EP2401289A1 - Altération et modulation de l'activité protéique en faisant varier la modification post-traductionnelle - Google Patents

Altération et modulation de l'activité protéique en faisant varier la modification post-traductionnelle

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Publication number
EP2401289A1
EP2401289A1 EP10746882A EP10746882A EP2401289A1 EP 2401289 A1 EP2401289 A1 EP 2401289A1 EP 10746882 A EP10746882 A EP 10746882A EP 10746882 A EP10746882 A EP 10746882A EP 2401289 A1 EP2401289 A1 EP 2401289A1
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EP
European Patent Office
Prior art keywords
enzyme
seq
protein
purified
isolated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10746882A
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German (de)
English (en)
Other versions
EP2401289A4 (fr
Inventor
David N. Thompson
David W. Reed
Vicki S. Thompson
Jeffrey A. Lacey
William A. Apel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Battelle Energy Alliance LLC
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Battelle Energy Alliance LLC
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Publication date
Priority claimed from PCT/US2009/035307 external-priority patent/WO2010051049A2/fr
Priority claimed from US12/655,993 external-priority patent/US8969033B2/en
Application filed by Battelle Energy Alliance LLC filed Critical Battelle Energy Alliance LLC
Publication of EP2401289A1 publication Critical patent/EP2401289A1/fr
Publication of EP2401289A4 publication Critical patent/EP2401289A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases

Definitions

  • the present invention relates generally to the field of biotechnology. More specifically, embodiments of the present invention relate to post-translational modification of proteins
  • Embodiments of the invention include methods of alte ⁇ ng the enzymatic activity of an extremophihc enzyme or other protein via means of chemical glycosylation and/or isolated or partially pu ⁇ fied glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.
  • Embodiments of the invention include methods of post-translationally modifying proteins.
  • the post-translational modification may occur using means of glycosylation (including chemical glycosylation), pegylation, phosphorylation, methylation or other forms of post-translational modification and/or be isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or m cells comprising one or more glycosyltransferases and/or post-translational modification proteins.
  • glycosylation including chemical glycosylation
  • pegylation including chemical glycosylation
  • phosphorylation methylation or other forms of post-translational modification
  • methylation or other forms of post-translational modification and/or be isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or m cells comprising one or more glycos
  • Embodiments of the invention include post-translationally modified proteins including, but not limited to, SEQ ID NOS'307 (celB), 331 (an Endoglucanase C), 333 (a Peptidoglycan N-acetylglucosamme deacetylase), 335 (a Beta-galactosidase), 337 (an arabmofuranosidase), and 338 (an alpha-xylosidase).
  • Embodiments thus include glycosylated versions of the aforementioned proteins.
  • a first aspect of the present invention relates to an enzyme isolated from an extremophihc microbe that displays optimum enzymatic activity at a temperature of greater than about 80 0 C, and a pH of less than about 2
  • Another aspect of the present invention relates to a hemicellulase that was derived from Alicyclobacillus acidocaldanus (ATCC 27009)
  • Another aspect of the present invention relates to an enzyme that is useful in the degradation of complex biomolecules. Still further, another aspect of the present invention relates to an enzyme that may be useful m a simultaneous saccharification and fermentation process to convert a biomass sugar into an end product.
  • Yet another aspect of the present invention relates to a method for the treatment of a biomass that includes the steps of providing a source of a biomass having a biomass sugar, pretreatmg the biomass with a water soluble hemicellulase derived from Alicyclobacillus acidocaldanus (ATCC 27009) to produce an end product.
  • a method for the treatment of a biomass that includes the steps of providing a source of a biomass having a biomass sugar, pretreatmg the biomass with a water soluble hemicellulase derived from Alicyclobacillus acidocaldanus (ATCC 27009) to produce an end product.
  • Another aspect of the present invention relates to a method for the preparation of a hemicellulase that includes the steps of providing a source of Alicyclobacillus acidocaldanus (ATCC 27009); cultivating the Alicyclobacillus acidocaldanus (ATCC 27009) in a microbial nutrient medium having a supernatant; separating the cells of the Alicyclobacillus acidocaldanus from the nutrient medium supernatant; and recovering and purifying the hemicellulase derived from the Alicyclobacillus acidocaldanus (ATCC 27009) from the nutrient medium supernatant.
  • another aspect of present invention relates to a method for hydrolyzmg a polysaccharide that includes the steps of providing a water soluble hemicellulase derived from a microbe, and conducting hydrolysis of a polysaccharide with the water soluble hemicellulase at a pH of less than about 2
  • FIG. 1 depicts a sequence alignment between SEQ E) NO: 1 (RAACOO 164) and ref
  • FIG. 2 depicts a sequence alignment between SEQ ID NO: 18 (RAAC00517) and ref
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ":"
  • FIG. 3 depicts a sequence alignment between SEQ E) NO:35 (RAAC00650) and ref
  • FIG. 4 depicts a sequence alignment between SEQ E) NO: 52 (RAAC00991) and ref]ZP_02327412.11, refjYP_OO 1487207.11. ref
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ":”.
  • FIGs. 5A and 5B depict a sequence alignment between SEQ E) NO:69 (RAACOl I lO) and ref
  • FIGs. 6A and 6B depict a sequence alignment between SEQ E) NO: 86 (RAACOl 166) and gb
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ":”.
  • FIG. 7 depicts a sequence alignment between SEQ E) NO: 103 (RAACO 1167) and ref
  • FIGs. 8A and 8B depict a sequence alignment between SEQ ID NO: 120 (RAACOl 170) and ref
  • FIG. 9 depicts a sequence alignment between SEQ ED NO: 137 (RAAC01248) and reflZP_02170160.1
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ":”.
  • FIGs. 1OA and 1OB depict a sequence alignment between SEQ ID NO: 154 (RAAC01348) and refjZPJ) 1665289.11, ref
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ":”.
  • FIG. 11 depicts a sequence alignment between SEQ ID NO:171 (RAAC01377) and ref
  • FIG. 12 depicts a sequence alignment between SEQ ID NO: 188 (RAAC01611) and ref
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ":”.
  • FIGs. 13A and 13B depict a sequence alignment between SEQ ID NO.205 (RAAC01612) and ref
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ":”.
  • FIGs. 14A and 14B depict a sequence alignment between SEQ ID NO:222 (RAAC01926) and ref
  • FIG. 15 depicts a sequence alignment between SEQ ID NO:239 (RAAC01998) and ref
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ".”.
  • FIG. 16 depicts a sequence alignment between SEQ ID NO:256 (RAAC02011) and ref]YP_754819.1
  • FIGs. 17A and 17B depict a sequence alignment between SEQ ID NO:273 (RAAC02381) and ref
  • Ammo acids conserved among all sequences are indicted by a "*” and generally conserved ammo acids are indicated by a ":”.
  • FIG. 18 depicts a sequence alignment between SEQ ID NO290 (RAAC02421) and ref
  • FIG. 19 is a graph depicting an effect of temperature on xylanase activity, as provided by the present invention.
  • FIG. 20 is a graph depicting an effect of temperature on cellulase activity, as provided by the present invention at a pH of 4.0.
  • FIG. 21 is a graph depicting an effect of pH on cellulase activity of the present invention at a temperature of 60 0 C.
  • FIG. 22 is a graph depicting an effect of pH on xylanase activity of the present invention at a temperature of 60 0 C.
  • FIG. 23 is a graph depicting the xylanase activity of SEQ ID NO:307 as determined using wheat arabmoxylan (WAX) at various pH and temperature levels. The enzyme was isolated from Alicyclobacillus acidocaldanus (black bars) or produced m E coli (white bars). There is no available data for enzyme isolated from Alicyclobacillus acidocaldanus at pH 5.5 (6O 0 C and 8O 0 C).
  • FIG. 24 is a graph depicting cellulase activity of SEQ ID NO:307 as determined using carboxymethyl cellulose (CMC) at various pH and temperature levels. The enzyme was isolated from Ahcyclobacillus acidocaldarius (black bars) or produced in E. coli (white bars). There is no available data for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5 (6O 0 C and 8O 0 C).
  • FIG. 25 is a graph depicting a ratio of cellulose/xylanase activity of SEQ ID NO:307 as determined from FIGs. 23 and 24 at various pH and temperature levels.
  • the enzyme was isolated from Alicyclobacillus acidocaldarius (black bars) or produced m E. coli (white bars). There is no available data for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5 (6O 0 C and 8O 0 C). The tops of the bars at pH 5.5 for the enzyme produced m E. coli are left open to indicate that the ratio was greater than 10.
  • FIG. 26 is a graph depicting xylanase activity of SEQ ID NO:307 as determined using wheat arabmoxylan (WAX) at various pH and temperature levels.
  • the enzyme was produced m Pichia pastoris (black bars) or produced m E coli (white bars).
  • FIG. 27 is a graph depicting cellulase activity of SEQ ID NO:307 as determined using carboxymethyl cellulose (CMC) at various pH and temperature levels. The enzyme was produced in Pichia pastoris (black bars) or produced m E coli (white bars).
  • FIG. 28 is a graph depicting a ratio of cellulose/xylanase activity of SEQ ID NO:307 as determined from FIGs. 26 and 27 at various pH and temperature levels. The enzyme was produced in Pichia pastoris (black bars) or produced m E. coli (white bars). The tops of the bars at pH 5.5 for the enzyme produced in E. coli are left open to indicate that the ratio was greater than 10.
  • FIG. 29 is a graph depicting xylanase activity of SEQ ID NO:307 as determined using wheat arabmoxylan (WAX) at various pH and temperature levels.
  • the enzyme was isolated from Alicyclobacillus acidocaldarius (black bars) or produced in Pichia pastoris (white bars). There is no available data for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5 (6O 0 C and 8O 0 C).
  • FIG. 30 is a graph depicting cellulase activity of SEQ ID NO:307 as determined using carboxymethyl cellulose (CMC) at various pH and temperature levels.
  • the enzyme was isolated from Alicyclobacillus acidocaldarius (black bars) or produced in Pichia pastoris (white bars). There is no available data for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5 (6O 0 C and 8O 0 C).
  • FIG. 31 is a graph depicting a ratio of cellulose/xylanase activity of SEQ ID NO:307 as determined from FIGs. 29 and 30 at various pH and temperature levels.
  • the enzyme was isolated from Alicyclobacillus acidocaldarius (black bars) or produced in Pichia pastoris (white bars). There is no available data for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5 (6O 0 C and 8O 0 C).
  • FIG. 32 is a graph depicting xylanase activity of SEQ ED NO:307 lacking the C-termmal 203 ammo acids as determined using wheat arabmoxylan (WAX) at various pH and temperature levels.
