An enzymatic future

Inter-departmental MU team aims to improve enzyme use and recovery for spectrum of industrial, medical and military applications

By Phillip Sitter | Bond LSC

Cylindrical bioreactor model

A mostly-finished cylindrical bio-reactor site sits in a 3D printer after the printing has stopped. With a 3D printer in-house, Chung-Ho Lin said that the inter-departmental team he is part of can generate four or five different prototypes a day to test in their bio-reactor model, instead of having to order from different fabrication companies. A basic printer like this used to cost $8,000, but within the last year prices dropped to only about $1,000. Lin is a research assistant professor at MU’s Center for Agroforestry, and the team and project are coordinated by Hsinyeh Hsieh, a veterinary pathobiology research scientist in George Stewart’s Bond LSC lab, where the team also does most of its work. | Phillip Sitter, Bond LSC

As Sagar Gupta watched a 3-D printer on a lab countertop construct a jumbo pencil eraser-sized, white plastic cylinder of what looked like a shell holding inter-woven letter Xs, he remarked that the only limitation to what you can print is the size of the printer.

“The timing is perfect, otherwise we wouldn’t have been able to afford it,” Chung-Ho Lin said of the availability of cheaper 3-D printers within the past couple years.

The two men were acutely aware, as the printer continued its methodical manufacture, that they may be architects of the first steps in a bio-chemical revolution.

It’s a revolution that could be hugely profitable financially and may help to save lives on battlefields, clean up some kinds of pollution and enable humans to venture further into space for a cheaper cost, among other things.

To understand how this cross-disciplinary team working in George Stewart’s lab at the Bond Life Sciences Center got there, we have to back up a little bit.

Sagar Gupta

Sagar Gupta holds a vial of carbon solution. Most of the team’s prototype designs for bio-reaction sites are made of carbon, and some are even bio-degradable. | Phillip Sitter, Bond LSC

From a bottleneck to a bioreactor

Their work began three years ago with a project to develop technology to reduce the cost of converting cellulose into glucose for biofuels — essentially the process by which raw plant fiber from wood or leaves is turned into a sugar that can be more efficiently burned to produce energy.

“That has been the bottleneck for the biofuel industry,” said Lin.

The team — consisting of Lin, a research assistant professor at MU’s Center for Agroforestry; Stewart, Hsinyeh Hsieh and several undergraduate and recently graduated students including Gupta — already developed E. coli bacteria that can mass-produce engineered enzymes to convert cellulose into glucose.

These enzymes speed up the reactions and reduce the cost because they have linkers attached to them — protein hooks that let them be recovered after a single use as catalysts in biological reactions, rather than having to throw them out. Hsieh said she developed this with Stewart’s input, and the assistance of a recently graduated student, Che-Min Su.

However, the team needed a platform for the linkers to hook onto — something they could continuously use to reel in their catch.

The answer in their search for the correct platform arrived when affordable 3-D printing technology came onto the market. With their own 3-D printer in-house, they custom-designed different platforms for their experiments and completely bypassed having to shop around with different fabrication companies.

All of the ingredients were there with that plastic cylinder Gupta and Lin watched print. The team now had a cheap way to mass produce and repeatedly recover enzymes. With this capability, they could produce a more efficient bioreactor — a controlled, isolated system in which desired reactions can take place with higher outputs of quantity and quality of a desired product.

It’s much like the more familiar concept of a nuclear reactor, which controls and isolates a nuclear chain reaction to harvest the most energy possible. The catalysts in that reaction are radioactive particles that give off heat as they decay. In a physical reaction, the heat released boils liquid water into gaseous steam, and the steam turns a turbine generator that makes electricity.

But in the team’s bioreactors, catalysts are enzymes that chemically react with cellulose and transform it into glucose instead of electricity. The glucose can be fermented further into butanol that can ultimately be used for liquid fuels to power vehicles.

