ixs'raaousar or mcnooacamsns
`
`30‘)
`
`monly used for this antibiotic, but it. is also known by the more chemical
`name chlm‘alupheuicol. The compound contains chlorine and is pro-
`duced by a streptomyces, hence it
`is quite apparent how the word
`Chloroniycctin came to be devised. The Inierlmrganisln producing
`Chlorolnycetin is called Streptoaiyccs vcncznetm’, an obviously poor name
`since it denotes geographical origin and not an inherent characteristic.
`The microorganism was obtained from a sample of soil from Venezuela.
`It has also been found in soils from Illinois and Japan and is probably
`widely distributed in nature.
`It is produced industrially both by fermentation and by synthesis.
`To date it is the only commercial antibiotic that is produced synthetically
`as well as by fermentation.
`The most distinctive feature about the chemical structure of Chloro-
`
`myeetin is the nitro group. Few organic compounds in nature contain
`:1 nitro group.
`It also contains chlorine, which, though not common,
`occurs in aureomycin and a. number of mold products, e.g., erdiu. The
`presence of an amide linkage relates it to peptides and explains its
`hydrolysis by enzymes found in cells of Proteus vulgaris. Chleromyectin
`contains no acidic or basic groups, hence it does not form salts.
`It is
`a neutral compound that crystallizes as colorless needles or elongated
`plates.
`Chloromyectin is relatively inactive against gram-positive bacteria,
`but is very potent against the gram-negative bacteria associated with
`intestinal diseases such as typhoid fever and dysentery.
`It is active
`against the same rickcttsial and viral diseases as anrccmycin and terra-
`mycin.
`Chlorornyeetin is relatively stable to acids and alkali, is rapidly ab-
`sorbed from the gastro-intcstinal tract, and hence is usually given by
`mouth. Liver and kidney tissues reduce the -—N02 group to an —NH2
`group. About 90 per cent of the administered dose is excreted as an
`inactive compound and 10 per cent as unchanged Chloromycetin in 24
`hours.
`
`Terramycin, aureomycin, and Chloromycetin are alike in bacterial spec-
`trum and appear to be similar in their mode of action;
`they interfere
`strongly with protein synthesis but are much less effective in stopping
`nucleic acid synthesis. A more specific reaction in protein metabolism
`has been observed for Chloromycctin.
`It acts as an antagonist against
`phenylalanine, but the antagonism is noncompetitive. Only low con-
`centrations of Chloromycetin can be overcome by addition of phenyl-
`alanine. At higher concentrations its effect cannot be reversed by
`adding more phenylalanine. This makes Chloromycctin a particularly
`effective antimetabolite since the inhibited organism cannot counteract
`the Chloromycetin by producing more phenylalanine.
`Bacitrncin, Polymyxin, and Tyrothrictn. These three antibiotics are
`all polypeptides and are produced respectively by Bacillus lichcniforinis,
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 373
`Petitioner Microsoft Corporation - EX. 1032, p. 373
`
`