  • the enzyme was produced in Pichia pastoris (black bars) or produced m E, coli (white bars).
  • FIG. 33 is a graph depicting cellulase activity of SEQ ID NO:307 lacking the C-termmal 203 ammo acids as determined using carboxymethyl cellulose (CMC) at various pH and temperature levels.
  • the enzyme was produced in Pichia pastons (black bars) or produced m E coli with an N-termmal His tag (white bars).
  • FIG. 34 is a graph depicting a ratio of cellulose/xylanase activity of SEQ ED NO:307 lacking the C-terrmnal 203 ammo acids as determined from FIGs. 32 and 33 at various pH and temperature levels.
  • the enzyme was produced in Pichia pastons (black bars) or produced in E coli (white bars).
  • FIG. 35 is a graph depicting a ratio of arabmofuranosidase activity of RAAC00307 (SEQ ID NO:337) produced in E. coli. Activity at 5O 0 C (diamonds), 6O 0 C (squares), 7O 0 C (triangles), 8O 0 C ("x"s), and 9O 0 C ("*"s) at various pH levels are shown. The enzyme had no activity at pH 2.
  • FIG. 36 is a graph depicting a ratio of beta xylosidase activity of RAAC00307 (SEQ ID NO:337) produced in E coli. Activity at 5O 0 C (diamonds), 6O 0 C (squares), 7O 0 C (triangles), 80 0 C ("x"s), and 9O 0 C ("*"s) at various pH levels are shown. The enzyme had no activity at pH 2
  • FIG. 37 is a graph depicting a ratio of beta xylosidase activity of RAAC00307 (SEQ ID NO:337) produced in Pichia pastons. Activity at 6O 0 C (diamonds) and 8O 0 C (squares), at various pH levels are shown.
  • One aspect of the present invention relates, in part, to enzymes isolated from an extremophilic microbe that display optimum enzymatic activity at a temperature of greater than about 80 0 C, and an optimum pH of less than about 2.
  • the enzyme may be a hemicellulase and/or xylanase that was derived from Alicyclobacillus acidocaldanus, where the organism is further identified as ATCC 27009.
  • the enzyme appears to display enzymatic activity at a pH of about 1. Still further, this same enzyme has a molecular weight of at least about 120 kDa.
  • the enzyme as disclosed, may be useful in a simultaneous saccha ⁇ fication and fermentation process and/or a sequential hydrolysis and fermentation process to convert a biomass sugar into an end product. Still further, the enzyme, as described herein, may be useful m the pretreatment of a biomass slurry to degrade a water-soluble or water-insoluble oligomer and/or polysaccharide that is present in the biomass slurry to produce an end product.
  • extreme microbe means an organism that can live and thrive under conditions that humans would consider extreme, such as boiling water, ice, battery acid or at the bottom of the ocean.
  • microbes include, but are not limited to, Pyrolobus fumani that grows at temperatures up to 235°F (113°C), Psychrobacter cryopegella that survives at temperatures of
  • the cultivation is preferably conducted at temperatures above 40 0 C and a pH below about 5, and more preferably above 50 0 C and below a pH of 4, and most preferably above 55°C and below a pH of 3.5, and under anaerobic, aerobic, and/or micro-aerophihc conditions. While the cultivation period varies depending upon the pH, temperature and nutrient medium used, a period of 12 hours to several days will generally give favorable results.
  • Alicyclobacillus acidocaldanus is defined as a microorganism that can be obtained from the American Type Culture Collection (ATCC), Manassas, Virginia, and that is identified as Alicyclobacillus acidocaldanus (ATCC 27009)
  • enzyme activity means the reaction an enzyme causes to occur. Enzymes are proteins produced by all living organisms that mediate, cause and/or promote reactions that change a chemical into another type of chemical without themselves being altered or destroyed.
  • the word "optimum,” when used in combination with the te ⁇ n "enzymatic activity,” means the most favorable conditions that allow the enzyme to work the best and the fastest for a given end result. The optimum enzymatic activity may be affected by conditions that include temperature, pH, and salt concentrations
  • xylanase means an enzyme that breaks apart hemicellulose by breaking the chemical bonds between the xylose sugars that make up the backbone of the hemicellulose molecule, or by breaking bonds between xylose sugars m the hemicellulose side chains.
  • polysaccharide as used hereinafter shall mean a chain of sugars (can be the same sugars or different sugars) that are linked together by chemical bonds Polysaccharides can consist of straight chains of these sugars with or without side chains Examples of polysaccharides include starch, pectm, cellulose, and hemicellulose.
  • biomass sugar shall mean sugars that have come from the breakdown of biomass components, such as cellulose and hemicellulose.
  • biomass sugars include, but are not limited to, saccharides, glucose, xylose, galactose, mannose, arabmose, as well as combinations, oligomers, and/or modified or substituted forms thereof.
  • saccha ⁇ fication and fermentation process shall mean hereinafter a process for making a fuel or chemical such as ethanol from a biomass that may or may not have been pretreated by chemical means, and where cellulase and/or hemicellulase enzyme(s) are used to break down biomass polysaccharides into sugars (saccha ⁇ f ⁇ cation); and the sugars are fermented by source(s) of microorgamsm(s) into the product fuel or chemical (fermentation). These two processes occur at the same time, in the same reaction vessel (simultaneous).
  • end product as used in the present application shall mean hereinafter the chemical(s) that is/are produced by a chemical or enzymatic reaction.
  • Examples of end products contemplated by the present invention include simpler saccharides and sugars (e.g., monomers, dimers, t ⁇ mers, oligomers, etc.), alcohols, fuels, and/or other products of an enzymatic reaction.
  • biomass m the context of the present invention shall mean plant and other lignocellulosic material such as corn stalks, wheat straw, and wood by-products, such as sawdust and the like.
  • pretreatment of a biomass slurry shall mean, m the context of the present application, the preparation of a biomass for its subsequent conversion to fuels, such as ethanol.
  • This pretreatment includes the steps of grinding the biomass to a powder or small particles, and adding water (this constitutes a slurry).
  • This slurry is then treated by a number of methods designed to partially or completely remove the hgnm from the biomass, and convert the hemicellulose and cellulose into a form that can be more easily degraded into their component sugars using enzymes such as cellulases and hemicellulases.
  • Some pretreatments degrade hemicellulose to its component sugars while leaving the cellulose as part of the solid residue.
  • This treatment step is called a "pretreatment” because it occurs before both the enzymatic degradation step and before the fermentation step that converts the sugars into ethanol.
  • water-soluble in the context of the present invention shall mean a chemical or other substance that can be dissolved completely in water without leaving any solid residue.
  • hemicellulose in the context of the present invention means one component of a plant (the other two being cellulose and hgnm), that is made of a linear chain of sugars such as xylose, or mannose that are connected by a chemical bond. This linear chain also has branches consisting of sugars and other chemicals along the chain.
  • hemicellulase in the context of the present invention means a class of enzymes that can break hemicellulose into its component sugars and other chemical monomers
  • hemicellulases include, but are not limited to, xylanases, mannanases, glucuronidases, and arabmofuranosidases.
  • the phrase "sequential hydrolysis and fermentation process" in the context of the present invention shall mean a process for making a fuel or chemical from the biomass, such as ethanol, and where the biomass is treated physically or with a reactive chemical or solvent, or mixtures thereof, to remove the hgnm, and to convert the cellulose and hemicellulose present in the biomass into their component sugars or into a form that can be more easily degraded into their component sugars using enzymes such as cellulases and hemicellulases. Examples of these include grinding, milling, acids, alkalis, organosolvents, and the like. These treatments can be performed at temperatures ranging from ambient to 300 0 C or more, and at pressures ranging from ambient to 2000 psig or more.
  • the sugars which are dissolved m water, are then cooled, and the pH adjusted to neutral, and then subsequently fermented by microorganisms of various types into a product fuel(s) or chemical(s) (fermentation). These two processes occur in separate reaction vessels with the hydrolysis step conducted first, and the fermentation step conducted second (e.g., sequential).
  • the phrase "cultivating Alicyclobacillus acidocaldarms" in the context of the present invention shall mean providing the aforementioned microbe with a food source (soluble or insoluble lignocellulose or other source of polysaccharides or sugars) and various vitamins and minerals dissolved in water (this constitutes the nutrient medium), and giving the microbe the proper conditions that allow it to grow (a temperature of 14O 0 F (6O 0 C), a pH of 3.5, and oxygen).
  • a food source soluble or insoluble lignocellulose or other source of polysaccharides or sugars
  • various vitamins and minerals dissolved in water this constitutes the nutrient medium
  • separating the cells of the Alicyclobacillus acidocaldarms shall include means for removing the bacterial cells from the nutrient medium by, for example, centnfugation.
  • the phrase "recovering and purifying the hemicellulase" m the context of the present invention shall mean separating the hemicellulase enzyme from the nutrient medium.
  • a process called cation exchange was used to separate hemicellulase from the nutrient medium.
  • the nutrient medium (with hemicellulase) was pumped through the cation exchange material.
  • the hemicellulase When brought into contact with the cation exchange material, the hemicellulase will attach itself to the cation exchange material, but the nutrient medium will pass through.
  • the hemicellulase enzyme is then removed from the cation exchange material and is purified.
  • microbial nutrient medium in the context of the present application means a food source for the microbe ⁇ Alicyclobacillus acidocaldarms) and vitamins and minerals, all dissolved in water and adjusted to the pH needed by the microbe to grow. More specifically, the microbial nutrient medium includes about 1 gram per liter of Xylan; about 10 mM NH 4 Cl; about 5.2 mM K 2 HPO 4 ; about 0.8 mM
  • the inventors have isolated and characterized temperature and acid stable endoglucanase and/or xylanases that demonstrate activity at elevated temperatures, and low pH, and that show stability when incubated under these conditions for extended pe ⁇ ods of time.
  • the inventors recognize that heat and acid stable hemicellulases and cellulases, as described hereinafter, have particular value m, or as an accessory to, processes that would lead, on the one hand, to the reduction m the severity of pretreatment processes, earlier described, and/or the elimination of these limitations in various processes.
  • the inventors screened numerous organisms from Yellowstone National Park and various culture collections for microbes that had the ability to produce enzymes that were stable at both high temperature, and low pH
  • water and sediment samples were collected from six springs m the Norris Geyser Basm of Yellowstone National Park. These samples were inoculated into a liquid mineral salt medium having a pH 3.5, and further containing either 0.5 grams per liter of oat spelt xylan, or 0.5 grams per liter of ground corn cobs. The subsequent cultures were incubated at 80 0 C and were observed daily for growth, both visually and microscopically.