Bioreactor column

A bio-reactor column stands packed with carbon fibers submerged in enzymes. If the column were hooked up a continous flow system, substrate would be pumped through it to spur bio-chemical reactions on the surface of the carbon fibers, or whatever other type of site is packed inside. | Phillip Sitter, Bond LSC

Money and blood

While only at a bench-top, proof-of-concept scale, the team’s first bioreactor has lasted more than four months. With prospects to increase its size, they “could be saving at least $10 to $12 million per year on an industrial scale,” said Gupta. Gupta graduated in May from MU with an MBA, and now works for Lin.

That estimate is just for one individual bioreactor. Begin to multiply it, and the cost-savings add up very quick.

“Nowadays, probably a majority of pharmaceutical companies have already switched their manufacturing process into the enzymatic process. One thing nice about the enzymatic process is that it can eliminate [the need for] a lot of hazardous chemicals. They also tend to have a better yield,” Lin explained.

Lin added that there is a bonus of complexity within this kind of 3-D platform system. Individual enzymes have different linkers, and this allows for multiple enzymes to catalyze reactions and be recovered on the platform at the same time. This is especially cost-saving because the conversion of cellulose into glucose requires three different kinds of enzymes.

“Because of this high specificity, we don’t need any enzymatic purification process,” he said.

Once the enzymes hooked to a platform start to naturally decay, the team can simply remove the decayed enzymes by a hot water bath and soak it in a new batch of enzymes, just like swapping out an empty printer cartridge for a full one with fresh ink.

While their primary focus is on biofuels, they are very aware that more efficient and cheaper bioreactors could have huge implications for a broad spectrum of industries.

One use they are developing could effectively transform one blood type into another using enzymes.

“This is not a completely new technology, but in the past, I would say back in the 90’s, some people tried some clinical trials and they ran into a problem, because a lot of times after the conversion, [loose] enzymes would get into the recipients’ bloodstream and cause an auto-immune reaction,” said Lin.

However, by being able to immobilize enzymes with their linkers on this 3-D device, they should be able to get around that problem, he said.

“I think there’s great potential for the soldier on the battlefield,” Lin cited as an application for the technology. A field doctor or medic wouldn’t have to worry about waiting on a certain type of blood for a transfusion, because they could convert another batch of blood into a universal-donor type.

Another team member, Hien Huynh explained that the more enzyme you add in ratio to the substrate, in this instance blood, the faster the conversion process will go — “maybe just 30 minutes.”

Hsieh wrote that “Blood type conversion would be the ultimate challenge for our bioreactor, because it has so many clinical aspects to be concerned [about] and conquered. It is a challenge but our [multi-disciplinary] team is willing to take it on and make it work.”

Lin said that the team has already submitted a letter of intent to the U.S. Department of Defense, “hopefully to secure some support for the blood-conversion application.”

Hien Huynh

Hien Huynh packs a bio-reactor column with carbon fiber bio-reactor sites that look like feathers. The sites are coated in enzyme before being packed into the column. | Phillip Sitter, Bond LSC

Enzymes in action

There are other potentially massive implications for the battlefields of the future.

“You can immobilize anti-microbial, anti-fungal and anti-inflammatory enzymes on a surface to use as a wound-healing patch,” Lin said, noting that such a patch could be used on the battlefield, as well as for cosmetic surgery recovery.

But the applications don’t stop there. Other uses could use enzymes to clean up TNT residues leeching out of unexploded ordinance like cluster bomblets, mortars, rocket-propelled grenades and landmines buried in the ground before the toxic residues contaminate groundwater.

Even within the confines of biofuels, there’s a strong military market. By 2020, the Navy wants 50 percent of its total energy consumption to come from alternative sources as opposed to petroleum-based fuels — part of a broader strategy to go green. The U.S. military in the near future wants to reduce the cost of its energy consumption and secure a stable domestic supply of energy.

According to the U.S. Government Accountability Office, from fiscal years 2007 to 2014, the Department of Defense bought 32 billion gallons of petroleum-based fuels at a cost of $107.2 billion.

