, the researchers produced hybrid solid catalysts for artificial photosynthesis. These catalysts exhibit high activity, stability, and durability, highlighting the potential of the proposed innovative approach.

Protein crystals, like regular crystals, are well-ordered molecular structures with diverse properties and a huge potential for customization. They can assemble naturally from materials found within cells, which not only greatly reduces the synthesis costs but also lessens their environmental impact.

Although protein crystals are promising as catalysts because they can host various functional molecules, current techniques only enable the attachment of small molecules and simple proteins. Thus, it is imperative to find ways to produce protein crystals bearing both natural enzymes and synthetic functional molecules to tap their full potential for enzyme immobilization.

Against this backdrop, a team of researchers from Tokyo Institute of Technology (Tokyo Tech) led by Professor Takafumi Ueno has developed an innovative strategy to produce hybrid solid catalysts based on protein crystals. As explained in their paper published in Nano Letters on 12 July 2023, their approach combines in-cell engineering and a simple in vitro process to produce catalysts for artificial photosynthesis.

The building block of the hybrid catalyst is a protein monomer derived from a virus that infects the Bombyx mori silkworm. The researchers introduced the gene that codes for this protein into Escherichia coli bacteria, where the produced monomers formed trimers that, in turn, spontaneously assembled into stable polyhedra crystals (PhCs) by binding to each other through their N-terminal -helix (H1). Additionally, the researchers introduced a modified version of the formate dehydrogenase (FDH) gene from a species of yeast into the E. coli genome. This gene caused the bacteria to produce FDH enzymes with H1 terminals, leading to the formation of hybrid H1-FDH@PhC crystals within the cells.

The team extracted the hybrid crystals out of the E. coli bacteria through sonication and gradient centrifugation, and soaked them in a solution containing an artificial photosensitizer called eosin Y (EY). As a result, the protein monomers, which had been genetically modified such that their central channel could host an eosin Y molecule, facilitated the stable binding of EY to the hybrid crystal in large quantities.

Through this ingenious process, the team managed to produce highly active, recyclable, and thermally stable EYH1-FDH@PhC catalysts that can convert carbon dioxide (CO2) into formate (HCOO) upon exposure to light, mimicking photosynthesis. In addition, they maintained 94.4% of their catalytic activity after immobilization compared to that of the free enzyme. "The conversion efficiency of the proposed hybrid crystal was an order of magnitude higher than that of previously reported compounds for enzymatic artificial photosynthesis based on FDH," highlights Prof. Ueno. "Moreover, the hybrid PhC remained in the solid protein assembly state after enduring both in vivo and in vitro engineering processes, demonstrating the remarkable crystallizing capacity and strong plasticity of PhCs as encapsulating scaffolds."

Overall, this study showcases the potential of bioengineering in facilitating the synthesis of complex functional materials. "The combination of in vivo and in vitro techniques for the encapsulation of protein crystals will likely provide an effective and environmentally friendly strategy for research in the areas of nanomaterials and artificial photosynthesis," concludes Prof. Ueno.

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Towards artificial photosynthesis with engineering of protein crystals ... - Science Daily

This is a guest post for the Computer Weekly Developer Network written Deepak Goel, CTO of D2iQ.

With many pressures being put on them, DevOps teams simply cant keep up with the Ops demands.

This is why platform engineering is gaining momentum, as it advocates for building a centrally managed developer platform shared by multiple teams, instead of having each team build and run its own platform.

This approach ensures that critical infrastructure tasks like security, governance and observability are done once and done right, instead of being haphazard and duplicate efforts.

To maximise developer productivity, the platform engineering team must manage the infrastructure and create an internal platform for developers.

This removes much of the operational burden from the developers, allowing them to focus on building business applications. Platform engineering solves the complexity and skills gap challenges by providing a ready-made internal developer platform and golden path for DevOps teams, enabling them to devote their labor to creating business value rather than struggling to build a container management platform.

The workforce is no longer dependent on the infrastructure and there is one process for managing an organisations fleet instead of operational silos and duplicate efforts.

Deepak Goel, CTO of D2iQ.

Concurrently, the internal developer platform provides the abstraction and automation that helps developers build, test and deploy applications easily. Consider a scenario where there are multiple DevOps teams working on different projects within an organisation.