`

`3?”
`
`METABOLISM OF MlCltOOltGANIa‘MS
`
`Bacillus polymer-a, and Bacillus turn-1's. The amino acid content of
`baeitraein is given in Table. 5—4 and presents no unusual features. There
`are several polymyxins, A, 15, C, D, and 1'). and each one Contains large
`amounts {mm-e than 50 per cent] of the unusual amino acid, L-u,y-(ll-
`aminolmtyrie acid. A second distinctive feature is the presence in the
`molecule of a nine-carbon fatty acid, probably ti—methyloctanoie acid.
`Tyl'otln'icin is not a lannogentmus substance but consists mainly of
`gramieidin, a neutral cyclic polypeptide.
`tin hydrolysis gramicidin gives
`live amino acids and cthanolamine. Nll2CllgCllgOH.
`
`Baeitracin resembles penicillin in being most active against grain-posi-
`tive bacteria.
`It causes kidney (Inning! (evidenced by albumin in the
`urinel. Because of this toxic elleet, its use is limited to combating local
`infections.
`
`The polymyxins are very potent against gram-negative bacteria, in-
`cluding the very resistant- Proteus and I’sendomoaas bacteria.
`l'nfortu-
`nately, the polymyxins cause more or less kidney dauiage, so their use.
`will probably he limited to refractory infections that do not respond to
`other treatments.
`
`'l‘yrotln'icin is the oldest commercial antibiotic, but probably the least
`used of
`the commercial products.
`It acts on grain-positive bacteria
`but is not suitable for injection or oral administration.
`It is used only
`for topical purposes, that is, where it can be brought into direct contact
`with the infecting organism, 8.9., surface abscesses.
`Because of the millions of gallons of media that must be used for the
`
`the fermentation is done in deep tanks of
`produetion of antibiotics,
`5—1:},000 gallon capacity. Sterile air is forced through the medium at
`the rate of about one-half volume of air per volume of medium per minute.
`The medium is also stirred vigorously to increase aeration. Deep tank
`fermentation was first developed for the production of penicillin and
`later applied to the production of other antibiotics and vitamins.
`Vitamin 1322 is produced simultaneously with streptomycin, aurcomycin,
`and terrainycin. Hence producers of
`these antibiotics obtain a second
`valuable product in the same lermcntation. Vitamin B13 is also pro-
`duced commercially by a mixed aerobacter-proteus type of fermentation.
`The vitamin is formed by many different kinds of bacteria and molds.
`Yeasts produce little, if any, of it.
`
`By yeast
`
`Baker’s yeast can grow under both aerobic and anaerobic conditions.
`If an abundance of air and a low concentration of sugar (3.9., 0.5 per
`cent) are supplied, the end products of metabolism are mainly carbon
`dioxide and yeast (50 per cent of the weight of sugar is obtained as dry
`weight of yeast} ; there is practically no alcohol. See Fig. 14—2.
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 374
`Petitioner Microsoft Corporation - EX. 1032, p. 374
`
`

`

`METABOLISM or ancnooncamsns
`
`371
`
`If the sugar content of the medium is raised to 5 per cent, the yield of
`yeast‘is markedly reduced, and much alcohol is formed.
`In other words,
`an anaerobic type of metabolism comes to the front, although an abun-
`dance of air is present. The explanation for this is that ordinary yeast
`
`
`
`Courtesy of Dr. Charles N. Frey, Fleischmann Laboratories.
`Fig. 1+2. Budding yeast cells.
`
`If
`has a weak aerobic enzyme system and a strong anaerobic system.
`more sugar is present than can be metabolized aerobically, the anaerobic
`system begins to operate.
`
`By molds
`
`Molds cannot grow in the absence of air; carbon dioxide and water are
`the usual products of metabolism. However, many species convert a
`large percentage of the sugar in the medium into other carbon products.
`Examples of products that make up more than 40 per cent by weight of
`the sugar consumed and the molds producing them are given in Table
`14—1. The highest yields of compounds in the table are for gluconic
`acid. Actually over 100 per cent has been obtained,
`if allowance is
`made for glucose going to lnycelium. This yield is possible since one
`oxygen is added per mole of glucose, which amounts to 196 g. of gluconic
`acid from 180 .g. of glucose, or 109 per cent.
`Citric Acid. This acid has been obtained many times in yields of
`80—90 per cent, but the usual yields, without allowing for glucose going
`to mycelium, are around 70 per cent. Very special conditions have to
`he maintained to keep the mold growth low. Such conditions are low
`concentrations of metals, Particularly manganese, high concentrations
`of sugar, and low pH in the medium. An explanation for the effect of
`manganese is that this nietal serves as a cofactor for some enzyme system
`that functions in the breakdown and oxidation of citric acid.
`If there
`
`is a deficiency of manganese, the enzyme cannot operate, and then citric
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 375
`Petitioner Microsoft Corporation - EX. 1032, p. 375
`
`

