Nav: Home

Modified enzyme can increase second-generation ethanol production

June 14, 2019

One of the main challenges of second-generation biofuel production is identifying enzymes produced by microorganisms for use in a "cocktail" of enzymes to catalyze biomass hydrolysis, in which the enzymes act together to break down the carbohydrates in sugarcane trash and bagasse, for example, and convert them into simple sugars for fermentation.

A group of researchers at the University of Campinas (UNICAMP), working in partnership with colleagues at the Brazilian Biorenewables National Laboratory (LNBR) in Campinas, São Paulo State, Brazil, have discovered that Trichoderma harzianum, a fungus found in the Amazon, produces an enzyme with the potential to play a key role in enzyme cocktails.

The enzyme, which is called β-glucosidase and belongs to glycoside hydrolase family 1 (GH1), acts in the last stage of biomass degradation to produce free glucose for fermentation and conversion into ethanol. In the laboratory, however, the researchers observed that high levels of glucose inhibited the activity of β-glucosidase.

"We also found that the enzyme's optimal catalytic activity occurred at 40 °C. This represented another obstacle to use of the enzyme because in an industrial setting, the enzymatic hydrolysis of biomass is performed at higher temperatures, typically around 50 °C," said Clelton Aparecido dos Santos, a postdoctoral researcher at UNICAMP's Center for Molecular Biology and Genetic Engineering (CBMEG) with a scholarship from FAPESP.

Based on an analysis of the enzyme's structure combined with genomics and molecular biology techniques, the researchers were able to modify the structure to solve these problems and considerably enhance its biomass degradation efficiency.

The study resulted from a project with a regular research grant from FAPESP and a Thematic Project also supported by FAPESP. The findings are published in the journal Scientific Reports.

"The modified protein we developed proved far more efficient than the unmodified enzyme and can be used to supplement the enzyme cocktails sold today to break down biomass and produce second-generation biofuels," Santos told.

To arrive at the modified protein, the researchers initially compared the crystal structure of the original molecule with structures of other wild-type β-glucosidases in the GH1 and GH3 glycoside hydrolase families. The results of the analysis showed that glucose-tolerant GH1 glucosidases had a deeper and narrower substrate channel than other β-glucosidases and that this channel restricted glucose access to the enzyme's active site.

Less glucose-tolerant β-glucosidases had a shallower but wider active site entrance channel, allowing more of the glucose produced by these enzymes to enter the last stage of biomass degradation. Retained glucose blocks the protein's channel and reduces its catalytic activity.

Based on this observation, the researchers used a molecular biology technique known as site-directed mutagenesis to replace two amino acids that might be acting as "gatekeepers" at the entrance to the enzyme's active site, letting in glucose or blocking it. Analysis of their experiments showed that the modification narrowed the channel to the active site.

"The mutant enzyme's active site shrank to a similar size to that of the glucose-tolerant GH1 β-glucosidases," Santos said.

Enhanced efficiency

The researchers conducted a number of experiments to measure the improved protein's performance in breaking down biomass, especially sugarcane bagasse, an agroindustrial waste with vast potential for profitable use in Brazil. During a research internship abroad with a scholarship from São Paulo Research Foundation - FAPESP, Santos worked with a research group led by Paul Dupree, a professor at the University of Cambridge in the UK, on an analysis of the tailored enzyme's glucose release efficiency when different sources of plant biomass were converted.

The analysis showed that the catalytic efficiency of the modified enzyme was 300% higher than that of the wild-type enzyme in terms of glucose release. Moreover, it was more glucose-tolerant, so more glucose was released from all the tested plant biomass feedstocks. The mutation also enhanced the enzyme's thermal stability during fermentation.

"Mutation of the two amino acids at the active site made the enzyme superefficient. It's ready for industrial application," said Anete Pereira de Souza, a professor at UNICAMP and principal investigator for the project. "One of the enzyme's advantages is that it's produced in vitro and not from a modified fungus or other organism, so it can be mass-produced at relatively low cost."
-end-
About São Paulo Research Foundation (FAPESP)

The São Paulo Research Foundation (FAPESP) is a public institution with the mission of supporting scientific research in all fields of knowledge by awarding scholarships, fellowships and grants to investigators linked with higher education and research institutions in the State of São Paulo, Brazil. FAPESP is aware that the very best research can only be done by working with the best researchers internationally. Therefore, it has established partnerships with funding agencies, higher education, private companies, and research organizations in other countries known for the quality of their research and has been encouraging scientists funded by its grants to further develop their international collaboration. You can learn more about FAPESP at http://www.fapesp.br/en and visit FAPESP news agency at http://www.agencia.fapesp.br/en to keep updated with the latest scientific breakthroughs FAPESP helps achieve through its many programs, awards and research centers. You may also subscribe to FAPESP news agency at http://agencia.fapesp.br/subscribe.