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen Und Zellkulturen
  • hemicellulase and/or cellulase activities were presumptively assumed present if growth occurred in the presence of xylan.
  • Cultures where growth occurred were harvested after approximately three days incubation. Cells were removed from the culture by cent ⁇ fugation. The culture supernatant was concentrated at about 1000-2000 fold using an AMICON® ultrafiltration cell with a 10000 MWCO membrane. The subsequent supernatant concentrate was then tested for hemicellulase and cellulase activity using arsenomolybdate reducing sugar acid assay (previously described by Somogyi (1952), J. Biol. Chem.
  • the enzyme activities were measured at a pH ranging from 1 to about 8 to determine the optimum pH for the enzyme activity.
  • the reducing sugar assay was modified by preparing the assay components in the appropriate pH buffer (pH 1-2, 50 mM sodium maleate or 50 mM glycine; pH 2-6, 50 mM sodium acetate; pH 6-8, 50 mM sodium phosphate; and pH 8-9, 50 mM T ⁇ s).
  • stabilities of the hemicellulases and cellulases as a function of temperature and pH were examined by incubating the supernatant concentrate at a temperature of about 70 0 C, and a pH of 2.0.
  • a layer of mineral oil was placed over the concentrate to limit evaporation during this exam.
  • Alicyclobacillus acidocaldarius (ATCC 27009) is capable of growth on a xylan substrate, and further produces extracellular hemicellulase and cellulase activity, which are both water-soluble and display significant hemicellulase activity at a pH of about 2, and which further has a molecular weight of at least about 120 kDa.
  • ATCC 27009 the Alicyclobacillus acidocaldarius
  • Aspergillus kawachu that has a pH optimum of 2.0 and a temperature optimum between 50 0 C to 6O 0 C.
  • Acid Stable Xylanases from Aspergillus kawachu, K Ito, H Ogasawara, T. Sugomoto, and T. Ishikawa, Bioscience Biotechnology and Biochemistry 56 (4).547-550, April 1992.
  • xylanases have been reported with pH optima in the range of 4 to 5
  • numerous xylanases have been reported that have temperature optima up to 100 0 C.
  • the enzyme as described hereinafter is the first enzyme known that has activities at such a low pH, and at such a high temperature, as claimed herein.
  • a xylanase has been purified from the same organism, that is, Ahcyclobacillus acidocaldanus (ATCC 27009), that is reported to have xylanase activity associated with it. It is reported that this enzyme had a pH optimum of 4.0, and a temperature optimum of about 80 0 C.
  • thermoacidophilic endoglucanase and/or xylanase (celB) from Ahcyclobacillus acidocaldanus (ATCC 27009) displayed high sequence similarity to arabmofuranosidases belonging to Family 51 of glycoside hydrolases (K. Eckert and E. Schneider, European Journal of Biochemistry, 270 (17):3593-3602, September, 2003).
  • the aforementioned cellulase precursor as described in this prior art reference is best understood by a study of SEQ ID NO:307, which is shown below:
  • SEQ ID NO:307 may be glycosylated. In further embodiments, SEQ ID NO: 307 may be glycosylated at least at positions 174, 193, 297, 393, and 404.
  • the new enzyme that was isolated from an extremophihc microbe has an N-termmal sequence comprising SEQ ID NO:326 as shown below: DVVSTPISMEIQV.
  • an enzyme as contemplated by the present invention and that is isolated from an extremophihc microbe comprises that which is seen in SEQ ID NO:327, which is provided below:
  • SEQ ID NO:327 aligns/corresponds to positions 166-248 of the celB sequence as seen in SEQ ID NO:307. It should be noted, that SEQ ID NO:327 includes changed amino acids at positions 207, 208, 212, 229, and 231; and added amino acids at positions 209, 213, and 230, respectively.
  • the enzyme of the present invention may be further characterized, and is best understood by a study of SEQ ID NO:328 below:
  • SEQ ID NO:328 aligns/corresponds to positions 258-304 of SEQ ED NO:307. It should be noted that SEQ ID NO:328 has a changed amino acid at position number 295, and an additional amino acid at position 296.
  • the enzyme as contemplated by the present invention further comprises the SEQ ED NO:329 as seen below:
  • SEQ ID NO:329 aligns/corresponds to positions 379-415 of SEQ ED NO:307. It should be understood that with respect to the earlier SEQ ID NO:307, the present SEQ ED NO:329 has changes in amino acids at positions 409, 411, and 413, respectively. Still further, additional amino acids are located at positions 412, 414, and 415, respectively.
  • the enzyme isolated from the extremophilic microbe of the present invention that displays optimum enzymatic activities at temperatures equal to or greater than 80 0 C and at a pH of less than 2, is considered to be water-soluble, and further has been isolated from cell supernatant.
  • the present invention is also directed to a method for the preparation of a hemicellulase that includes the steps of providing a source of Alicyclobacillus acidocaldarius (ATCC 27009); cultivating the Alicyclobacillus acidocaldarius (ATCC 27009) in a microbial nutrient medium having a supernatant; separating the cells of the Alicyclobacillus acidocaldarius from the nutrient medium supernatant; and recovering and purifying the hemicellulase derived from the Alicyclobacillus acidocaldarius (ATCC 27009) from the nutrient medium supernatant.
  • the methodology produces a hemicellulase that is water-soluble and displays significant enzymatic activity at a pH of less than about 2, and at temperatures greater than about 80 0 C.
  • the hemicellulase comprises the sequence as depicted in SEQ ED NOS:326, 327, 328, and/or 329.
  • the nutrient medium that is utilized further includes about 1 gram per liter of Xylan, about 10 itiM NH 4 Cl, about 5.2 mM K 2 HPO 4 , about 0.8 mM MgSO 4 -7 H 2 O, about 1.74 mM Na 2 SO 4 , about 25 mg per liter MgCl 2 , about 6.6 mg per liter of CaCl 2 , about 2.0 mg per liter MnSO 4 , about 0.5 mg per liter ZnSO 4 , about 0.5 mg per liter of boric acid, about 5 mg per liter of FeCl 3 , about 0.15 mg per liter of CuSO 4 , about 0.025 mg per liter OfNaMoO 4 , about 0.05 mg per liter Of CoNO 3 , about 0.02 mg per liter of NiCl 2 , about 0.08 mg per liter of pyridoxme hydrochloride, about 0.01 mg per liter of folic acid, about 0.1 mg per
  • the methodology includes the step of supplying the recovered and purified hemicellulase to a simultaneous saccha ⁇ fication and fermentation process to facilitate the conversion of a biomass polysaccharide into an end product.
  • One process for using the enzyme as noted above includes a step of pretreatmg a biomass slurry with the recovered and purified hemicellulase or with a crude enzyme preparation prepared from the organism containing a majority of the protein comprised of the hemicellulase to degrade an oligomer and/or polysaccharide that is present in the biomass slurry to produce an end product.
  • the enzyme that has been isolated from the extremophilic microbe may be used in a method for hydrolyzmg a polysaccharide, which includes the step of providing a water-soluble hemicellulase derived from an extremophilic microbe; and conducting hydrolysis of a polysaccharide with the water-soluble hemicellulase at a pH of less than about 2.
  • the water-soluble hemicellulase has an optimal enzymatic activity at a temperature of about 80 0 C.
  • an enzyme isolated from extremophilic microbe that displays optimum enzymatic activity at a temperature of about 8O 0 C and a pH of less than about 2 is best understood by a study of SEQ ID NOS.327-329, respectively.
  • the enzyme that has been isolated is useful in a method for treating a biomass, which includes the steps of providing a source of a biomass having a biomass sugar; pretreatmg the biomass with a water-soluble hemicellulase derived from Alicyclobacillus acidocaldanus (ATCC 27009) to produce an end product
  • the biomass sugar comprises a polysaccharide
  • the hemicellulase hydrolyzes the polysaccharide.
  • the hemicellulase displays enzymatic activity at a pH of less than about 2, and at temperature of greater than about 80 0 C.
  • the hemicellulase as contemplated by the present invention, has a molecular weight of about 120 kDa.
  • the methodology includes additional steps of pretreatmg the biomass in the presence or absence of the hemicellulase, providing a sequential hydrolysis and fermentation process to convert the biomass sugar into the end product; and supplying the hemicellulase to the sequential hydrolysis and fermentation process to facilitate the conversion of the biomass sugar into the end product.
  • the method includes a further step of providing a simultaneous saccha ⁇ fication and fermentation process to convert the biomass sugar into the end product; and supplying the hemicellulase to the simultaneous saccha ⁇ fication and fermentation process to facilitate the conversion of the biomass sugar into the end product.
  • the present enzyme and methodology avoids many of the shortcomings attendant with the prior art enzymes and practices employed heretofore, and further provides a convenient means for producing various desirable end products, while simultaneously reducing the severity of pretreatment steps that had the propensity for generating various deleterious waste products, as well as for increasing the cost of the overall process through the requirement of high temperatures, pressures and quantities of acid to attain the high pretreatment seventy.
  • Embodiments of the invention include methods of post-translationally modifying proteins
  • the post-translational modification may occur using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or m cells comprising one or more glycosyltransferases and/or post-translational modification proteins.
  • Glycosyltransferases and/or post-translational modification proteins may be, without limitation, of the following classes: UDP beta-glucosephosphotransferases, Dohchol-phosphate mannosyltransferases, and Glycosyltransferases.
  • the glycosyltransferases and/or post-translational modification proteins may be those of a thermoacidophilic organism.
  • thermoacidophiles include, but are not limited to, Alicyclobacillus acidocaldanus, and those organisms belonging to the genera Acidianus, Alicyclobacillus, Desulfurolobus, Stygwlobus, Sulfolobus, Sulfunsphaera, Sulfur ococcus, Thermoplasma, and Picrophilus
  • Examples of glycosyltransferases and/or post-translational modification proteins include, but are not limited to, those provided by SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, and 290, as well as those available from the NCBI at accession numbers XP_002490630.1, Q54IL5.1, ABN66322.2, XP
  • Embodiments of the invention also include proteins glycosylated according to methods of the invention.
  • proteins include, but are not limited to, glycosylated forms of the proteins of SEQ ID NOS:307, 331, 333, 335, 337, and 338.
  • Embodiments of the invention include methods of altering the physical and/or kinetic properties of an endoglucanase and/or xylanase from a thermoacidophile.