`

`372
`
`METABOLISM or MICROORGANISMS
`
`acid accumulates. Associated with citric acid production is sparse spore
`formation. The mycclium presents a beaded or braided appearance, and
`this appearance supports the idea that accumulation of citric acid is an
`abnormal type of metabolism.
`One of the theoretical problems connected with citric acid production
`is how to harmonize the high yield with the conventional system of inter-
`mediary metabolism that operates in yeast and animals (1). 331). This
`system would require three 2-carbon pieces, or one and one-half moles
`of glucose per mole of citric acid. On a percent-age basis only 71 per
`cent of citric acid could be obtained. Many theories have been proposed
`to account for higher yields. The current and best explanation is the
`uptake of carbon dioxide by pyruric acid to form oxalacetic acid {Wood-
`Werkman reaction) and condensation of this acid with acetic acid to form
`citric acid:
`
`002 + CH3 - CO - COOH——> HOOC - CH3 ' CO ' COOH
`
`HOOC - CH3 ° CO - COOH + CH3 - COOH—> HOOC' CH3 - C(OH) ' COOH
`
`lC
`
`H2 - COOH
`
`Uptake of isotopic carbon dioxide has been shown to take place, but
`whether this is adequate to account for the high yields is still not certain.
`The mechanism of citric acid formation is under active investigation in
`both animal and mold studies, and many of the questions now unanswered
`should be cleared up in the near future.
`Penicillin. Besides the major products mentioned in Table 14—1,
`molds produce hundreds of other compounds in amounts from a fraction
`of a per cent to 10 per cent. The best known of these products is peni-
`cillin. Approximately 20 tons of penicillin are produced every month
`in the United States alone. Yields of 1 g. of penicillin per liter of medium
`are usual, and about 70 per cent of the penicillin in the broth is recovered
`as finished product. A typical medium is:
`lactose, 3 per cent; corn
`steep solids {the concentrate of the water extract obtained in the indus—
`trial manufacture ef starch, gluten, and other com products), 3 per cent;
`calcium carbonate, 0.5 per cent; sodium sulfate, 0.1 per cent; phenyl—
`acetic acid, 0.3 per cent. The medium is sterilized, inoculated with a
`high-yielding strain of Penicillin-m chrysogcnnm, and aerated and stirred
`vigorously during the fermentation period, 34 days. The penicillin is
`extracted from the acidified broth with now] acetate, transferred to a
`buffer, purified, and finally crystallized as the sodium, potassium, or
`procaine salt.
`Since penicillin is an acid, many different salts can be
`made, but the above three are those in commercial use. More than a
`dozen companies are producing penicillin in this country, and the market
`value of the yearly product has been more than 100 million dollars for
`several years. See Fig. 14—3.
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 376
`Petitioner Microsoft Corporation - EX. 1032, p. 376
`
`

`

`
`
`Courtesy of Myron I’. Backus.
`(:1)
`
`Cuurlusy of Ahhott Laboratories.
`(C)
`
`the high-yielding
`(:1) Colony of
`Fig. 14-3. Production of penicillin.
`penicillin mold, Pcnicillium clifysflgrmmn. Wis. Q1?6. This culture was
`used industrially for many ymrs to produce penir'illin.
`(h) Fermmitulion
`tanks of 6000 gal. cupnuii)’ used in the submevgml production of pmiioillin.
`(v) Crystals of the sodium salt. of penicillin.
`373
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 377
`Petitioner Microsoft Corporation - EX. 1032, p. 377
`
`