Each DevOps team will choose its own infrastructure, from the cloud to on-premise based on their needs. They will use their own tools and scripts to manage the lifecycle of the infrastructure, including provisioning and upgrades. They will also have their own measures for security, resulting in a structure that creates a self-service environment.

However, without platform engineering and internal developer platforms, it often leads to redundancy in operational efforts.

Each team has to execute the same lifecycle operations for their own infrastructure, creating silos that ultimately lead to the uneconomical use of infrastructure resources. Including DevOps within the framework of platform engineering brings standardisation and consistency, while maintaining the self-service environment DevOps teams have come to expect. In the above scenario, platform engineering teams centralise many of the operations needed to manage the lifecycle of the infrastructure, ensuring optimal use of the infrastructure resources by sharing them across various teams.

In addition, this approach provides a self-service internal developer platform environment within the security guardrails established by the platform engineering team.

Platform engineering makes developers more productive while simultaneously avoiding any pitfalls, improving an organizations overall ROI.

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CWDN series: DevEx D2iQ: The platform engineering experience - ComputerWeekly.com

Discovery of a CaPETase with high PET hydrolytic activity and thermostability

To discover a PET hydrolase, we performed a sequence homology analysis using the National Center for Biotechnology Information (NCBI) database and selected 10 PETase candidates (see Methods for details). A phylogenetic tree was constructed for the 10 selected PETase candidates and 17 reported PET hydrolases. The 27 enzymes were divided into two groups: one group contained mesophilic enzymes, such as IsPETase, and the other group contained thermophilic enzymes, such as TfCut2 and LCC (Fig.1a, Supplementary Fig.1 and Supplementary Table1). The phylogenetic tree was further separated into 10 subgroups, and 3 selected PETase candidates, namely, RZL00883.1, KOX11336.1, and SHM40309.1, formed discrete lineages with low phylogenetic relationships to the reported PET hydrolases (Fig.1a). To characterize the 10 selected PETase candidates (PCs), we first attempted to produce them in a signal peptide-truncated form. Eight of the 10 PETase candidates were successfully produced, except for KOX11336.1 and MAM88718.1. We also measured the melting temperature (Tm) of the eight PETase candidates to determine the thermostability of these enzymes. The candidates exhibited a range of Tm values from 38.6C to 70.5C (Fig.1b and Supplementary Fig.2). We then checked PET hydrolytic activity of the eight PETase candidates at a wide range of temperatures from 30 to 60C using several PET samples, such as post-consumer transparent PET powder (PC-PETTransparent), semi-crystalline PET powder (Cry-PET, Goodfellow Cambridge Ltd) (Cat. No. ES306000), and amorphous PET film (AF-PET, Goodfellow Cambridge Ltd) (Cat. No. ES301445). Among these candidates, PC3, PC7, PC8 and PC10 showed relatively low or undetectable levels of PET hydrolytic activity across the tested conditions (Fig.1c and Supplementary Fig.3). PC6, which have the highest Tm value of 70.5C, produced only negligible amounts of PET hydrolysis products at 30C, but showed the optimal PET hydrolytic activity at 50C (Fig.1b, c and Supplementary Figs.2, 3). PC4 and PC5 showed relatively high PET hydrolytic activity at 50C and 40C, respectively (Fig.1c and Supplementary Fig.3). Surprisingly, compared to other PETase candidates, PC2 exhibited significantly high PET hydrolytic activity across a broad range of reaction conditions. Notably, PC2 exhibited superior activity at 30C and produced the highest amount of PET hydrolysis products across all three PET substrates conditions compared to other candidates. (Fig.1c and Supplementary Fig.3). In addition, the pH profile results for the eight PETase candidates also showed that PC2 showed the highest level of PET hydrolytic activity (Supplementary Fig.4). It is noteworthy that PC2 showed remarkable PET hydrolytic activity compared with the other enzymes, and also exhibited high thermostability with a Tm value of 66.8C (Fig.1b, c and Supplementary Fig.2). Moreover, PC2 had the highest soluble expression level compared with the other enzymes (Fig.1b). These results indicate that PC2 has excellent properties for efficient PET degradation, including enzyme activity, thermostability, and protein expression levels. Thus, we selected PC2 (accession code: SHM40309.1, PETase from Cryptosporangium aurantiacum, CaPETase) as the most robust PET hydrolase among the eight PETase candidates tested. The measurements of changes of the Tm value and activity by addition of metal ions showed that PC2 is not a metal ion-dependent enzyme (Supplementary Fig.5).

