Overview
Enzyme production is a significant problem for enzyme producers in Indonesia as the high cost of the downstream processes and the inefficiency of the process due to the unconcentrated protein yield makes them unable to produce significantly to meet the domestic enzyme needs and to compete globally. Indonesia still has to import 90% of its enzyme demands even though enzymes on its own are in a very high demand worldwide. Therefore, with this system, we hope to tackle these problems using synthetic biology by providing an easier method to harvest enzymes from microbes with a more concentrated yield. It is expected for Indonesian enzyme producers to be able to produce in a more efficient and cheaper way by using this system and domestic enzyme producers can support themselves independently and to export globally.
Other applications for OMV and this Omp fusion would be for the drug and vaccine delivery as it increases the display efficiency on the OMVs, aside from being cell-free. This idea needs further analysis since different methods of harvesting would be required. For more technical future prospect explanations, go to our Implementation page. Environmentally, the use of enzyme would lower the carbon offset by industries to the environment see our Sustainability Page During our talks and discussions with several SDG stakeholders, we found that the enzyme production systems that are used in Indonesia still have many several setbacks.
BPPT as a research body in Indonesia plays an important role in accelerating science in Indonesia. The opinions and concerns of experts in Indonesia are important to get an idea on how the reality and condition of enzyme production in Indonesia is. Our project is designed to be a bridge from the existing gap. From the discussion, we found that the methods currently used by domestic enzyme industries for their downstream processes are ultrafiltration and microfiltration. These are the obstacles which cause expensive and complicated methods due to them needing multiple steps of processes and this needs to be solved.
Stakeholder's Perspectives
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Dr.rer.nat Niknik Nurhayati
Indonesian Agency for Technology Assessment and Application
Dr.rer.nat Niknik Nurhayati is a researcher from the Indonesian Agency for Technology Assessment and Application who specializes in bacterial strain repair at the bioindustry technology laboratory, informed us that the most significant weak point in the current national enzyme production is the downstream or recovery process of enzymes. With the use of only wild-type strains that produce proteins extracellularly such as Bacillus, the option for recombinant protein production is still limited without the use of genetic engineering.
We also discovered that the present downstream procedure is expensive and requires many processing stages. Dr. Niknik further noted that owing to the media volume, enzyme yield would most likely be diluted, therefore a system that provided concentrated enzyme yield would be desirable. Other metabolites in the medium will also act as contaminants, making it difficult to obtain a clean purified yield. She stated that our system would be prospective, and that exceeding the yield of conventional systems would be considered a success. She emphasized that there should be an alternative to the existing ultrafiltration and microfiltration methods, which are both quite costly.
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Riahna Kembaren
Wageningen University Ph.D candidate
Riahna Kembaren is a PhD student who studies enzymes. We were able to obtain some information on the materials and procedures that we can utilize for more efficient lab work, as well as what components we may enhance in our design, thanks to Riahna Kembaren's advice and assistance.
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Safendrri Komara Ragamustari, Ph.D.
Amano Enzyme (2013-2015)
Dr. Safendrri is a researcher at Indonesian National Research and Innovation Agency. We asked for insight from Dr. Safendrri because he also worked at one of the biggest enzyme manufacturers, Amano Enzyme Research & Development Center Gifu in 2013-2015. With insights from Dr. Safendrri, we are able to understand what was really happening in the field for enzyme production. He stated several things that we found useful and gave us an idea of the situation in the field. There are some things we need to consider before designing and implementing our system. Different expression systems might need different types of promoters and codons. Different protein characteristics would also play a significant role in deciding which enzymes can be produced, as they may unfold and enter inclusion bodies. This can be predicted with simulations before being implemented, but the situation on the ground is often significantly different from what was expected in silico. He also highlighted the need of considering the pathways that convert a material to a finished product. He also informed us that lipase, amylase, and protease are the most commonly used enzymes in industries, in accordance with the papers we found on enzyme demands.
He also noted that getting the proper operational system and scaling up from the lab to the pilot plant might take some time. The majority of enzyme research takes two to five years to complete. This is because numerous parameters must be addressed, particularly while scaling up, such as aeration, pH, and a variety of other aspects.
Evidence
Apart from being responsible, we discovered that our project might be of considerable benefit to the world as a result of our engagement and conversations with key stakeholders. Stakeholders believe that this research has a promising future since greater and purified yield is essential in industrial enzyme applications, but the development required to achieve a stable, efficient system might take several years. This study might pave the way for the future development of improved and more efficient enzyme production systems.
Integrated Human Practices
We were able to create a clearer aim for ourselves in structuring our project and prioritizing what has to be solved as a result of our discussions with stakeholders. It aided us in developing our idea based on their feedback and taking into account additional things that we may have overlooked if we hadn't had the conversations.
Brainstorming & Designing
We believe that synthetic biology can address the majority of the current concerns about the enzyme manufacturing process. We also need to be efficient with our time, as well as thorough and accurate with our design, since we only have a limited amount of time in the lab to demonstrate our concept works due to the pandemic and the lack of laboratory access. We discovered that creating a cost-effective method while maintaining excellent quality is at the top of our priority as well as examining the properties of enzymes or proteins we can use for our proof of concept, based on the expectations and concerns of the stakeholders. For the need of higher and concentrated yield, we designed two constructs with the use of strong signal promoters for both universal and T7 expression to compare the effectiveness of both in recombinant protein expression. The promoter is expected to produce the outer membrane protein in a higher amount therefore giving a more concentrated yield. For easier and cost-effective downstream processes, we exploit the use of eCPX to make our eCPX-PoI fusion linked with a TEV protease-specific linker. This allows for specific cleaving of the PoI and the contaminants would already be long removed before the PoI is purified. Less steps on the purification would allow for a more cost-effective system. And the specific cleaving would allow for clean harvesting, providing a cleaner, purified yield of enzymes. The use of less expensive methods for purifying such as dialysis would also allow for lower cost of the downstream process. see more on our Engineering Page