The assignee for this patent, patent number 8652819, is
Reporters obtained the following quote from the background information supplied by the inventors: "The use of enzymes to saccharify lignocellulosic biomass is typically performed after other physical and/or chemical methods of pretreatment and can be accomplished prior to or in conjunction with fermentation. Pretreatment breaks down biomass to allow access to the enzymes, which can then hydrolyse the remaining cellulose, hemicellulose, and pectin polymers. Most enzymatic saccharification are performed with commercially available cell-free extracts of fungal cultures, or in some cases, bacterial cultures, designed to provide predominantly cellulase, xylanase, or pectinase hydrolysis of the lignocellulose. The fungal enzymes typically have optima of 45.degree. C. and pH 4.5, which can differ significantly from optimal fermentation conditions, especially when the ethanologen is a bacterium.
"Cellulose degradation can occur via free, secreted enzymes or by enzyme complexes attached to the surface of microorganisms (a cellulosome). While anaerobic organisms typically possess cellulosomes, aerobic bacteria and fungi typically employ free enzymes. The degradation of cellulose is achieved through the action of three types of enzymes: endo-glucanases, cellobiohydrolases (or exo-glucanases), and .beta.-glucosidases.
"Hemicellulases are either glycoside hydrolases (GHs) or carbohydrate esterases (CEs), and are classified into families based on their activity and homology of primary sequence. GH enzymes are responsible for the hydrolysis of glycosidic bonds, while ester linked acetate and ferulic acids side chains are cleaved by CE enzymes. As the structure of hemicellulose is very heterogeneous, a wide array of enzymes is necessary for hydrolysis. Additionally, many hemicellulases have carbohydrate-binding modules in addition to catalytic domains; as much of the hemicellulose structure can be insoluble, the carbohydrate-binding modules play a role in targeting of the enzymes to the polymers.
"Xylan is one major type of hemicellulose. Xylanases cleave the .beta.-1,4 glycosidic bonds of the xylose backbone, while xylosidases hydrolyze resultant oligomers to monomeric xylose. Ferulic acid esterases and acetyl-xylan esterases cleave the ester bonds of ferulic acid and acetate side chains, respectively. Arabinofuranosidases hydrolyze arabinofuranosyl side chains from the xylose backbone and can have varying specificity as to the location of the arabinofuranosyl group. Finally, glucuronidases are responsible for the cleavage of glucuronic acid side chain .alpha.-1,2-glycosidic bonds.
"A second form of hemicellulose is substituted .beta.-mannan, such as galactomannan. Much like xylanases, .beta.-mannanases are responsible for cleaving the mannose backbone to oligomers, which are then hydrolyzed to monomeric mannose by mannosidases. Side chain moieties, like galactose, are cleaved by respective GHs, and, in this case, by .alpha.-galactosidases.
"Pectinases can be divided into three general activity groups: protopectinases, which act on insoluble pectic polymers; esterases, which de-esterify methyl and acetyl moieties from pectin; and depolymerases, which either cleave or hydrolyze glycosidic bonds with polygalacturonic acid polymers. Protopectinases are usually unnecessary for degradation of lignocellulose if physical and/or chemical pretreatment methods have been employed prior to enzymatic saccharification.
"Pectin methylesterases are well described in bacteria and fungi and are responsible for the hydrolysis of the ester linkages from the polygalacturonic acid backbone. Pectin acetylesterases, which act in the same manner as pectin methylesterases to remove acetyl groups, have been described in plants and fungi; however, this type of enzyme has been found in only one bacterium, Erwinia chrysanthemi 3937. Pectin esterases are particularly important because many depolymerases cannot act upon methylated or acetylated pectin.
"Pectin depolymerases act upon the polygalacturonate backbone and belong to one of two families: polygalacturonases or lyases. Polygalacturonases are responsible for the hydrolytic cleavage of the polygalacturonate chain, while lyases cleave by .beta.-elimination giving a .DELTA.4,5-unsaturated product. For pectin polymers with a rhamnogalacturonan-I backbone, other hydrolases are also necessary; rhamnosidases hydrolyze rhamnose from the backbone, and arabinofuranosidases and galactosidases cleave arabinose and galactose, respectively, from substituted rhamnose subunits."