Away from the military sphere, Lin detailed other uses for cheaper, higher quality enzymes. It could purify and recycle urine into clean water on space flights on for astronauts or convert waste into energy with an ammonia fuel cell that’s already available.

Mass-produced enzymes can be used for water treatment on earth, too. Pollutants like dioxin and herbicides like atrazine that contaminate soil can be bio-remediated in the same way that TNT residues can be cleaned up.

The food industry already uses enzymes as flavor removers to remove strong tastes from products like beer.

Minh Ma

Minh Ma simulates the end result of a successful operation of the bio-reactor. She extracts and separates samples of real glucose product produced by the reactions in the column. A stronger yellow color in the solution indicates a higher concentration of glucose. | Phillip Sitter, Bond LSC

A bright bioreactor future

To call the team’s work revolutionary might be a bit premature.

There is a whole process ahead of them, including patent filing and university reviews, before the team can approach investors with the assurance their discoveries are legally protected. And, future investors will ultimately help determine how the technology is used.

But, Lin and the others might just have found themselves in the right place at the right time to make major breakthroughs, and that’s not all due to just advancements in technology.

“We have identified new directions and found a new niche to be competitive. I think the most important resource we have is people, and their brains,” Lin said.

Hsieh wrote that “To assemble a successful team is to put the right talent in the proper position and to inspire them to challenge themselves. I was lucky to come across so many young, talented students who are eager to learn and work hard for their bright future on MU’s campus.”

Hsinyeh Hsieh, a veterinary pathobiology research scientist in George Stewart’s  Bond LSC lab, coordinates this project. Hsieh is an expert in gene fusion, enzyme production and characterization and enzymatic blood type conversion. Stewart is a medical bacteriologist, McKee Professor of Microbial Pathogenesis and chair of Veterinary Pathobiology at MU.

Lin works with Stewart and Hsieh to develop concepts, design prototypes and assemble the rest of the team — students and recent graduates — that optimizes the enzymatic reactions and the physical and chemical aspects of their bioreactor system. Minh Ma is a junior studying bio-chemistry. Mason Schellenberg studies bio-engineering, will be a senior and worked to find the most efficient platform design that the team’s 3-D printer could produce. Hien Huynh is a recent graduate who works on immobilizing enzymes. In addition to his MBA, Gupta also has a background that includes nano-technology, molecular engineering and financing. He concentrates on the feasibility and market potential of the team’s work.

Tiger Energy Solutions, LLC is the team’s industry partner — a spinoff startup from the team’s research project . Their focus in the development of a cheaper and higher quality method of converting cellulose into glucose for biofuels is to produce aviation biofuel. Tiger Energy serves as the interface between the team and industry while the team’s work is scaled-up for commercialization.

Minh Ma, Hien Huynh and Sagar Gupta

From left to right: Minh Ma, Hien Huynh and Sagar Gupta are three of the teams members, standing here in George Stewart’s lab. Ma is junior studying bio-chemistry. Huynh is a recent graduate who works on enzyme immobilization. Gupta is also a recent graduate — he obtained his MBA in May — and he focuses on the financial feasibility and market potential of the team’s work. | Phillip Sitter, Bond LSC

Saturday Morning Science returns to Bond LSC

Saturday Morning Science

This past weekend not only ushered in Mizzou’s first home game of the season, but the return of Saturday Morning Science. The weekly lecture series connects the Columbia community with MU scientists and their research, from bio-engineering to volcanology to anthropology and linguistics.

Elizabeth G. Loboa, dean of the College of Engineering, kicked off the semester with her talk on tissue engineering in the age of drug-resistant bacteria.

Tissue engineering is about turning cells into tissues and organs, for example, fat-derived stem cells into muscle, bone and cartilage. The tissues take shape on tiny scaffolds that are bio-compatible and biodegradable.