  • the ratio of cellulase activity to xylanase activity of an endoglucanase and/or xylanase from a thermoacidophile is altered by post-translational modification of the endoglucanase and/or xylanase.
  • the post-translational modification may be glycosylation.
  • the solubility of an endoglucanase and/or xylanase from a thermoacidophile is altered by post-translational modification of the endoglucanase and/or xylanase.
  • the endoglucanase and/or xylanase is an endoglucanase and/or xylanase of Alicyclobacillus acidocaldarius .
  • the endoglucanase and/or xylanase is celB (SEQ ID NO:307).
  • Embodiments of the invention include modification of the ratio of cellulase activity to xylanase activity of SEQ ED NO:307 through glycosylation of SEQ ID NO:307.
  • Glycosylation of SEQ ID NO:307 may be performed, by way of non-hmitmg example, by expression of a sequence encoding SEQ ID NO:307 in an organism capable of glycosylating SEQ ID NO:307, by exposure of SEQ ID NO:307 to an enzyme having glycosylating activity or a cell producing an enzyme having glycosylating activity; or by chemical methods known m the art.
  • the ratio of cellulase activity to xylanase activity of an endoglucanase and/or xylanase may be altered based on the level and location of post-translational modification.
  • the level and location of post-translational modification may be manipulated, by way of non-hmitmg examples, through the use of different enzymes having glycosylating activity, different cells capable of glycosylating a protein, different chemical methods of glycosylation, and/or by varying the amount of glycosylating activity or time the endoglucanase and/or xylanase is exposed to.
  • post-translational modification of the endoglucanase and/or xylanase results in increased xylanase activity at an acidic pH.
  • the modified form of the endoglucanase and/or xylanase has increased xylanase activity compared to an un-modified form of the endoglucanase and/or xylanase at, by way of non-hmitmg example, pH of less than about 5, pH of about 5, pH of about 3.5, and pH of about 2.
  • post-translational modification of the endoglucanase and/or xylanase results in greater solubility at an acidic pH.
  • the modified form of the endoglucanase and/or xylanase is more soluble that the un-modified form of the endoglucanase and/or xylanase at, by way of non-limiting example, pH of less than about 5, pH of about 5, pH of about 3.5, and pH of about 2.
  • Embodiments of the invention include genes and associated proteins related to the glycosylation and/or post-translational modification of proteins of the thermoacidophile Alicyclobacillus acidocaldanus.
  • Coding sequences for genes related to these processes were determined from sequence information generated from sequencing the genome of Alicyclobacillus acidocaldanus. These genes and proteins may represent targets for metabolic engineering of Alicyclobacillus acidocaldanus or other organisms. Non-limitmg examples of nucleotide sequences found withm the genome of Alicyclobacillus acidocaldanus, and ammo acids coded thereby, associated with glycosylation and/or post-translational modification of proteins are listed in Table 1.
  • Glycosyltransferases and/or post-translational modification proteins may be, without limitation, of the following classes: UDP beta-glucosephosphotransferases, Dolichol-phosphate mannosyltransferases, and Glycosyltransferases; and others.
  • Embodiments of the invention relate in part to the gene sequences and/or protein sequences comprising genes and/or proteins of Alicyclobacillus acidocaldanus.
  • Genes and proteins included are those which play a role m glycosylation and/or post-translational modification of proteins.
  • Intracellular enzyme activities may be thermophilic and/or acidophilic m nature and general examples of similar genes are described in the literature.
  • Classes of genes, sequences, enzymes and factors include, but are not limited to, those listed in Table 1.
  • the present invention relates to nucleotides sequences comprising isolated and/or purified nucleotide sequences of the genome of Alicyclobacillus acidocaldarius selected from the sequences of SEQ ID NOS.2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340, or one of their fragments
  • the present invention likewise relates to isolated and/or purified nucleotide sequences, characterized in that they comprise at least one of. a) a nucleotide sequence of at least one of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 or one of their fragments, b) a nucleotide sequence homologous to a nucleotide sequence such as defined in a), c) a nucleotide sequence complementary to a nucleotide sequence such as defined in a) or b), and a nucleotide sequence of their corresponding RNA; d) a nucleotide sequence capable of hybridizing under stringent conditions with a sequence such as defined m a), b) or c); e) a nucleotide sequence comprising
  • aspects of the invention relate nucleotide sequences which it has been possible to isolate, purify or partially purify, starting from separation methods such as, for example, ion-exchange chromatography, by exclusion based on molecular size, or by affinity, or alternatively, fractionation techniques based on solubility in different solvents, or starting from methods of genetic engineering such as amplification, cloning, and subclonmg, it being possible for the sequences of the invention to be carried by vectors.
  • separation methods such as, for example, ion-exchange chromatography, by exclusion based on molecular size, or by affinity, or alternatively, fractionation techniques based on solubility in different solvents, or starting from methods of genetic engineering such as amplification, cloning, and subclonmg, it being possible for the sequences of the invention to be carried by vectors.
  • nucleotide sequence fragment according to the invention will be understood as designating any nucleotide fragment of the genome of Alicyclobacillus acidocaldarius, and may include, by way of non-limiting examples, length of at least 8, 12, 20 25, 50, 75, 100, 200, 300, 400, 500, 1000, or more, consecutive nucleotides of the sequence from which it originates.
  • nucleotide sequence designating any nucleotide fragment of the genome of Alicyclobacillus acidocaldarius, having, after alignment and comparison with the corresponding fragments of genomic sequences of Alicyclobacillus acidocaldarius, at least one nucleotide or base of different nature.
  • An homologous isolated and/or purified nucleotide sequence in the sense of the present invention is understood as meaning an isolated and/or purified nucleotide sequence having at least a percentage identity with the bases of a nucleotide sequence according to the invention of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99 6%, or 99.7%, this percentage being purely statistical and it being possible to distribute the differences between the two nucleotide sequences at random and over the whole of their length.
  • Specific homologous nucleotide sequence m the sense of the present invention is understood as meaning a homologous nucleotide sequence having at least one nucleotide sequence of a specific fragment, such as defined above.
  • Said "specific" homologous sequences can comprise, for example, the sequences corresponding to the genomic sequence or to the sequences of its fragments representative of variants of the genome of Ahcyclobacillus acidocaldarius .
  • These specific homologous sequences can thus correspond to variations linked to mutations withm strains of Ahcyclobacillus acidocaldarius, and especially correspond to truncations, substitutions, deletions and/or additions of at least one nucleotide.
  • Said homologous sequences can likewise correspond to variations linked to the degeneracy of the genetic code.
  • degree or percentage of sequence homology refers to "degree or percentage of sequence identity between two sequences after optimal alignment" as defined m the present application.
  • Two ammo-acids or nucleotidic sequences are said to be "identical” if the sequence of ammo-acids or nucleotidic residues, in the two sequences is the same when aligned for maximum correspondence, as described below.
  • Sequence comparisons between two (or more) peptides or polynucleotides are typically performed by comparing sequences of two optimally aligned sequences over a segment or "comparison window" to identify and compare local regions of sequence similarity.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Ad. App. Math 2:482 (1981), by the homology alignment algorithm of Neddleman and Wunsch, J. MoI.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the peptide or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical ammo-acid residue or nucleic acid base occurs m both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity is the definition that would be used by one of skill in the art.
  • the definition by itself does not need the help of any algorithm, said algorithms being helpful only to achieve the optimal alignments of sequences, rather than the calculation of sequence identity. From the definition given above, it follows that there is a well defined and only one value for the sequence identity between two compared sequences which value corresponds to the value obtained for the best or optimal alignment.
  • BLAST 2 sequence software which is available at the web site worldwideweb.ncbi.nlm.mh.gov/gorf/bl2.html, and habitually used by the inventors and m general by the skilled person for comparing and determining the identity between two sequences, gap cost which depends on the sequence length to be compared is directly selected by the software (i.e., 11 2 for substitution matrix BLOSUM-62 for length>85).
  • Complementary nucleotide sequence of a sequence of the invention is understood as meaning any DNA whose nucleotides are complementary to those of the sequence of the invention, and whose orientation is reversed (antisense sequence).
  • Hybridization under conditions of stringency with a nucleotide sequence according to the invention is understood as meaning hybridization under conditions of temperature and ionic strength chosen in such a way that they allow the maintenance of the hybridization between two fragments of complementary DNA.
  • the hybridization is carried out at a preferential temperature of 65°C m the presence of SSC buffer, 1 x SSC corresponding to 0.15 M NaCl and 0.05 M Na citrate.
  • the washing steps can be the following: 2 x SSC, at ambient temperature followed by two washes with 2 x SSC, 0.5% SDS at 65°C; 2 x 0.5 x SSC, 0.5% SDS; at 65°C for 10 minutes each.
  • the conditions of intermediate stringency using, for example, a temperature of 42 0 C m the presence of a 2 x SSC buffer, or of less stringency, for example a temperature of 37°C in the presence of a 2 x SSC buffer, respectively, require a globally less significant complementarity for the hybridization between the two sequences.
  • nucleotide sequences according to the invention are those that can be used as a primer or probe in methods allowing the homologous sequences according to the invention to be obtained.
  • methods such as the polymerase chain reaction (PCR), nucleic acid cloning, and sequencing, are well known to a person skilled in the art.
  • nucleotide sequences according to the invention are again preferred which can be used as a primer or probe in methods allowing the presence of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340, one of their fragments, or one of their variants such as defined below to be diagnosed.
  • the nucleotide sequence fragments according to the invention can be obtained, for example, by specific amplification, such as PCR, or after digestion with appropriate restriction enzymes of nucleotide sequences according to the invention, these methods m particular being described in the work of
  • Modified nucleotide sequence will be understood as meaning any nucleotide sequence obtained by mutagenesis according to techniques well known to the person skilled in the art, and containing modifications with respect to the normal sequences according to the invention, for example mutations in the regulatory and/or promoter sequences of polypeptide expression, especially leading to a modification of the rate of expression of said polypeptide or to a modulation of the rephcative cycle.
  • Modified nucleotide sequence will likewise be understood as meaning any nucleotide sequence coding for a modified polypeptide such as defined below.
  • the present invention relates to nucleotide sequence comprising isolated and/or purified nucleotide sequences of Alicyclobacillus acidocaldanus, characterized in that they are selected from the sequences SEQ ED NOS'2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 or one of their fragments.