a Maximum likelihood phylogenetic tree and percentage identity matrix of the 10 selected PETase candidate (PC1PC10) and 17 reported PET hydrolase sequences. Bootstrap values for 1000 replications are shown at the branching edges. The colored bar represents the level of the percent identity of the enzymes, and detailed percent identity values are listed in Supplementary Fig.31. b Protein yield and the Tm values of the 8 PCs. c PET hydrolytic activity of the eight PCs. The reaction was performed with post-consumer transparent PET powder (PC-PETTransparent, 15mgmL1 with 500nM enzyme), semi-crystalline PET powder (Cry-PET, 15mgmL1 with 2M enzyme), and amorphous PET film (AF-PET, 15mgmL1 with 2M enzyme) in 50mM Glycine-NaOH pH 9.0 buffer at various temperatures (30C, 40C, 50C, 60C) for 3 days. Reactions were performed in triplicate; Data are presented as mean valuesSD. d Comparison of the PET hydrolytic activity of CaPETase, IsPETase, LCC, and TfCut2. The reaction was conducted with Cry-PET (15mgmL1 with 2M enzyme) in 50mM Glycine-NaOH (pH 9.0) at various temperatures (30C, 40C, 50C, 60C) for 12h. Reactions were performed in triplicate; Data are presented as mean valuesSD.

Next, we compared the PET hydrolytic activity of CaPETase with that of well-known PET hydrolases, such as IsPETase, TfCut2, and LCC, over a broad temperature range from 30 to 60C using Cry-PET as a substrate. In reactions at 30C, CaPETase showed significantly higher PET hydrolytic activity than LCC and TfCut2 (Fig.1d and Supplementary Fig.3). Moreover, CaPETase exhibited 1.4-fold higher activity than IsPETase, which is known to have the highest PET hydrolytic activity at ambient temperature among the reported PET hydrolases (Fig.1d)17. In particular, the PET hydrolytic activity of CaPETase was 3.1-fold higher than that of IsPETase at 40C (Fig.1d), likely because CaPETase has much higher thermostability than IsPETase. However, the PET hydrolytic activity of CaPETase at 60C dramatically decreased and reversed compared with that of LCC at temperatures of 50C and 60C (Fig.1d). These results indicate that CaPETase is a promising PET hydrolase that exhibits high PET decomposition ability and thermostability. Considering that it is important to make improvements without the loss of enzyme activity and thermostability in the development of superior PET-degrading enzymes37, we propose that CaPETase represents a more efficient template enzyme for enzyme engineering than other enzymes with extreme mesophilic and thermophilic properties, such as IsPETase and LCC, respectively.