`

`374
`
`METABOLISM OF MICROORGANISMS
`
`The yield of penicillin has been increased about a thousandfold over
`that obtained in the beginning of its production. The high yield has
`been attained largely by selection of better cultures. The best of these
`have been obtained by treating the mold spores with x-ray, ultraviolet
`light, or N-mustard gas to give high-yielding mutants.
`Other factors in obtaining high yields have been the use of better
`media and better methods of aeration and agitation of the media. The
`
`improvement in penicillin yields is strikingly similar to the development
`in wheat-raising. Penicillin production might be called factory farming,
`for the principles Operating are the same as in wheat production.
`Molds produce at least a half dozen different types of penicillin in
`the same medium. These differ only in the B group part of the mole—-
`mule. Today, only one type of penicillin is wanted in commerce, that
`is the benzyl or G penicillin, which has the formula
`
`S
`11
`can—Cinuc—Nn—Cn—C/ “C—{Cfioz
`= -—— — —-COOH
`O C
`N
`CH
`
`0
`
`R grasp
`
`Penicillin G
`
`If a suitable precursor, c.g., phenylacctie acid, is added to the medium,
`the mold obligingiy responds by incorporating this compound into the mol-
`ecule. Other R groups are: in F penicillin, pentenyl (CH3 - CHE - CH :-
`CH ' GHQ—l ; in K penicillin, hcptyl (GIL-g ' CH2 ' CHg ' CHg ' CH2 - CH2 '
`Olly—L More than 20 penicillins have been obtained by addition of
`the appropriate precursors.
`Penicillin acts on gram-positive bacteria, and in exceedingly low con-
`ccntrations. For example, 0.03 units per milliliter will inhibit the growth
`of the assay organism ilIir-rocor-ens pyogcncs vanaureos (formerly called
`Staphylococcus nitrous). Since a unit of penicillin is 0.6 pg., 0.03 unit
`is less than 0.02 pg. per milliliter or 2 mg. per 100 l. of medium. A
`clinical dose of 100,000 Units is only 60 mg. Unfortunately, strains of
`microorganisms that are resistant to penicillin are beginning to appear.
`These resistant strains probably come from patients who have been re—
`cently treated with penicillin.
`is that al—
`The most obvious effect of penicillin on the microbial cell
`though the cell grows larger, it does not divide. This shows that formation
`of cell constituents, e.g., proteins and nucleic acids, continues for some
`time after the penicillin enters the cell. Eventually the enlarged cell
`bursts.
`
`Interference with absorption of amino acids, protein synthesis, nucleic
`acid synthesis, and phosphorylation reactions have all been attributed
`to penicillin.
`It is difficult to determine which of these are primary
`effects and which are secondary manifestations. Metabolism is a series
`of events, and interference at one place will show 11p in all subsequent
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 378
`Petitioner Microsoft Corporation - EX. 1032, p. 378
`
`

`

`METABOLISM OF MICHOORGANISMS
`
`37“;
`
`places and eventually reflect back to the original point of iinerl'erence.
`It would probably be more correct
`to think of metabolism as a cycle
`rather than a chain of events.
`
`Penicillin is specifically and irreversibly bound by tartan-positive bac-
`teria.
`For example, 0.49 units of penicillin per gram of dry weight
`are bound by cells of Bacillus ccreua. Extracts of the cells also bind
`the penicillin, and it should be possible to identify the substance in the
`extract that possesses binding power.
`It may be that the reaction between
`penicillin and this cell constitutcnt
`is the primary reaction and other
`effects are secondary.
`A well-defined effect of penicillin on nucleic acid metalmlism is reported
`by Park. This is the accumulation of uridine-5'1iyrophosphate conn‘dexes
`in cells of Staphylococcus oto'cns that. have been treated with penicillin.
`In addition to uracil, ribose. and phosphoric acid, one of these complexes
`contains an X~acetyl amino sugar. A second complex contains L-alanine
`in addition to the other four components, and a third has attached to
`it a peptide made up of nL-alanine, L-lysine, and n-glntainie acid. Prob-
`ably the accunmlation of these complexes is a secondary effect caused
`by the blocking of some reaction that utilizes the uracil compounds.
`From the discussion given. it is evident that
`the. specific effect of
`
`penicillin is still undetermined. However, since many able investigators
`are attacking the problem, distinct progress toward its solution may be
`expected.
`Tetronie Acids. The prodturtion of a series of compounds closely
`related in structure is a characteristic feature of mold metabolism. Be-
`
`sides the penicillins, another such series is the tetronie acids.
`
`3
`30—0
`
`
`
`(Ts—nor)
`(n)11~cn2—(ELO/C=O
`n
`
`1. y-Mcthyl tetronie acid, Pe-aicillftmi clicrlcsfi.
`2. Carolinic acid, P. chm'lesia'.‘
`It’ :- COlCIIghCOOII [succinyI group}.
`3. Carolie aeid {+Hgol, P. ehorlcsii:
`R' : ("OtCIIghCHgOI-I (-3;-hydroxyhutyryl].
`4. Terrestric acid [+1120], P. terrestrc:
`R’ : C'OlCI-Ig)ng-IOII - 02H,-I
`{an ethyl derivative of the R' group in canolie acid).
`Dchydrocarolic acid ( +H20), Pcnieillimn cinernsecns:
`CII3— of carnlic acid is replaced by (31-12:
`6. Ca rlic acid, P. clmrlcsii:
`I? : HOOC; 11' = COICHQECHQOH (Thydroxybutyryl)
`7. Ca rlnsie acid, P. chorlcsii:
`It : IIOOC; R’ = C0 ' CH20II2CI'13 {butyryl}.
`
`UI
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 379
`Petitioner Microsoft Corporation - EX. 1032, p. 379
`
`