  • Embodiments of the invention likewise relate to isolated and/or purified nucleotide sequences characterized in that they comprise a nucleotide sequence selected from: a) at least one of a nucleotide sequence of SEQ ID NOS-2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 or one of their fragments; b) a nucleotide sequence of a specific fragment of a sequence such as defined in a); c) a homologous nucleotide sequence having at least 80% identity with a sequence such as defined in a) or b); d) a complementary nucleotide sequence or sequence of RNA corresponding to a sequence such as defined m a), b) or c); and e) a nucleotide sequence modified by a sequence such as defined in a), b
  • nucleotide sequences are the nucleotide sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340, or fragments thereof and any isolated and/or purified nucleotide sequences which have a homology of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identity with the at least one of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291,
  • Said homologous sequences can comprise, for example, sequences corresponding to genomic sequences Alicyclobacillus acidocaldanus.
  • these specific homologous sequences can correspond to variations linked to mutations within strains of Alicyclobacillus acidocaldanus and especially correspond to truncations, substitutions, deletions and/or additions of at least one nucleotide.
  • homologues are easily created and identified using standard techniques and publicly available computer programs such as BLAST. As such, each homologue referenced above should be considered as set forth herein and fully described.
  • Embodiments of the invention comprise the isolated and/or purified polypeptides coded for by a nucleotide sequence according to the invention, or fragments thereof, whose sequence is represented by a fragment.
  • Ammo acid sequences corresponding to the isolated and/or purified polypeptides may be coded by one of the three possible reading frames of at least one of the sequences of SEQ ID NOS:2, 19, 36, 53,
  • Embodiments of the invention likewise relate to the isolated and/or purified polypeptides, characterized in that they comprise a polypeptide selected from at least one of the ammo acid sequences of SEQ ID NOS.1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338 or one of their fragments.
  • isolated and/or purified polypeptides are the isolated and/or purified polypeptides of ammo acid sequence SEQ ID NOS: 1, 18, 35, 52, 69, 86, 103,
  • Embodiments of the invention also relate to the polypeptides, characterized m that they comprise a polypeptide selected from: a) a specific fragment of at least five ammo acids of a polypeptide of an ammo acid sequence according to the invention; b) a polypeptide homologous to a polypeptide such as defined in a); c) a specific biologically active fragment of a polypeptide such as defined m a) or b); and d) a polypeptide modified by a polypeptide such as defined in a), b) or c).
  • a polypeptide selected from: a) a specific fragment of at least five ammo acids of a polypeptide of an ammo acid sequence according to the invention; b) a polypeptide homologous to a polypeptide such as defined in a); c) a specific biologically active fragment of a polypeptide such as defined m a) or b); and d) a polypeptide modified by a
  • polypeptide In the present description, the terms polypeptide, peptide and protein are interchangeable.
  • the isolated and/or purified polypeptides according to the invention may be glycosylated, pegylated, and/or otherwise post-translationally modified.
  • glycosylation, pegylation, and/or other post-translational modifications may occur in vivo or in vitro and/or may be performed using chemical techniques.
  • any glycosylation, pegylation and/or other post-translational modifications may be N-lmked or O-lmked.
  • any one of the isolated and/or purified polypeptides according to the invention may be enzymatically or functionally active at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/or may be enzymatically or functionally active at a pH at, below, and/or above 8, 7, 6, 5, 4, 3, 2, 1, and/or 0.
  • glycosylation, pegylation, and/or other post-translational modification may be required for the isolated and/or purified polypeptides according to the invention to be enzymatically or functionally active at a pH at or below 8, 7, 6, 5, 4, 3, 2, 1, and/or 0 or at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius.
  • aspects of the invention relate to polypeptides that are isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or alternatively by chemical synthesis and that they may thus contain unnatural ammo acids, as will be described below.
  • polypeptide fragment according to the embodiments of the invention is understood as designating a polypeptide containing at least five consecutive ammo acids, preferably ten consecutive ammo acids or 15 consecutive amino acids.
  • a "specific polypeptide fragment” is understood as designating the consecutive polypeptide fragment coded for by a specific fragment of a nucleotide sequence according to the invention.
  • homologous polypeptide will be understood as designating the polypeptides having, with respect to the natural polypeptide, certain modifications such as, m particular, a deletion, addition, or substitution of at least one ammo acid, a truncation, a prolongation, a chimeric fusion, and/or a mutation.
  • homologous polypeptides those are preferred whose ammo acid sequence has at least 80% or 90%, homology with the sequences of amino acids of polypeptides according to the invention.
  • Specific homologous polypeptide will be understood as designating the homologous polypeptides such as defined above and having a specific fragment of a polypeptide according to the invention.
  • substitution In the case of a substitution, one or more consecutive or nonconsecutive ammo acids are replaced by "equivalent” ammo acids.
  • the expression "equivalent” ammo acid is directed here at designating any ammo acid capable of being substituted by one of the ammo acids of the base structure without, however, essentially modifying the biological activities of the corresponding peptides and such that they will be defined by the following.
  • substitutions are easily created and identified using standard molecular biology techniques and publicly available computer programs such as BLAST.
  • each substitution referenced above should be considered as set forth herein and fully described.
  • These equivalent ammo acids may be determined either by depending on their structural homology with the amino acids which they substitute, or on results of comparative tests of biological activity between the different polypeptides, which are capable of being carried out.
  • substitutions capable of being carried out without resulting in an extensive modification of the biological activity of the corresponding modified polypeptides
  • the replacement for example, of leucine by valine or isoleucme, of aspartic acid by glutamic acid, of glutamine by asparagme, of argmine by lysine etc., the reverse substitutions naturally being envisageable under the same conditions.
  • substitutions are limited to substitutions m ammo acids not conserved among other proteins which have similar identified enzymatic activity.
  • one of ordinary skill m the art may align proteins of the same function m similar organisms and determine which ammo acids are generally conserved among proteins of that function
  • One example of a program that may be used to generate such alignments is available on the Internet at worldwideweb.cha ⁇ te de/biomf/strap/ in conjunction with the databases provided by the NCBI
  • substitutions or mutations may be made at positions that are generally conserved among proteins of that function.
  • nucleic acid sequences may be mutated or substituted such that the ammo acid they code for is unchanged (degenerate substitutions and/mutations) and/or mutated or substituted such that any resulting ammo acid substitutions or mutations are made at positions that are generally conserved among proteins of that function
  • the specific homologous polypeptides likewise correspond to polypeptides coded for by the specific homologous nucleotide sequences such as defined above and, thus, comprise m the present definition the polypeptides that are mutated or correspond to variants which can exist in Ahcyclobacillus acidocaldanus , and which especially correspond to truncations, substitutions, deletions, and/or additions of at least one ammo acid residue
  • Specific biologically active fragment of a polypeptide will be understood m particular as designating a specific polypeptide fragment, such as defined above, having at least one of the characteristics of polypeptides according to the invention.
  • the peptide is capable of behaving as at least one of the types of proteins outlined m Table 1
  • polypeptide fragments can correspond to isolated or purified fragments naturally present m Ahcyclobacillus acidocaldanus or correspond to fragments which can be obtained by cleavage of said polypeptide by a proteolytic enzyme, such as trypsin or chymotrypsm or collagenase, or by a chemical reagent, such as cyanogen bromide (CNBr).
  • a proteolytic enzyme such as trypsin or chymotrypsm or collagenase
  • CNBr cyanogen bromide
  • Such polypeptide fragments can likewise just as easily be prepared by chemical synthesis, from hosts transformed by an expression vector, according to the invention, containing a nucleic acid allowing the expression of said fragments, placed under the control of appropriate regulation and/or expression elements.
  • Modified polypeptide of a polypeptide according to an embodiment of the invention is understood as designating a polypeptide obtained by genetic recombination or by chemical synthesis as will be described below, having at least one modification with respect to the normal sequence. These modifications may or may not be able to bear on ammo acids at the origin of specificity, and/or of activity, or at the origin of the structural conformation, localization, and of the capacity of membrane insertion of the polypeptide according to the invention. It will thus be possible to create polypeptides of equivalent, increased, or decreased activity, and of equivalent, narrower, or wider specificity. Among the modified polypeptides, it is necessary to mention the polypeptides m which up to five or more ammo acids can be modified, truncated at the N- or C-termmal end, or even deleted or added.
  • the preceding modified polypeptides can be obtained by using combinatorial chemistry, in which it is possible to systematically vary parts of the polypeptide before testing them on models, cell cultures or microorganisms, for example, to select the compounds that are most active or have the properties sought.
  • Chemical synthesis likewise has the advantage of being able to use unnatural ammo acids, or nonpeptide bonds.
  • ammo acids for example in D form
  • ammo acid analogs especially sulfur-contammg forms
  • nucleotide sequences coding for a polypeptide according to the invention are likewise part of the invention.
  • the invention likewise relates to nucleotide sequences utihzable as a primer or probe, characterized in that said sequences are selected from the nucleotide sequences according to the invention.
  • the present invention in various embodiments, likewise relates to specific polypeptides of Alicyclobacillus acidocaldarius , coded for by nucleotide sequences, capable of being obtained by purification from natural polypeptides, by genetic recombination or by chemical synthesis by procedures well known to a person skilled m the art and such as described in particular below.
  • the labeled or unlabeled mono- or polyclonal antibodies directed against said specific polypeptides coded for by said nucleotide sequences are also encompassed by the invention.
  • Embodiments of the invention additionally relate to the use of a nucleotide sequence according to the invention as a primer or probe for the detection and/or the amplification of nucleic acid sequences.
  • nucleotide sequences according to embodiments of the invention can thus be used to amplify nucleotide sequences, especially by the PCR technique (Polymerase Chain Reaction) (Erlich, 1989, Innis et al , 1990, Rolfs et al., 1991; and White et al., 1997).
  • PCR technique Polymerase Chain Reaction
  • oligodeoxyribonucleotide or oligo ⁇ bonucleotide primers advantageously have a length of at least eight nucleotides, preferably of at least twelve nucleotides, and even more preferentially at least 20 nucleotides.
  • amplification techniques of the target nucleic acid can be advantageously employed as alternatives to PCR.
  • the nucleotide sequences of the invention can likewise be employed m other procedures of amplification of a target nucleic acid, such as: the TAS technique (Transcription-based Amplification System), described by Kwoh et al. m 1989; the 3SR technique (Self-Sustained Sequence Replication), described by Guatelh et al. in 1990; the NASBA technique (Nucleic Acid Sequence Based Amplification), described by Kievitis et al. in 1991; the SDA technique (Strand Displacement Amplification) (Walker et al., 1992); and the TMA technique (Transcription Mediated Amplification).