To provide a structural basis for high PET hydrolytic activity of CaPETase, we determined its crystal structure at a resolution of 1.36 (Supplementary Table2). CaPETase shows an / hydrolase fold and a nine-stranded -sheet at the center surrounded by six -helices and two 310-helices (Fig.2a and Supplementary Fig.6). Sequence-independent pairwise superposition of CaPETase with three distinctive PET hydrolases, namely, IsPETase, LCC, and TfCut2, generated global root mean square deviation values of 0.69, 0.65, and 0.53, respectively. Formation of one conserved disulfide bond (DS, C279/C297) and lack of an extended loop in the substrate binding site of CaPETase suggest that the enzyme originated from an ancestor of TfCut2 and LCC rather than IsPETase (Supplementary Fig.7). Interestingly, CaPETase exhibits a somewhat different backbone structure at the active site compared with other PET hydrolases (Supplementary Fig.8). Because the structural comparison using a Cartesian coordinate system is known to be subjective for distinguishing the detailed structural differences of the main chains38, we further analyzed the - torsion angles of the main chains of these four PETases (Supplementary Fig.9) and found that there were local differences in backbone torsion angles between CaPETase and other PET hydrolases (Fig.2a and Supplementary Fig.10). Interestingly, CaPETase also showed significant differences in the backbone torsion angles at the five connecting loops (31, 42, 67, 74, and 85) that form an active site, whereas comparisons of the corresponding loops between the other three PET hydrolases exhibited less differences (Fig.2a and Supplementary Fig.11), suggesting that CaPETase has a unique active site conformation. There were some differences in the network of residues extending from the active site to the nearby spatial environment compared with that of the other PET hydrolases. Among them, we observed unique differences affecting the backbone torsion angles of these loops. Near the 31 loop, distinct residues positioned in the 42 loop and a W105L108G124 network force, which form a unique side-chain internal network, appear to influence the conformation of the 31 loop (Supplementary Fig.12). In fact, the 31 loop has high root mean square fluctuation values near the active sites of other PET hydrolases in molecular dynamic simulations39,40. A unique A192G212F248 network is formed under the 85 loop, where catalytic H246 is located (Supplementary Fig.13). At the corresponding F248 position in CaPETase, LCC and TfCut2 have an alanine residue, whereas IsPETase has a cysteine residue that forms a second disulfide bond. Therefore, the positioning of a bulky F248 might cause significant torsional differences in the 85 loop and 74 loop of CaPETase (Supplementary Fig.13). Finally, an R176W200F209 network appears to trigger conformational differences in the 3-helix and 67 loop (Supplementary Fig.14). Importantly, the 3-helix contains the catalytic S169, and the 67 loop was previously annotated as a wobbling tryptophan-containing loop in PETase from Rhizobacter gummiphilus (Supplementary Fig.14)41. We further analyzed backbone fluctuations of these four PET hydrolases using molecular dynamic simulations and CaPETase exhibits quite unique backbone fluctuation profile (Fig.2b and Supplementary Fig.15). CaPETase has more stable 67 and 74 loops than mesophilic IsPETase, and particularly, the enzyme shows high stability at the front region of the 85 catalytic loop where H246 is located (Fig.2b). However, the end region of 85 which corresponds to the extended loop of IsPETase, and the front region of 31 loop showed the highest and lowest flexibility among the four homologs, respectively (Fig.2b). To our interest, the differences of the backbone fluctuation profile was localized exactly to the unique internal network affecting the backbone torsion angles of these loops. Thus, we believe that the unique backbone conformation of CaPETase allows the enzyme to maintain high activity while stabilizing several flexible loops of the mesophilic PET hydrolase.

a Comparison of the backbone torsion angle differences between CaPETase and IsPETase, LCC, and TfCut2. The structure of five connecting loops forming the active site of CaPETase is displayed as a putty tube representation of the same diameter in PyMoL. The structure is colored according to the Euclidean distance values between the two Ramachandran points of the aligned residues. Colors of white to red designate low to high Euclidean distance values, respectively. The catalytic triad of CaPETase is shown as a stick model with a cyan-color circle. b MD simulations show unique backbone fluctuation profile of CaPETase. C atom root-mean-square fluctuations (RMSF, ) of the CaPETase, IsPETase, TfCut2, LCC during MD simulations. c Comparison of the residues forming the substrate binding cleft of CaPETase, LCC, and TfCut2. The highlighted residues are shown as a stick model. d Distinct residues in the substrate binding site of CaPETase. Distinct and conserved residues are presented in magenta and light blue, respectively. e PET hydrolytic activity of the variants. PC-PETTransparent (15mgmL1) were incubated with 500nM enzymes at 40C for 24h in 50mM Glycine-NaOH buffer pH 9.0. Total amount of released products and the Tm value of the variants are shown as bars and red-colored dots, respectively. Reactions were performed in triplicate; Data are presented as mean valuesSD.

In addition to the unique backbone conformation at the active site, residues forming the substrate binding cleft of CaPETase showed significant differences compared with other thermophilic PET hydrolases (Fig.2c, d). In the vicinity of the wobbling W194, CaPETase possesses unique G196 and L133 residues, where highly conserved residues are located in other PET hydrolases (Fig.2c, d and Supplementary Fig.7). Mutating these residues to the corresponding residues in other PET hydrolases, such as G196L, L133Y, and L133Q, had a negative effect on enzyme activity and/or thermostability (Fig.2e). However, the G196T mutation exhibited enhanced thermostability (Fig.2e), which may result from the formation of a hydrogen bond between G196T and N195. CaPETase also contains a unique I102 residue in the 31 loop showing the largest torsion differences, whereas other PET hydrolases contain a highly conserved threonine residue at the corresponding position, which probably enables CaPETase to form a relatively wider substrate binding cleft (Fig.2c, d and Supplementary Fig.16). Replacement of I102 with threonine resulted in decreased enzyme activity, confirming that the residue contributes to high enzyme activity (Fig.2e). Furthermore, CaPETase has unique residues, such as Q107, W168, and T250, at the regions of the 31 loop, 85 loop, and 3, whereas most of the corresponding residues are highly conserved in other thermophilic PET hydrolases (Fig.2c, d and Supplementary Fig.7). Mutating these residues to the conserved residues in other thermophilic PET hydrolases decreased enzymatic activity and/or stability, indicating that the combined positioning of these residues is necessary to create an optimal substrate binding site for CaPETase with a unique shape and polarity (Fig.2e). One exception was the Q107S mutation, which resulted in no noticeable differences in enzyme activity or thermostability (Fig.2e). Taken together, we suggest that along with unique backbone torsion angles, the positioning of distinct residues at the substrate binding site enable CaPETase to form an optimal substrate binding site for high PET hydrolytic activity.