`

`376
`
`METABOLISM OF Micaooaeamsns
`
`Formulas with (+H20) mean that their peculiar structure is present
`only in water. These compounds crystallize as anhydridcs. y-Methyl
`tetronic acid may be regarded as the parent substance, and the others,
`as substitution products. Carolinic acid has a succiny] group in place
`of the hydrogen on the u-carbon. Carolic acid has a carbinol group
`instead of a carboxyl group in the side chain.
`In earlosic acid the end
`group in the R' side chain is methyl. These three compounds clearly
`represent different degrees of Oxidation.
`In carlosic acid and carlie acid
`there are also carboxyl groups replacing the hydrogen at R in the formula.
`All of these compounds are produced in only small amounts, in the order
`of 1 to 2 per cent of the sugar fermented.
`It is of special interest that
`five of the compounds are produced by the same mold. These must be
`interrelated in the metabolism of P. Charleen.
`
`Many other such series of compounds have been described by Raistrick
`as characteristic of mold metabolism, e.g., a citric, an anthraquinonc,
`and a tropolone series. Over 200 mold product-s have been isolated in
`more than a quarter century of research work by Raistrick and co-workers.
`From this wealth of material many interesting features of mold metabolism
`have been discovered. For more information see his review paper listed
`in the references at the end of this chapter.
`Another noteworthy aspect. of mold metabolism is the formation of
`organic chlorine compounds. Examples of such compounds are crdin
`(Cu-.H1007Clg), and geodin (C13H1207Cig). These two compounds are
`produced by the same mold, Aspergi‘ttus terreas, and are closely related
`in structure.
`
`leather, hay,
`Spoilage of such Commercial products as wood, paper,
`grain, bread, etc., constitutes a debit side of mold activities. An
`inhibitor of mold growth, propionic acid,
`is widely used in the bread
`industry. For use on nonfnod materials, there are a number of mold
`inhibitors, (3.9., pentachlorphenol, CnCerH.
`
`ANAEROBIC METABOLISM OF CARBOHYDRATES
`
`By bacteria
`
`The anerobic metabolism of bacteria is probably more diversified than
`the aerobic and probably results in a larger number of products.
`{See
`Table 14—4.) The principal
`types of anaerobic fermentations can be
`classified by their major end products, as follows:
`1. The homolactic type of fermentation [e.g., by S. toetis) accounts for
`more than 90 per cent of the glucose as lactic acid. Thus
`
`osnmofl—r eon, - enou -c00n
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 380
`Petitioner Microsoft Corporation - EX. 1032, p. 380
`
`