  • the polynucleotides of the invention can also be employed m techniques of amplification or of modification of the nucleic acid serving as a probe, such as: the LCR technique (Ligase Cham Reaction), described by Landegren et al. in 1988 and improved by Barany et al. in 1991, which employs a thermostable ligase; the RCR technique (Repair Cham Reaction), described by Segev m 1992; the CPR technique (Cycling Probe Reaction), described by Duck et al. m 1990, the amplification technique with Q-beta rephcase, described by Miele et al. in 1983 and especially improved by Chu et al. in 1986, Lizardi et al.
  • LCR technique Liigase Cham Reaction
  • RCR technique Repair Cham Reaction
  • CPR technique Cycling Probe Reaction
  • the target polynucleotide to be detected is possibly an RNA, for example an mRNA
  • the target polynucleotide to be detected is possibly an RNA, for example an mRNA
  • the cDNA obtained will thus serve as a target for the p ⁇ mer(s) or the probe(s) employed in the amplification or detection procedure according to the invention.
  • the detection probe will be chosen in such a manner that it hybridizes with the target sequence or the amplicon generated from the target sequence.
  • a probe will advantageously have a sequence of at least 12 nucleotides, in particular of at least 20 nucleotides, and preferably of at least 100 nucleotides.
  • Embodiments of the invention also comprise the nucleotide sequences utihzable as a probe or primer according to the invention, characterized m that they are labeled with a radioactive compound or with a nonradioactive compound.
  • the unlabeled nucleotide sequences can be used directly as probes or primers, although the sequences are generally labeled with a radioactive isotope (32P, 35S, 3H, 1251) or with a nonradioactive molecule (biotm, acetylammofluorene, digoxigenm, 5-bromodeoxyu ⁇ dme, fluorescein) to obtain probes which are utihzable for numerous applications.
  • a radioactive isotope 32P, 35S, 3H, 1251
  • a nonradioactive molecule biotm, acetylammofluorene, digoxigenm, 5-bromodeoxyu ⁇ dme, fluorescein
  • the hybridization technique can be carried out in various manners (Matthews et al., 1988).
  • the most general method consists m immobilizing the nucleic acid extract of cells on a support (such as nitrocellulose, nylon, polystyrene) and in incubating, under well-defined conditions, the immobilized target nucleic acid with the probe. After hybridization, the excess of the probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, of the fluorescence or of the enzymatic activity linked to the probe).
  • the invention in various embodiments, likewise comprises the nucleotide sequences according to the invention, characterized in that they are immobilized on a support, covalently or noncovalently.
  • the latter can be used immobilized on a support and can thus serve to capture, by specific hybridization, the target nucleic acid obtained from the biological sample to be tested. If necessary, the solid support is separated from the sample and the hybridization complex formed between said capture probe and the target nucleic acid is then detected with the aid of a second probe, a so-called detection probe, labeled with an easily detectable element.
  • Another aspect of the present invention is a vector for the cloning and/or expression of a sequence, characterized in that it contains a nucleotide sequence according to the invention.
  • the vectors according to the invention characterized in that they contain the elements allowing the integration, expression and/or the secretion of said nucleotide sequences in a determined host cell, are likewise part of the invention.
  • the vector may then contain a promoter, signals of initiation and termination of translation, as well as appropriate regions of regulation of transcription.
  • the vector may be able to be maintained stably m the host cell and can optionally have particular signals specifying the secretion of the translated protein. These different elements may be chosen as a function of the host cell used.
  • the nucleotide sequences according to the invention may be inserted into autonomous replication vectors within the chosen host, or integrated vectors of the chosen host.
  • Such vectors will be prepared according to the methods currently used by a person skilled in the art, and it will be possible to introduce the clones resulting therefrom into an appropriate host by standard methods, such as, for example, lipofection, electroporation, and thermal shock.
  • the vectors according to the invention are, for example, vectors of plasmid or viral origin.
  • a vector for the expression of polypeptides of the invention is baculovirus. These vectors are useful for transforming host cells in order to clone or to express the nucleotide sequences of the invention.
  • the invention likewise comprises the host cells transformed by a vector according to the invention.
  • These cells can be obtained by the introduction into host cells of a nucleotide sequence inserted into a vector such as defined above, then the cultu ⁇ ng of said cells under conditions allowing the replication and/or expression of the transfected nucleotide sequence.
  • the host cell can be selected from prokaryotic or eukaryotic systems, such as, for example, bacterial cells (01ms and Lee, 1993), but likewise yeast cells (Buckholz, 1993), as well as plant cells, such as Arabidopsis sp., and animal cells, in particular the cultures of mammalian cells (Edwards and
  • Aruffo 1993
  • Chinese hamster ovary (CHO) cells but likewise the cells of insects in which it is possible to use procedures employing baculoviruses, for example Sf9 insect cells (Luckow, 1993)
  • Embodiments of the invention likewise relate to organisms comprising one of said transformed cells according to the invention.
  • transgenic organisms according to the invention expressing one or more of the genes of Alicyclobacillus acidocaldanus , or part of the genes, may be carried out m, for example, rats, mice, or rabbits according to methods well known to a person skilled in the art, such as by viral or nonviral transfections It will be possible to obtain the transgenic organisms expressing one or more of said genes by transfection of multiple copies of said genes under the control of a strong promoter of ubiquitous nature, or selective for one type of tissue It will likewise be possible to obtain the transgenic organisms by homologous recombination m embryonic cell strains, transfer of these cell strains to embryos, selection of the affected chimeras at the level of the reproductive lines, and growth of said chimeras
  • transformed cells as well as the transgenic organisms according to the invention are utihzable in procedures for preparation of recombinant polypeptides.
  • transformation relate to the introduction of nucleic acids into a cell, whether prokaryotic or eukaryotic. Further, “transformation” and “transformed,” as used herein, need not relate to growth control or growth deregulation.
  • the preparation procedures employing a vector, and/or a cell transformed by said vector and/or a transgenic orgamsm comprising one of said transformed cells, containing a nucleotide sequence according to the invention coding for a polypeptide of Alicyclobacillus acidocaldanus .
  • a variant according to an embodiment of the invention may consist of producing a recombinant polypeptide fused to a "carrier" protein (chimeric protein).
  • a carrier protein chimeric protein
  • an embodiment of the invention relates to a procedure for preparation of a polypeptide of the invention comprising the following steps: a) culture of transformed cells under conditions allowing the expression of a recombinant polypeptide of nucleotide sequence according to the invention; b) if need be, recovery of said recombinant polypeptide.
  • An embodiment of the invention also relates to a polypeptide which is capable of being obtained by a procedure of the invention such as described previously.
  • the invention also comprises a procedure for preparation of a synthetic polypeptide, characterized in that it uses a sequence of ammo acids of polypeptides according to the invention
  • a further embodiment of the invention likewise relates to a synthetic polypeptide obtained by a procedure according to the invention.
  • polypeptides according to embodiments of the invention can likewise be prepared by techniques which are conventional m the field of the synthesis of peptides. This synthesis can be carried out m a homogeneous solution or in a solid phase
  • This method of synthesis comprises successively condensing, two by two, the successive ammo acids m the order required, or m condensing ammo acids and fragments formed previously and already containing several ammo acids m the appropriate order, or alternatively several fragments previously prepared in this way, it being understood that it will be necessary to protect beforehand all the reactive functions carried by these ammo acids or fragments, with the exception of amine functions of one and carboxyls of the other or vice-versa, which must normally be involved m the formation of peptide bonds, especially after activation of the carboxyl function, according to the methods well known in the synthesis of peptides
  • the invention additionally relates to hybrid polypeptides having at least one polypeptide according to the invention, and a sequence of a polypeptide capable of inducing an immune response in man or animals.
  • the antigenic determinant is such that it is capable of inducing a humoral and/or cellular response.
  • Such a determinant will be possible for such a determinant to comprise a polypeptide according to the invention in glycosylated, pegylated, and/or otherwise post-translationally modified form used with a view to obtaining immunogenic compositions capable of inducing the synthesis of antibodies directed against multiple epitopes.
  • hybrid molecules can be formed, in part, of a polypeptide carrier molecule or of fragments thereof according to the invention, associated with a possibly immunogenic part, in particular an epitope of the diphtheria toxin, the tetanus toxm, a surface antigen of the hepatitis B virus (French Patent 7921811), the VPl antigen of the poliomyelitis virus or any other viral or bacterial toxm or antigen.
  • the procedures for synthesis of hybrid molecules encompass the methods used in genetic engineering for constructing hybrid nucleotide sequences coding for the polypeptide sequences sought. It will be possible, for example, to refer advantageously to the technique for obtainment of genes coding for fusion proteins described by Mmton m 1984.
  • hybrid nucleotide sequences coding for a hybrid polypeptide, as well as the hybrid polypeptides according to the invention are so characterized m that they are recombinant polypeptides obtained by the expression of said hybrid nucleotide sequences, which are likewise part of the invention.
  • the invention likewise comprises the vectors characterized in that they contain one of said hybrid nucleotide sequences.
  • the host cells transformed by said vectors, the transgenic organisms comprising one of said transformed cells as well as the procedures for preparation of recombinant polypeptides using said vectors, said transformed cells and/or said transgenic organisms are, of course, likewise part of the invention.
  • polypeptides according to the invention, the antibodies according to the invention described below and the nucleotide sequences according to the invention can advantageously be employed in procedures for the detection and/or identification of Ahcyclobacillus acidocaldanus, m a sample capable of containing them.
  • These procedures, according to the specificity of the polypeptides, the antibodies and the nucleotide sequences according to the invention which will be used, will in particular be able to detect and/or to identify Alicyclobacillus acidocaldarius
  • polypeptides according to the invention can advantageously be employed in a procedure for the detection and/or the identification of Alicyclobacillus acidocaldarius m a sample capable of containing them, characterized in that it comprises the following steps: a) contacting of this sample with a polypeptide or one of its fragments according to the invention (under conditions allowing an immunological reaction between said polypeptide and the antibodies possibly present in the biological sample), b) demonstration of the antigen-antibody complexes possibly formed
  • Any conventional procedure can be employed for carrying out such a detection of the antigen-antibody complexes possibly formed.
  • a method according to various embodiments brings into play lmmunoenzymatic processes according to the ELISA technique, by immunofluorescence, or radioimmunological processes (RIA) or their equivalent.
  • the invention likewise relates to the polypeptides according to the invention, labeled with the aid of an adequate label, such as, of the enzymatic, fluorescent or radioactive type.