Although CaPETase has high PET hydrolytic activity and thermostability, its performance is still insufficient for industrial applications. We conducted rational protein engineering of CaPETase to further enhance the PET hydrolytic activity and thermostability of the enzyme using various strategies, such as introducing disulfide bonds and hydrogen bonds and modifying the protein surface charge (Supplementary Fig.17). The thermostability of the variants was monitored by measuring Tm values, and the PET hydrolytic activity of the variants was measured using post-consumer transparent PET powder (PC-PETTransparent) at ambient temperature (40C). We introduced four disulfide bonds, namely, G76C/A143C (DS1), L180C/A202C (DS2), T204C/R233C (DS3), and R242C/S291C (DS4). The DS2 and DS4 mutations increased the Tm value by approximately 3C, whereas the DS1 and DS3 mutations decreased the Tm value compared with CaPETaseWT (Fig.3a and Supplementary Fig.18). Moreover, the introduction of the DS2 and DS4 mutations increased PET hydrolytic activity by more than 20% compared with CaPETaseWT (Fig.3a and Supplementary Fig.18). These results indicate that the DS2 and DS4 mutations were successfully formed in CaPETaseWT and exerted positive effects on enzyme activity and thermostability. We also attempted to improve the thermostability of CaPETase by introducing noncovalent bonds, such as hydrogen bonds and salt bridges, and designed seven mutations, namely, V129T (NC1), P136S (NC2), A192T (NC3), R198K (NC4), V203T (NC5), A252N (NC6), and A257S(NC7). Of these, the NC1 and NC4 mutations increased Tm values by approximately 2C and enhanced PET hydrolytic activity by 30% compared with CaPETaseWT (Fig.3a and Supplementary Fig.18). Finally, in an attempt to improve the protein adsorption ability to the PET surface by modifying the protein surface charge, we designed five mutations to render the protein surface hydrophobic, i.e., N109A (HP1), R151A (HP2), R157A (HP3), R160A (HP4), and R233A (HP5), and four mutations to render the protein surface positive, i.e., T86R (SC1), A155R (SC2), T275R (SC3), and M294R (SC4). Unfortunately, most mutations did not show significant changes or even negative effects on thermostability or enzyme activity; however, the HP1 mutation increased the Tm value by 3.2C, and the SC2 mutation enhanced PET hydrolytic activity by 20% compared with CaPETaseWT (Fig.3a and Supplementary Fig.18). Taken together, we introduced eight point-mutations that resulted in improved thermostability and PET hydrolytic activity, i.e., DS2, DS4, NC1, NC4, HP1, and SC2, among the 20 rationally designed mutations tested (Fig.3a and Supplementary Fig.18). There were also ambiguous mutations that only improved enzyme activity or thermostability, such as NC2, HP2, and HP5. We excluded these mutations from the final selection to develop a much superior variant without compromising enzymatic activity or thermostability (Fig.3a and Supplementary Fig.18)37.

a Single-point mutations of CaPETase. Released PET hydrolysis products and the Tm values of the single-point mutations are presented. The reactions were performed using 500nM enzymes with post-consumer transparent PET powder (PC-PETTransparent, 15mgmL1) as the substrate in 50mM Glycine-NaOH buffer (pH 9.0) for 24h at 40C. Reaction was carried out in triplicate; error bars represent the s.d. of the replicate measurement. b Combinatorial mutations of CaPETase. PET hydrolytic activity of the variants generated using the combinatorial strategy. Released PET hydrolysis products per hour and the Tm values of the combinatorial variants are presented. The reactions were performed using 500nM enzymes with PC-PET (15mgmL1) as the substrate in 100mM Glycine-NaOH buffer (pH 9.0) at 40C for 24h and 60C for 6h, respectively. Reaction was carried out in triplicate; error bars represent the s.d. of the replicate measurement. c Comparison of PET hydrolysis activity between CaPETaseM9 and LCCICCG at various temperatures. The reactions were carried out at different temperatures using PC-PETTransparent (12.5mgmL1) with 1M enzyme and Cry-PET (12.5mgmL1) with 4M enzyme under the 200mM Glycine-NaOH buffer pH 9.0. Reactions were performed in triplicate; Data are presented as mean valuesSD.