In addition to obtaining background information on this patent, NewsRx editors also obtained the inventors' summary information for this patent: "Provided herein are methods for producing ethanol. In some aspects the methods include fermenting a composition that includes a pretreated lignocellulosic material, wherein the fermenting includes contacting the composition with an ethanologenic microbe and a Paenibacillus spp., such as P. amylolyticus. The pretreated lignocellulosic material may be present at a concentration of at least 10% solids, and the fermenting may be a simultaneous saccharification and fermentation. In some aspects the ethanologenic microbe may be a yeast, such as Saccharomyces cerevisiae, or a prokaryotic microbe, such as E. coli. The pretreated lignocellulosic material may be pine, such as Pinus taeda. The Paenibacillis spp. may produce an enzyme having saccharifying activity when incubated on a medium that includes inorganic salts and a carbon source selected from glucose, mannose, xylose, arabinose, cellulose, pectin, starch, xylan, carboxymethylcellulose, or a combination thereof.
"In some aspects, the Paenibacillis spp. may produce an antimicrobial, such as a polymyxin, and in other aspects the Paenibacillus spp. is genetically modified to not produce an antimicrobial. The contacting may include inoculating the composition with the Paenibacillis spp. before inoculating the composition with the ethanologenic microbe, for instance, at least 12 hours before the composition is inoculated with the ethanologenic microbe. The contacting may include inoculating the composition with the Paenibacillis spp. and the ethanologenic microbe at substantially the same time. The method may further include adding pretreated lignocellulosic material to the composition after the fermenting has begun, for instance, at least 12 hours after the fermenting has begun.
"Also provided herein are methods that include providing a composition that includes spent hydrolysates and culturing a Paenibacillus spp., such as Paenibacillus amylolyticus, in the composition under conditions suitable for the production of an enzyme having saccharifying activity. In some aspects, the Paenibacillis spp. may produce an antimicrobial, such as a polymyxin, and in other aspects the Paenibacillus spp. is genetically modified to not produce an antimicrobial. The spent hydrolysates may be obtained from fermentation of a pretreated lignocellulosic material. The Paenibacillis spp. may produce an enzyme having saccharifying activity when incubated on a medium that includes inorganic salts and a carbon source selected from glucose, mannose, xylose, arabinose, cellulose, pectin, starch, xylan, carboxymethylcellulose, or a combination thereof. The method may further include mixing the composition that includes the Paenibacillis spp. and an enzyme having saccharifying activity with a composition that includes a pretreated lignocellulosic material to result in a fermentation composition. This fermentation composition may be contacted with an enthanologenic microbe including a yeast, such as Saccharomyces cerevisiae, or a prokaryotic microbe, such as E. coli.
"Further provided herein are methods including providing a first composition obtained from culturing a Paenibacillus spp., such as P. amylolyticus, in a second composition having spent hydrolysates under conditions suitable for the production of an enzyme having saccharifying activity, and mixing the first composition with a third composition that includes a pretreated lignocellulosic material to result in a fermentation composition. The fermentation composition may be contacted with an ethanologenic microbe including a yeast, such as Saccharomyces cerevisiae, or a prokaryotic microbe, such as E. coli. In some aspects, the Paenibacillis spp. may produce an antimicrobial, such as a polymyxin, and in other aspects the Paenibacillus spp. is genetically modified to not produce an antimicrobial.
"Also provided herein are methods that include culturing a Paenibacillus spp. in a composition that includes spent hydrolysates under conditions suitable for the production of an enzyme having saccharifying activity, wherein the culturing results in a second composition that includes the Paenibacillis spp. and an enzyme having saccharifying activity. The Paenibacillus spp. may be substantially removed from the second composition. In some aspects, the Paenibacillis spp. may produce an antimicrobial, such as a polymyxin, and in other aspects the Paenibacillus spp. is genetically modified to not produce an antimicrobial.
"Provided herein are genetically modified Paenibacillus amylolyticus that have been genetically modified to not produce an antimicrobial. The antimicrobial may be polymyxin E, and the genetically modified P. amylolyticus may include a transposon mutation that prevents expression of polymyxin E
"The term 'and/or' means one or all of the listed elements or a combination of any two or more of the listed elements.
"The words 'preferred' and 'preferably' refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
"The terms 'comprises' and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
"Unless otherwise specified, 'a,' 'an,' 'the,' and 'at least one' are used interchangeably and mean one or more than one.
"Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
"For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
"The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list."
For more information, see this patent:
Keywords for this news article include: Antimicrobial Cationic Peptides, Esterases, Hydrolases, Polymyxins, Escherichia, Cyclic Peptides, Membrane Proteins, Enterobacteriaceae, Gammaproteobacteria, Enzymes and Coenzymes,
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