  • Such methods comprise, for example, the following acts deposition of determined quantities of a polypeptide composition according to the invention m the wells of a microtiter plate, introduction into said wells of increasing dilutions of serum, or of a biological sample other than that defined previously, having to be analyzed, incubation of the microtiter plate, introduction into the wells of the microtiter plate of labeled antibodies directed against pig immunoglobulins, the labeling of these antibodies having been carried out with the aid of an enzyme selected from those which are capable of hydrolyzing a substrate by modifying the absorption of the radiation of the latter, at least at a determined wavelength, for example at 550 nm, detection, by comparison with a control test, of the quantity of hydrolyzed substrate.
  • polypeptides according to embodiments of the invention enable monoclonal or polyclonal antibodies to be prepared which are characterized in that they specifically recognize the polypeptides according to the invention It will advantageously be possible to prepare the monoclonal antibodies from hyb ⁇ domas according to the technique described by Kohler and Milstem m 1975. It will be possible to prepare the polyclonal antibodies, for example, by immunization of an animal, in particular a mouse, with a polypeptide or a DNA, according to the invention, associated with an adjuvant of the immune response, and then purification of the specific antibodies contained m the serum of the immunized animals on an affinity column on which the polypeptide which has served as an antigen has previously been immobilized.
  • polyclonal antibodies according to the invention can also be prepared by purification, on an affinity column on which a polypeptide according to the invention has previously been immobilized, of the antibodies contained in the serum of an animal immunologically challenged by Alicyclobacillus acidocaldarius, or a polypeptide or fragment according to the invention.
  • the invention in various embodiments, likewise relates to mono- or polyclonal antibodies or their fragments, or chimeric antibodies, characterized m that they are capable of specifically recognizing a polypeptide according to the invention
  • the antibodies of embodiments of the invention may be labeled in the same manner as described previously for the nucleic probes of the invention, such as a labeling of enzymatic, fluorescent or radioactive type.
  • An embodiment of the invention is additionally directed at a procedure for the detection and/or identification of Alicyclobacillus acidocaldarius in a sample, characterized in that it comprises the following steps: a) contacting of the sample with a mono- or polyclonal antibody according to the invention (under conditions allowing an immunological reaction between said antibodies and the polypeptides of Alicyclobacillus acidocaldarius possibly present in the biological sample); b) demonstration of the antigen-antibody complex possibly formed.
  • the present invention likewise relates to a procedure for the detection and/or the identification of Alicyclobacillus acidocaldarius in a sample, characterized in that it employs a nucleotide sequence according to the invention.
  • the invention relates to a procedure for the detection and/or the identification of Alicyclobacillus acidocaldarius in a sample, characterized in that it contains the following steps, a) if need be, isolation of the DNA from the sample to be analyzed; b) specific amplification of the DNA of the sample with the aid of at least one primer, or a pair of primers, according to the invention; c) demonstration of the amplification products.
  • nucleic probe can be detected, for example, by the technique of molecular hybridization utilizing a nucleic probe according to the invention.
  • This probe will advantageously be labeled with a nonradioactive (cold probe) or radioactive isotope.
  • DNA of the biological sample or "DNA contained m the biological sample” will be understood as meaning either the DNA present in the biological sample considered, or possibly the cDNA obtained after the action of an enzyme of reverse transcriptase type on the RNA present in said biological sample.
  • a further embodiment of the invention comprises a method, characterized m that it comprises the following steps: a) contacting of a nucleotide probe according to the invention with a biological sample, the DNA contained in the biological sample having, if need be, previously been made accessible to hybridization under conditions allowing the hybridization of the probe with the DNA of the sample, b) demonstration of the hybrid formed between the nucleotide probe and the DNA of the biological sample.
  • the present invention also relates to a procedure according to embodiments of the invention, characterized in that it comprises the following steps: a) contacting of a nucleotide probe immobilized on a support according to the invention with a biological sample, the DNA of the sample having, if need be, previously been made accessible to hybridization, under conditions allowing the hybridization of the probe with the DNA of the sample; b) contacting of the hybrid formed between the nucleotide probe immobilized on a support and the DNA contained m the biological sample, if need be after elimination of the DNA of the biological sample which has not hybridized with the probe, with a nucleotide probe labeled according to the invention; c) demonstration of the novel hybrid formed in step b).
  • this is characterized m that, prior to step a), the DNA of the biological sample is first amplified with the aid of at least one primer according to the invention.
  • Embodiments of methods include glycosylating or post-translationally modifying a first polypeptide using a second polypeptide selected from the group consisting of a polypeptide having at least 90% sequence identity to SEQ DD NOS: 1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338.
  • Further embodiments of methods include methods of modulating protein stability, solubility, degradation, activity profile, and/or externahzation of a first polypeptide, the methods comprising glycosylating or post-translationally modifying the first polypeptide via a second polypeptide selected from the group consisting of a polypeptide having at least 90% sequence identity to SEQ DD NOS: 1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338.
  • inventions of methods include placing a cell producing or encoding a recombinant, purified, and/or isolated nucleotide sequence comprising a nucleotide sequence selected from the group consisting of a nucleotide sequences having at least 90% sequence identity to at least one of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 and/or a recombinant, purified, and/or isolated polypeptide selected from the group consisting of a polypeptide having at least 90% sequence identity to at least one of the sequences of SEQ ID NOS: 1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and
  • the present invention provides cells that have been genetically manipulated to have an altered capacity to produce expressed proteins.
  • the present invention relates to Gram-positive microorganisms, such as Bacillus species having enhanced expression of a protein of interest, wherein one or more chromosomal genes have been inactivated, and/or wherein one or more chromosomal genes have been deleted from the Bacillus chromosome.
  • one or more indigenous chromosomal regions have been deleted from a corresponding wild-type Bacillus host chromosome.
  • the Bacillus is an Alicyclobacillus sp. or Alicyclobacillus acidocaldanus .
  • methods of glycosylating and/or post-translationally modifying a polypeptide at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/or at a pH at, below, and/or above 8, 7, 6, 5, 4, 3, 2, 1, and/or 0 via a recombinant, purified, and/or isolated nucleotide sequence comprising a nucleotide sequence selected from the group consisting of a nucleotide sequences having at least 90% sequence identity to at least one of the sequences of SEQ ED NOS.2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 and/or a recombinant, purified, and/or isolated polypeptide selected from the group consisting of a polypeptide having
  • any one of the isolated and/or purified polypeptides according to the invention may be enzymatically or functionally active at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/or may be enzymatically or functionally active at a pH at, below, and/or above 8, 7, 6, 5, 4, 3, 2, 1, and/or 0.
  • glycosylation, pegylation, and/or other post-translational modification may be required for the isolated and/or purified polypeptides according to the invention to be enzymatically or functionally active at pH at or below 8, 7, 6, 5, 4, 3, 2, 1 , and/or 0 or at a temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius.
  • Example 1 Glycosylation Using Nucleotide and Amino Acid Sequences from Alicyclobacillus acidocaldarius
  • SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 are a nucleotide sequences isolated from Alicyclobacillus acidocaldarius and coding for the polypeptides of SEQ ID NOS: 1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338, respectively.
  • the nucleotide sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 are placed into expression vectors using techniques standard in the art.
  • the vectors are then provided to cells such as bacteria cells or eukaryotic cells such as Sf9 cells or CHO cells.
  • the vectors comprising SEQ ID NOS-2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 produce the polypeptides of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338.
  • polypeptides of SEQ ID NOS: 1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338 are then isolated and/or purified.
  • the isolated and/or purified polypeptides of SEQ ID NOS: 1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338 are then each demonstrated to have one or more of the activities provided m Table 1 or some other activity.
  • the isolated and/or purified polypeptides of SEQ ID NOS: 1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290 are demonstrated to have activity in glycosylating other proteins in conjunction with other proteins or cellular components.
  • Example 2 Modulating Protein Stability, Solubility, Degradation, Activity Profile, and/or Externalization of a First Polypeptide Using Nucleotide and Amino Acid Sequences from Alicyclobacillus acidocaldarius
  • Example 1 The polypeptides and nucleotide sequence of Example 1 are used to post-translationally modify one or more other proteins through glycosylation or other post-translational modification.
  • the modified proteins are demonstrated to have altered protein stability, solubility, degradation, activity profile, and/or externalization m comparison to non-modified proteins of the same or similar ammo acid sequence.
  • Polypeptide RAAC02676 (SEQ ID NO: 307) was obtained via the following protocol. Alicyclobacillus acidocaldarius was cultured on wheat arabinoxylan and harvested after three days. The culture was centrifuged to remove cells and the resulting supernatant was filtered with a 0.22 micron filter to remove any remaining debris. The filtered supernatant was concentrated to approximately 1 mL by ultrafiltration through a 10,000 Da molecular weight cutoff membrane. The resulting concentrated filtered supernatant was additionally purified by trapping proteins on a cation exchange column, elutmg them with a salt gradient, reloading them on a second cation exchange column and elutmg with a second salt gradient.
  • Alicyclobacillus acidocaldarius was cultured on wheat arabmoxylan and harvested after three days. The culture was centrifuged to remove cells and the resulting supernatant was filtered with a 0.22 micron filter to remove any remaining debris. The filtered supernatant was concentrated to approximately 1 mL by ultrafiltration through a 10,000 Da molecular weight cutoff membrane Several lanes of this concentrated material were run on a 12% SDS-PAGE gel along with a positive and negative control that are known glycosylated and non-glycosylated proteins using standard protocols.
  • the gel was cut in half vertically and one half stained using SIMPLY BLUETM SAFE STAIN and the other half using a glycoprotein detection kit from Sigma
  • the Alicyclobacillus acidocaldarius protein lanes revealed a band at approximately 120 kDa on the SIMPLY BLUETM stained gel which is the expected weight of one extracelluar protein of Alicyclobacillus acidocaldarius.
  • the same position on the glycoprotein stained gel showed pmk bands indicating a positive result for a glycosylated protein.
  • Example 5 Chemical Glycosylation of SEQ ID NO:307 Results in Greater Xylanase activity.
  • SEQ ID NO:307 was expressed in E coli and purified using a Co-resin system.
  • the nucleic acid encoding SEQ ID NO:307 was altered for optimal codon usage in E coli.
  • the purified SEQ ID NO.307 was chemically glycosylated using a mono(lactosylamido) mono(succmimidyl) suberate. This chemically reacts with amine groups on proteins to form an N-lmked modification with a terminal lactose on the protein
  • the glycosylation of the purified SEQ ID NO: 307 was verified using a glycosylation stain.
  • glycosylated and un-glycosylated SEQ ID NO:307 was tested for xylanase activity at pHs 2, 3.5, and 5
  • the glycosylated SEQ ID NO:307 had higher levels of activity at pH 2 and 3.5 that the ungylcosylated from of SEQ ID NO:307.