The Loboa lab does this, but they’ve added an extra layer to their research: Loboa’s scaffolds also act as pipelines that deliver wound-healing and anti-bacterial compounds to cells as they grow into tissue. The idea is to reduce infection, inflammation and scarring as the wound heals.

“We’re trying to kill these bacteria while helping these stem cells become the cells we want to create,” Loboa said, about her research at the University of North Carolina-Chapel Hill and North Carolina State University.

Using a process called electrospinning, Loboa’s group makes scaffolds shaped like porous fibers, sheaths, or hollow sheaths. Depending on their structure, these scaffolds act like faucet taps that control the rate and timing at which anti-bacterial compounds are released.

“I look at our fibers as delivery platforms,” Loboa said.

Saturday Morning Science takes place 10:30 a.m. Saturday at the Bond LSC’s Monsanto Auditorium. Coffee and bagels are available preceding the talks. This semester’s schedule is as follows:

9/17: Carolyn Orbann, Assistant Teaching Professor, Department of Health Sciences, “Historical Epidemics, Novel Techniques: Using Historical and Ethnographic Materials to Build Computer Simulation Models”

9/24: Michael Marlo: Associate Professor of English, “Documenting linguistic diversity: a view from the East African Great Lakes”

10/1: Steve Keller, Associate Professor of Chemistry, “The 20 Greatest Hits in Science…In an Hour”

10/8: Manuel Leal, Associate Professor of Biological Sciences, “Are Lizards Smarter Than Those Who Study them?”

10/15: Stephan Kanne: Executive Director and Associate Professor, Thompson Center for Autism & Neurodevelopmental Disorders, “What Do We Look For When We Diagnose Autism?”

10/29: Libby Cowgill, Assistant Professor Anthropology, “Fitness for the Ages: How to Lift Like a Neanderthal?”

11/5: Arianna Soldati, Ph.D. Candidate, Department of Geological Sciences, “Living in a Viscous World: A Volcanologist’s Perspective”

11/12: Frank Schmidt and Gavin Conant, Professor of Biochemistry (Schmidt); Associate Professor of Bioinformatics, Department of Animal Science (Conant), “Networks in Biology and Beyond”

12/3: Elizabeth King, Assistant Professor, Division of Biological Sciences, “What’s the Best Way to Divide up the Pie: The Price of Long Life”

Building blocks of life in the lab could revolutionize life for us all

NASA, NIH-funded work seeks to understand bio-chemical mechanisms of life on Earth, and among the stars
By Phillip Sitter | Bond LSC

Donald Burke-Agüero

Donald Burke-Agüero stands in his office in Bond LSC, holding a model of an RNA protein structure. Burke-Agüero studies the bio-chemical functions of RNA, and how those functions might be able to be artificially designed or replicated. | Phillip Sitter, Bond LSC

Any child obsessed with Legos knows the fun of creation bound only by imagination and the size or variety of the blocks within their pile.

For some scientists, that spirit extends into adulthood, but instead of plastic parts they think about arranging blocks of nucleic acids.

Scientists may not be able to create dinosaurs, dragons or mythical sea creatures the way kids with Legos can. Through the manipulation of nucleic acid building blocks though, they may be better able to understand how the processes of life on Earth work, as well as out among the stars.

“I have a lot of fun asking what is possible,” said Donald Burke, a Bond Life Sciences Center investigator who spends his time researching the building blocks of life.

Burke said he has been interested in the origins of life for 40 years, and he has been associated with NASA for about 20 years.

NASA’s interest in understanding the origins of life is pretty straightforward. It wants to know what clues to look for on other worlds to figure out if those planets also support life.

Many of Burke’s previous discoveries at Bond LSC are funded by NASA’s exobiology and evolutionary biology program.

“No, I have not thought of an excuse to fly anything up there. I’ve tried to think ‘which of my experiments would make sense to do in a micro-gravity or zero gravity environment?’” he explained of the prospect of sending some of his work into orbit, with a wry smile.