Fundação de Amparo à Pesquisa do Estado de São Paulo

Related Amino Acids Articles:

A natural amino acid could be a novel treatment for polyglutamine diseases
Researchers from Osaka University, National Center of Neurology and Psychiatry, and Niigata University identified the amino acid arginine as a potential disease-modifying drug for polyglutamine diseases, including familial spinocerebellar ataxia and Huntington disease.
Alzheimer's: Can an amino acid help to restore memories?
Scientists at the Laboratoire des Maladies Neurodégénératives (CNRS/CEA/Université Paris-Saclay) and the Neurocentre Magendie (INSERM/Université de Bordeaux) have just shown that a metabolic pathway plays a determining role in Alzheimer's disease's memory problems.
New study indicates amino acid may be useful in treating ALS
A naturally occurring amino acid is gaining attention as a possible treatment for ALS following a new study published in the Journal of Neuropathology & Experimental Neurology.
Breaking up amino acids with radiation
A new experimental and theoretical study published in EPJ D has shown how the ions formed when electrons collide with one amino acid, glutamine, differ according to the energy of the colliding electrons.
To make amino acids, just add electricity
By finding the right combination of abundantly available starting materials and catalyst, Kyushu University researchers were able to synthesize amino acids with high efficiency through a reaction driven by electricity.
Nanopores can identify the amino acids in proteins, the first step to sequencing
While DNA sequencing is a useful tool for determining what's going on in a cell or a person's body, it only tells part of the story.
Differentiating amino acids
Researchers develop the foundation for direct sequencing of individual proteins.
Simulating amino acid starvation may improve dengue vaccines
In a new paper in Science Signaling, researchers at the University of Hyderabad in India and the Cornell University College of Veterinary Medicine show that a plant-based compound called halofuginone improves the immune response to a potential vaccine against dengue virus.
CoP-electrocatalytic reduction of nitroarenes: a controllable way to azoxy-, azo- and amino-aromatic
The development of a green, efficient and highly controllable manner to azoxy-, azo- and amino-aromatics from nitro-reduction is extremely desirable both from academic and industrial points of view.
Origin of life insight: peptides can form without amino acids
Peptides, one of the fundamental building blocks of life, can be formed from the primitive precursors of amino acids under conditions similar to those expected on the primordial Earth, finds a new UCL study published in Nature.
More Amino Acids News and Amino Acids Current Events

Trending Science News

Current Coronavirus (COVID-19) News

Top Science Podcasts

We have hand picked the top science podcasts of 2020.
Now Playing: TED Radio Hour

Listen Again: The Biology Of Sex
Original broadcast date: May 8, 2020. Many of us were taught biological sex is a question of female or male, XX or XY ... but it's far more complicated. This hour, TED speakers explore what determines our sex. Guests on the show include artist Emily Quinn, journalist Molly Webster, neuroscientist Lisa Mosconi, and structural biologist Karissa Sanbonmatsu.
Now Playing: Science for the People

#569 Facing Fear
What do you fear? I mean really fear? Well, ok, maybe right now that's tough. We're living in a new age and definition of fear. But what do we do about it? Eva Holland has faced her fears, including trauma and phobia. She lived to tell the tale and write a book: "Nerve: Adventures in the Science of Fear".
Now Playing: Radiolab

The Wubi Effect
When we think of China today, we think of a technological superpower. From Huweai and 5G to TikTok and viral social media, China is stride for stride with the United States in the world of computing. However, China's technological renaissance almost didn't happen. And for one very basic reason: The Chinese language, with its 70,000 plus characters, couldn't fit on a keyboard.  Today, we tell the story of Professor Wang Yongmin, a hard headed computer programmer who solved this puzzle and laid the foundation for the China we know today. This episode was reported and produced by Simon Adler with reporting assistance from Yang Yang. Special thanks to Martin Howard. You can view his renowned collection of typewriters at: antiquetypewriters.com Support Radiolab today at Radiolab.org/donate.