We sequentially integrated the six mutations described above to develop a superior CaPETase variant with higher thermostability and PET hydrolytic activity. First, we combined the DS2 and DS4 mutations, and the resulting CaPETaseDS2/DS4 variant showed a synergistic effect on thermostability with a Tm value of 74.3C (Tm=7.4C) (Fig.3b and Supplementary Fig.19). Moreover, the variant enhanced PET hydrolytic activity by 1.35- and 4.45-fold at 40C and 60C, respectively, compared with CaPETaseWT (Fig.3b and Supplementary Fig.19). Next, we set up CaPETaseDS2/DS4 as a scaffold for the next combination. We integrated the NC1/NC4, HP1, and SC2 mutations individually into CaPETaseDS2/DS4 using our engineering strategy. The addition of the NC1/NC4 mutation increased the Tm value significantly by 3.9C and increased PET hydrolytic activity at both 40C and 60C (Fig.3b and Supplementary Fig.19). It showed 3.8- and 17-fold enhanced activity at 60C compared with CaPETaseDS2/DS4 and CaPETaseWT, respectively (Fig.3b and Supplementary Fig.19). The addition of the HP1 mutation increased the Tm value by 2.7C and enhanced PET hydrolytic activity by 1.7-fold at 60C compared with CaPETaseDS2/DS4 (Fig.3b and Supplementary Fig.19). When the SC2 mutation was integrated into the CaPETaseDS2/DS4 variant, we observed no noticeable improvements in activity and thermostability; however, there was a slight increase in PET hydrolytic activity at 60C (Fig.3b and Supplementary Fig.19).

These results suggest that all four mutations (NC1, NC4, HP1, and SC2) had a positive effect on the thermostability and activity of CaPETaseDS2/DS4; thus, we combined the four mutations into CaPETaseDS2/DS4 to generate CaPETaseDS2/DS4/NC1/NC4/HP1/SC2 (CaPETaseM8). Surprisingly, when all four mutations were added to CaPETaseDS2/DS4, a synergistic effect on thermostability and enzyme activity was observed, and CaPETaseM8 exhibited significantly enhanced thermostability with a Tm value of 80.7C and 1.5- and 25.8-fold enhanced PET hydrolytic activity at 40C and 60C, respectively, compared with CaPETaseWT (Fig.3b and Supplementary Fig.19).

As mentioned above, the G196T mutation resulted in positive effects on both thermostability and enzyme activity (Fig.2e); thus, we finally generated CaPETaseDS2/DS4/NC1/NC4/HP1/SC2/G196T (CaPETaseM9) by integrating the G196T mutation into CaPETaseM8. CaPETaseM9 exhibited a Tm value of 83.2C, which corresponds to a 16.7C increase in Tm compared with CaPETaseWT. Moreover, the PET hydrolytic activity of CaPETaseM9 increased by 1.7- and 31.2-fold at 40C and 60C, respectively, compared with CaPETaseWT (Fig.3b and Supplementary Fig.19). These results indicate a positive effect of G196T on CaPETaseWT was applied similarly to CaPETaseM8.

CaPETaseM9 showed much higher activity at all temperature conditions from 30 to 70C, and particularly, showed 41.7-fold higher specific activity at 60C than CaPETaseWT (Supplementary Fig.20). The result was also reproduced in a scale-up system of 50-mL shaking flasks (Supplementary Fig.21). These results demonstrated the improved enzyme activity and reinforced thermostability of CaPETaseM9. The improved thermostability of the variant was further verified through heat inactivation experiments, where CaPETaseM9 maintained its activity even after incubation at 60C for 12h, whereas CaPETaseWT showed complete loss of activity within an hour (Supplementary Fig.22).

We then compared the PET hydrolytic activity of CaPETaseM9 with LCCICCG towards PC-PET and Cry-PET at temperatures ranging from 30 to 60C. CaPETaseM9 showed significantly higher PET hydrolytic activity compared to LCCICCG, at 30C and 40C (Fig.3c). At 50C and 60C, CaPETaseM9 showed quite similar activity compared with LCCICCG (Fig.3c).