But, there’s even more to understanding the building blocks of life than looking for bio-chemical signatures out among the stars. Knowing how these parts are put together allows scientists like Burke to understand the origins and processes of Earth’s biology, and, conceivably create chemical and biological processes or even organisms not found in nature in the near future.

 

A quadrillion arrangements of blocks, one arrangement at a time

“Many of the molecules of life are built from strings of amino acids, or nucleotides or other building blocks,” Burke explained. He also noted that these buildings blocks are not just strings, but fold up into three dimensional shapes.

RNA, or ribonucleic acid, stands out as an essential building blocks in the bio-chemical processes of life.

Put simply, RNA is a kind of molecular structure of nucleic acids similar to DNA (deoxyribonucleic acid) that comes in many combinations. These combinations are at the core of every cell, and play a role in coding, decoding, regulating and expressing the basic operating instructions for each cell — its genes.

The molecules we’re talking about are almost unimaginably small. In one test tube, Burke said there can be one quadrillion of them — that’s a one with 15 zeroes after it. Put another way, that’s roughly equivalent to the estimated number of ants that live on Earth.

Burke’s work focuses on the end goal of being able to artificially create original RNA combinations. In what’s known as experimental evolution, “the population of molecules in the tube is evolving as a result of us imposing experimental constraints upon it.”

This artificial synthesis of RNA molecules looks to create random sequences or variations on natural RNA to create new ones non-existent in nature. A second route aims to selectively choose molecules with certain properties, and use them to build altogether new combinations.

“Their string-like properties allow us to copy them, and make more copies, and make more copies, and make more copies. Their shape-like properties allow us to observe the bio-chemical behaviors they may have,” Burke explained how he and other scientists interact with RNA’s structure in the lab.

“I don’t think we know what those limitations are yet,” he said of the capabilities of RNA.

The motivation for wanting to be able to intentionally design RNA molecules is so that it “can do the things we want it to do under the conditions where we want it to do those things,” he explained of the process of the process of selecting RNA sequences for specific properties.

“I want the ones that will bind a tumor cell. I want the ones that will bind a viral protein. I want the ones that will catalyze useful chemical reactions.”

 

RNA’s path to the future following in biology’s footsteps

The National Institutes of Health and other organizations recognize that engineered forms of RNA have the potential to fight diseases, and they have funded Burke’s work.

He has studied RNA that instructs human cells on how to defend themselves from HIV and is now looking at other RNA that interferes with the proteins of the Ebola virus.

The expectation is that such therapeutics would work in conjunction with other treatments. In the future, they could be expanded to help fight other viruses, cancers and other diseases.

RNA could also be used to start, or catalyze, chemical reactions. As Burke explained, catalysts remove barriers to chemical reactions — “they don’t make things happen that wouldn’t otherwise happen, but they speed up the process.”

Synthetic RNA could be used to accelerate removal of toxins from soil or to get the bacteria in our guts to recognize cancerous tumor cells and kick-start an immune response.

But, the future of RNA research may soon reveal a few different Holy Grail moments on its horizon.

One such Holy Grail that Burke said will definitely happen will be observations consistent with the presence of life on other worlds, based on evidence like an atmosphere having certain chemical compositions.

Another likelihood could involve construction of a self-replicating, fully-artificial organism, either created from scratch or reverse-engineered from other organisms.

For those of you already anticipating the plot of a low-budget sci-fi thriller, Burke offered to assuage your fears.

“The notion of it escaping out in the world and taking over Los Angeles is [only] good 1950’s B-movie” material, because the conditions under which this artificial organism would survive would probably be difficult to maintain even in the controlled environment of lab, he said.

Instead of B-movie science, Burke explained that “really, I’m thinking about what kinds of chemistries we want to see take place, and then building the enzymes that would make it possible.”

“Biology has had a few billion years to work on this, but we’re just starting to figure it out.”

Donald Burke-Agüero is a professor of molecular microbiology and immunology and joint professor of biochemistry and biological engineering.