Since the Japanese discovered the properties of persimmons in ancient times, fermented persimmon juice, kakishibu, has traditionally been used for protecting wood and paper and as an antimicrobial agent – without understanding why it worked. Now, researchers have studied kakishibu, a cinnamon extract and hundreds of other plant extracts to find substances that can improve how waste is reused in the green transition. The very processes that researchers find in the natural cycle may affect whether microorganisms can attack humans.
Walking through the forest, you are in the middle of an invisible long-term struggle for nutrients and a balanced symbiosis between majestic trees and tiny elusive microorganisms. A growing number of researchers are focusing on understanding these processes, to ensure that nature’s delicate balances remain undisturbed and to try to recreate some of nature’s processes in laboratories and then on a large scale in industry.
“Our research focuses on the processes that decompose the walls of plant cells in nature. For example, fungi use enzymes to break down biomass, such as peat soil and wood, to absorb nutrients. Recreating these processes in industrial biorefineries can create energy and materials from what we currently call waste. In our most recent studies, we found useful substances in persimmon and especially cinnamon that can help to regulate some of the key enzymes in the natural cycle,” explains Katja Salomon Johansen, Professor, Department of Geosciences and Natural Resource Management, University of Copenhagen.
Effects not specific enough
The fermented juice from unripe persimmons, kakishibu, has played a very important role in Asian culture, cooking, medicine and especially for protecting wood and paper. How it works, however, has been uncertain. It contains substances that can inhibit enzymes in fungi from breaking down cellulose in the cell walls of plants and trees. This preserves the structures of wood, but the mechanism has not been clearly defined.
“We studied how kakishibu affects enzymes called lytic polysaccharide monooxygenases (LPMOs) and a cocktail of cellulose hydrolases, which we know microorganisms use naturally to break down cellulose. By understanding the mechanisms that affect these enzymes, we hoped to find ways to switch these degrading enzymes on and especially off. This knowledge may be absolutely key to improving how plant biomass is used,” says Katja Salomon Johansen.
LPMOs are considered a breakthrough in the industrial degradation of cellulose in biomass because they accelerate the breakdown of long-chain glycosides in the presence of oxygen. LPMOs are, therefore, a key component in the enzyme cocktails used in producing bioethanol.
“We especially turned our attention to the tannin in kakishibu. Tannins or tannic acid, such as those found in red wine, influence and precipitate enzymes in saliva to create a bitter taste and a dry feeling. We, therefore, suspected that the tannins in kakishibu could also influence LPMOs,” explains Katja Salomon Johansen.
The researchers found that kakishibu and tannic acid affected the LPMOs similarly. They further showed that tannase, which can destroy the tannins, alleviates this inhibitory effect.
“This suggests that the interaction between the tannin and enzymes is key to the inhibitory effect of kakishibu. Unfortunately, the interaction between kakishibu and LPMOs seems to be too non-specific and may be excessively affected by external factors, such as acidity, for it to be used industrially, and the tannins in kakishibu are expected to interfere with other enzymes in an industrial cocktail too,” says Katja Salomon Johansen.
Large library of extracts
Although studying the kakishibu extracts did not provide a new effective tool for regulating industrial processes, the new knowledge can still be useful, since it is part of determining how LPMOs function.
“We know that microorganisms and plants have evolved with a remarkable ability to defend themselves against predators and competitors through a wealth of defence mechanisms. With this in mind, we investigated the hypothesis that some plants may produce compounds that inhibit LPMOs,” explains a main author of this study, Radina Tokin, postdoc at Section for Molecular Plant Biology, Department of Plant and Environmental Sciences, University of Copenhagen.
The large library of hundreds of extracts thus led the researchers to discover the first reported natural LPMO inhibitor, cinnamtannin B1, derived from cinnamon bark.
“Amazingly, cinnamon, a ubiquitous spice, was more potent that all other extracts. It has been used throughout the ages for its medicinal properties and its anti-inflammatory and antioxidant effects,” says Radina Tokin.
May play a role in infection
Unlike kakishibu, cinnamtannin B1 appears to have a much more specific effect. The researchers used protein crystallography to determine exactly where it binds to the surface of the LPMOs – close to the enzyme’s active site and at a pocket on the opposite side of the protein.
“Cinnamtannin B1 seems to be suitable to inhibit the processes in large bioindustrial tanks – a kind of stop button,” explains Radina Tokin.
The study emphasises that specific natural LPMO inhibitors exist in nature, so the hunt continues for compounds that can be used in various biotechnological contexts in the future, such as fuels, materials and food. In addition, studying LPMOs is absolutely key to understanding how fungi and other microorganisms in nature degrade carbonaceous compounds from plants and trees and thereby ensure balance in the carbon cycle in nature.
“Our understanding of how LPMOs function has expanded. We now know that they are more than just biomass-degrading enzymes. However, the question of how to control their activity remains unanswered. This is very important to investigate, since LPMOs have been suggested to play a role in infection by pathogenic microorganisms. Thinking about inhibiting LPMOs inspires several potential uses, such as developing medicine, sustainable pesticides for agriculture, adapted animal feed and even environmentally-sound material coatings,” concludes Katja Salomon Johansen.