To provide structural insights into the enhanced PET-degrading capacity of CaPETaseM9, we determined its crystal structure at a resolution of 1.53 (Fig.4 and Supplementary Table2). The formation of the introduced DS2 and DS4 disulfide bonds was clearly observed in CaPETaseM9, and the SS interatomic length of both disulfide bonds was within the optimal disulfide bond length range (Fig.4 and Supplementary Fig.23). Interestingly, DS4 was located in the vicinity of one of the calcium binding sites of Cut190 and the mutation point of IsPETase R280A42,43, and the formation of DS4 also caused significant changes in the surface electrostatic potential and neighboring region conformation (Fig.4 and Supplementary Figs.23 and 24). The side chain of the mutated V129T was flipped to form a hydrogen bond with the adjacent T131 and D132 residues, thereby further stabilizing the 43 connecting loop (Fig.4 and Supplementary Figs.23 and 25). With respect to R198K, the mutated lysine residue moved inward to form hydrogen bonds with the main chains of N195 and D222, thereby stabilizing the wobbly tryptophan-containing loop (Fig.4 and Supplementary Fig.23). The mutated G196T formed a water-mediated hydrogen bond with the adjacent N195 residue, resulting in further stabilization of the wobbly tryptophan-containing loop (Fig.4 and Supplementary Fig.23). The A155R mutation changed the hydrophobic surface to a positive charge, which seems to increase the attachment of the enzyme to the PET surface, as suggested by a previous report (Fig.4 and Supplementary Fig.23)44. Finally, the N109A mutation appeared to strengthen internal hydrophobic interactions (Fig.4 and Supplementary Figs.23 and 26).

The crystal structure of CaPETaseM9 is shown as a cartoon diagram, and the mutated residues are shown as a stick or a surface electrostatic potential model.

To evaluate the industrial applicability of CaPETaseM9, we conducted a PET decomposition experiment in a pH-stat bioreactor using PC-PETTransparent as a substrate (Supplementary Fig.27). The bioreactor was operated at 55C using 2.70 mgenzymegPET1, and the pH was continuously titrated at 8.0 by adding NaOH. The decomposition rate was measured by monitoring released amounts of MHET and TPA. After a short lag phase of an hour, which was required for initial hydrophilization, the PET degradation rate increased exponentially, and 50% of PC-PETTransparent was depolymerized within 4h (Fig.5a). In the second half of the reaction, the degradation rate slightly decreased because of a decrease in the amount of substrate; however, a final degradation rate of 94.1% was achieved after 12h (Fig.5a). This was a significant result in terms of showing that a high depolymerization rate of 90% or more could be achieved even at 55C, which is a temperature condition relatively lower than the Tg temperature. This result also indicated that CaPETaseM9 has significant PET hydrolytic activity and thermostability comparable to other benchmark biocatalysts. We also performed decomposition of a post-consumer colored PET powder (PC-PETColored), which is known to be relatively difficult to recycle because of the presence of colors, additives, multilayer structure, labels and other complexities45. Interestingly, the depolymerization rate of PC-PETColored was almost identical to that of PC-PETTransparent, showing 50% depolymerization within 4h (Fig.5b); however, the final depolymerization rate of PC-PETColored was 89.2% at 12h, which was slightly lower than that of PC-PETTransparent (Fig.5b). This is probably due to the impurities present in PC-PETColored. These results demonstrate that unlike other recycling methods, biorecycling of PET plastic can be achieved regardless of the color of PET plastic.

Decomposition of post-consumer transparent PET powder (PC-PETTransparent) (a) and post-consumer colored PET powder (PC-PETColored) (b) in a pH-stat bioreactor using CaPETaseM9. Reactions were performed in triplicate independently; Data are presented as mean valuesSD. c Complete degradation of a post-consumer PET container using CaPETaseM9 at 60C. Reactions were performed in triplicate; Data are presented as mean valuesSD.

Finally, we determined whether untreated post-consumer PET containers can be depolymerized by CaPETaseM9. As depolymerization proceeded, the PET film became opaque and thin, and the PET film disappeared completely in 3 days (Fig.5c). These results suggest that CaPETaseM9 can be utilized for decomposing PET plastics with various physical properties.

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