  • Un-glycosylated SEQ ID NO:307 displayed reduced solubility at pH 2 and 3.5.
  • Example 6 Expression of SEQ ID NO:307 in Pichiapastoris.
  • Nucleic acid encoding SEQ ID NO:307 was inserted into Pichia pastons and several clones had significant xylanase and cellulose activities at pH 3.5, but not at pH 2. The nucleic acid encoding SEQ ID NO:307 was altered for optimal codon usage in Pichiapastoris.
  • Example 7 Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307 Expressed in Alicyclobacillus acidocaldarius and E. coli.
  • Nucleic acid encoding SEQ ID NO:307 was inserted into E coli with an N-termmal His tag and the resultant protein purified The nucleic acid encoding SEQ ID NO.307 was altered for optimal codon usage m E coli
  • the resulting purified protein had no glycosylation Protein of SEQ ID NO 307 was also purified from Ahcyclobacillus acidocaldarius
  • the purified protein from Alicyclobacillus acidocaldarius was glycosylated as normal for protein produced from this organism
  • the purified proteins were tested for xylanase activity using wheat arabmoxylan (WAX) The results of the comparison are presented m FIG 23 There was no data available for enzyme purified from Ahcyclobacillus acidocaldarius at pH 5 5 As can be seen in FIG
  • FIG 24 There was no data available for enzyme purified from Ahcyclobacillus acidocaldarius at pH 5 5 As can be seen in FIG 24, the Ahcyclobacillus acidocaldarius purified enzyme (black bars) had significantly less cellulase activity at pH 3 5 than the E coli purified enzyme (white bars)
  • FIG 25 presents the ratio of cellulose/xylanase activity for the data presented in FIGs 23 and 24
  • enzyme purified from Ahcyclobacillus acidocaldarius had predominantly xylanase activity at pH 2 and 8O 0 C, while having predominantly cellulose activity at all other conditions tested (black bars)
  • the enzyme purified from E coli had predominantly cellulose activity at all conditions tested (white bars)
  • This data confirms that the glycosylation state of SEQ ID NO 307 varies the relative xylanase and cellulose activities of SEQ ID NO 307
  • Example 8 Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307 Expressed in
  • Nucleic acid encoding SEQ ID NO 307 was inserted into Pichia pastoris and the resultant protein purified The nucleic acid encoding SEQ ID NO 307 was altered for optimal codon usage in Pichia pastoris The purified protein from Pichia pastoris was glycosylated as normal for protein produced from this organism Nucleic acid encoding SEQ ID NO 307 was inserted into E coli with an N-terminal His tag and the resultant protein purified The nucleic acid encoding SEQ ID NO 307 was altered for optimal codon usage in E coli The resulting purified protein had no glycosylation The purified proteins were tested for xylanase activity using wheat arabmoxylan The results of the comparison are presented in FIG 26 As can be seen in FIG 26, the Pichia pastoris purified enzyme (black bars) had significantly more xylanase activity at all conditions other than pH 3 5 and 8O 0 C than the E coli purified enzyme (white bars)
  • FIG. 27 presents the ratio of cellulose/xylanase activity for the data presented in FIGs. 26 and 27.
  • Example 9 Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307 Expressed in Alicyclobacillus acidocaldarius and Pichia pastoris.
  • Nucleic acid encoding SEQ ID NO:307 was inserted into Pichia pastoris and the resultant protein purified The nucleic acid encoding SEQ ID NO.307 was altered for optimal codon usage m Pichia pastoris The purified protein from Pichia pastoris was glycosylated as normal for protein produced from this organism. Protein of SEQ ID NO:307 was also purified from Alicyclobacillus acidocaldarius. The purified protein from Alicyclobacillus acidocaldarius was glycosylated as normal for protein produced from this organism. The purified proteins were tested for xylanase activity using wheat arabmoxylan. The results of the comparison are presented m FIG.
  • the purified proteins were also tested for cellulase activity using carboxymethyl cellulose. The results of the comparison are presented m FIG. 30. There was no data available for enzyme purified from Alicyclobacillus acidocaldarius at pH 5 5 As can be seen in FIG. 30, the Alicyclobacillus acidocaldarius purified enzyme (black bars) had significantly less cellulase activity at all conditions other than pH 3.5 and 8O 0 C than the Pichia pastoris purified enzyme (white bars).
  • FIG. 31 presents the ratio of cellulose/xylanase activity for the data presented in FIGs. 29 and 30.
  • enzyme purified from Alicyclobacillus acidocaldarius had predominantly xylanase activity at pH 2 and 8O 0 C, while having predominantly cellulose activity at all other conditions tested (black bars).
  • the enzyme purified from Pichia pastoris also had predominantly xylanase activity at pH 2 and 8O 0 C, while having predominantly cellulase activity at all other conditions tested (white bars).
  • This data confirms that the glycosylation state of SEQ ID NO.307 varies the relative xylanase and cellulose activities of SEQ ID NO:307.
  • Example 10 Comparison of Xylanase and Cellulase Activity of Truncated SEQ ID NO:307 Expressed in E. coli and Pichia pastoris.
  • Nucleic acid encoding a truncated SEQ ID NO:307 without the 203 C-termmal ammo acids was inserted into Pichia pastoris with a C-termmal His tag and the resultant protein purified.
  • the nucleic acid encoding SEQ ID NO 307 was altered for optimal codon usage in Pichia pastoris .
  • the purified protein from Pichia pastons was glycosylated as normal for protein produced from this organism
  • Nucleic acid encoding a truncated SEQ ID NO 307 without the 203 C-termmal ammo acids and N-terminal 33 leader sequence ammo acids were inserted into E coli with an and the resultant protein purified
  • the nucleic acid encoding SEQ ID NO 307 was altered for optimal codon usage m E coli.
  • the resulting purified protein had no glycosylation.
  • the purified proteins were tested for xylanase activity using wheat arabmoxylan The results of the comparison are presented in FIG. 32 As can be seen in FIG.
  • FIG. 34 presents the ratio of cellulose/xylanase activity for the data presented in FIGs. 32 and 33. As can be seen therein, both purified enzymes had predominantly cellulase activity at all conditions.
  • Example 11 Activity of SEQ ID NO:338 Expressed in E. coli and Pichia pastons.
  • RAAC00568 (SEQ ID NO'338) was expressed in both E coli and Pichia pastons Codon usage in the encoding DNA was optimized for the particular organism.
  • E coli the enzyme expressed primarily as inclusion bodies
  • IMAC immobilized metal affinity chromatography
  • Example 12 Activity of SEQ ID NO:337 Expressed in E. coli and Pichia pastons.
  • RAAC00307 SEQ ID NO 337) was expressed in both E coli and Pichia pastons Codon usage m the encoding DNA was optimized for the particular organism
  • the E coli produced enzyme was purified via a His tag by immobilized metal affinity chromatography (EvIAC). This purified enzyme was tested for both alpha arabmofuranosidase (AFS) activity, as well as beta xylosidase (BXYL) activity The results of this testing are presented in FIGs. 35-37.
  • a soluble enzyme was expressed and purified by IMAC.
  • the optimum conditions for the E coli expressed AFS were pH 6.0 and 7O 0 C, while the optimum conditions for BXYL were pH 5.0 and between 70 0 C and 80 0 C (FIGs. 35 and 36)
  • the enzyme did not have activity at pH 2.0 for either AFS or BXYL activities (FIGs. 35 and 36).
  • the Pichia pastons expressed enzyme was screened at pH 2, 3.5, and 5.5 and at 60 0 C and 80 0 C. The results are presented in FIG. 37. The glycosylation modifications made by Pichia pastons during expression appeared to have shifted the activity to a lower pH.
  • the BXYL in the Pichia pastons expressed enzyme was 10 8 U/mg at pH 3.0, 6O 0 C, while it was only 1.1 U/mg for the E coli expressed enzyme
  • the Pichia pastons expressed enzyme also had some activity at pH 2.0 while there was no activity for the E coli expressed enzyme

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Abstract

Les modes de réalisation selon l'invention comprennent des procédés pour altérer l'activité enzymatique ou la solubilité d'une enzyme extrêmophile ou pour modifier après traduction une protéine d'intérêt au moyen de glycosyltransférases isolées ou partiellement purifiées et/ou de protéines de modification post-traductionnelles, d'extraits de cellules comprenant des glycosyltransférases et/ou des protéines de modification post-traductionnelles et/ou dans des cellules comprenant une ou plusieurs glycosyltransférases et/ou protéines de modification post-traductionnelles.
EP10746882A 2009-02-26 2010-02-26 Altération et modulation de l'activité protéique en faisant varier la modification post-traductionnelle Withdrawn EP2401289A4 (fr)

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PCT/US2009/035307 WO2010051049A2 (fr) 2008-02-27 2009-02-26 Gènes et enzymes de glycosylation thermophiles et thermoacidophiles provenant d'alicyclobacillus acidocaldarius et d'organismes apparentés, et procédés
US12/380,450 US9234228B2 (en) 2008-02-27 2009-02-26 Thermophilic and thermoacidophilic glycosylation genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods
US12/655,993 US8969033B2 (en) 2005-11-02 2010-01-12 Alteration and modulation of protein activity by varying post-translational modification
PCT/US2010/025521 WO2010099394A1 (fr) 2009-02-26 2010-02-26 Altération et modulation de l'activité protéique en faisant varier la modification post-traductionnelle alteration and modulation of protein activity by varying post-translational modification

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BIRGIT SCHWERMANN ET AL: "Purification, Properties and Structural Aspects of a Thermoacidophilic alpha-Amylase from Alicyclobacillus Acidocaldarius Atcc 27009. Insight into Acidostability of Proteins", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 226, no. 3, 1 December 1994 (1994-12-01), pages 981-991, XP55007469, ISSN: 0014-2956, DOI: 10.1111/j.1432-1033.1994.00981.x *
ECKERT K ET AL: "A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases", EUROPEAN JOURNAL OF BIOCHEMISTRY, BLACKWELL PUBLISHING, BERLIN, DE, vol. 270, no. 17, 1 September 2003 (2003-09-01), pages 3593-3602, XP002297030, ISSN: 0014-2956, DOI: 10.1046/J.1432-1033.2003.03744.X *
See also references of WO2010099394A1 *

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AU2010217847A1 (en) 2011-10-27
CN102317309A (zh) 2012-01-11
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BRPI1008486A2 (pt) 2018-01-16
WO2010099394A1 (fr) 2010-09-02
CA2752175A1 (fr) 2010-09-02

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