`

`METABOLISM OF MICROORCANISMS
`
`377
`
`2. The hetcrolactic fermentation [c.g.. by L. pentoocctieus} turns about
`half of the glucose into lactic acid and converts the other half mainly
`into carbon dioxide and ethyl alcohol. The equation is
`
`€611,206 —-> C11,, - coon - coon + cou + co, ~c1non
`
`Sometimes considerable amounts of acetic acid and small quantities of
`glycerol are formed.
`'
`These two types of lactic acid fermentation, homolactic and hetero-
`lactic, are important in the industrial production of lactic acid and in
`the making of cheese, sauerkraut, pickles, and silage.
`3. The propionic fermentation (c.g., by l". pcntosoccum} gives propionic
`acid, acetic acid, succinic acid, and carbon dioxide as major products,
`but under certain conditions considerable amounts of lactic acid are
`
`formed. The propioaic fermentation may be regarded as superimposed
`upon a lioniolactie fermentation, but it does not seem probable that lactic
`acid is an intermediate in the production of propionic acid.
`In this and
`the following fermentations the reactions are too complicated to be readily
`expressed by simple equations.
`4. The colon-acrogenes-typhoid bacteria, not only produce all of the
`compounds formed by the mixed lactics except glycerol, but in addition
`make formic acid, hydrogen, and butylcne glycol. This is a very hetero-
`geneous group of organisms, and the proportion of the products to one
`another varies greatly with the species of bacteria. Perhaps the most
`distinctive products are:
`formic acid by Eberthcllo typhi, acids and
`hydrogen by Escherichia cob", and acetoin and butylene glycol by Aero-
`bactcr acrowncs.
`5. The butyrie arid fermentation (3.9., by Ct. ocetobatylfcum) is char-
`acterized by the almost complete absence of lactic acid and the appear-
`ance of acetic acid, butyric acid, carbon dioxide, hydrogen, butyl alcohol,
`ethyl alcohol, and acetone.
`Isopropyi alcohol may replace acetone
`wholly or in part in certain butyric fcl‘mcntations. Some bacteria in
`this group do not form the last four compounds, collectively called
`solvents, while others produce them in large amounts.
`6. The naturally occurring methane. fermentation (e.g., by flfethono-
`bacterium omelionsfrii} involves a unique type of metabolism. The two
`extremes of oxidized and reduced carbon products, carbon dioxide and
`methane, meet here. This apparent contradiction did not appear 5o
`strange when it was discovered that methane arose. at. least partially,
`by reduction of carbon dioxide with hydrogen. Methane (also called
`marsh gas) occurs extensively in coal mines, stagnant waters, sewage,
`certain types of plants, and in the intestinal tract of animals.
`7. Fairly well-characterized polysaccharides have been obtained from
`more than 60 species of bacteria. Some of these give unusual products
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 381
`Petitioner Microsoft Corporation - EX. 1032, p. 381
`
`

`

`373
`
`METABOLISM OF MICROORCANISMS
`
`on hydrolysis such as glucnrmiie acid, o-arabinose, and inositol. Some
`of the most notable polysaccharides, their important characteristics, and
`the bacteria producing them are as follows:
`(a)
`(dell-atom. This is true cellulose, identical in chemical and physical
`properties with that found in higher plants.
`It is produced by .‘lrctoboctcr
`:cyh'mon. and other members of this genus.
`{b'l Polysaccharides with marked physiological and chemical proper-
`ties are produced by many pneumoeoeei. The polysaccharidc produced
`by Type III consists of alternate glucose and glucuronic acid units bound
`together through oxygen by a fi-linkage from carbon 4 of the glucose
`to carbon 1 of the glucuronic acid and a second fi-linliage between carbon
`3 of the glucuronie acid and carbon 1 of the glucose. There appear to
`be over 600 units each of glucose and glueuronic acid in the polysaccharide
`chain. The polysaccharide has marked antigenic properties; when in-
`jected into a rabbit, it evokes production of antibodies and innnunity
`against infection with Type III pnenmocoecus.
`{c} Dcxtrans. Many bacteria produce dextrans, but L. m-cscntcroidcs
`is the best known dextran-producer. One reason for the current interest
`in this bacterium is that it produces a dextran that is now being manu~
`factured as a substitute for blood plasma. L. incscntcroidcs is found as
`a contaminant in sugar factories, and the dextran produced interferes
`seriously with manufacturing operations. A 10 per cent. sucrose solu~
`tion is fermented in about 24 hours and gives a yield of 25455 per cent
`dextran, based on sucrose used. The dextran comes from the glucose
`part of the sucrose molecule, but glucose itself does not give any dcxtran,
`although the microorganism grows well on this sugar. On a glucose
`medium the microorganism behaves as a heterolaetic. The dextran
`appears to be formed from sucrose according to the following equation:
`
`”(Cichconl _’ (ceHmOslfl 'l‘ tilaniaocl
`Sucrose
`Dcxtran
`Fructose
`
`Some of the glucose and most of the fructose are fermented to lactic acid,
`acetic acid, ethyl alcohol, carbon dioxide, and mannitol. Potent enzyme
`preparations which bring about
`the rapid formation of dextran and
`fructose from sucrose have been obtained from the culture solution.
`
`The dextran has a branching structure with apparently o~l ,6—linkages in
`the main chain and u-l,4-linkages at the branching points (pp. 50 and 60] -
`The molecular weights of dextrans from different strains of L. meccntcr-
`aides are enormous, (3.9., 25 to 80 millions. These dcxtrans are too large
`for use directly as blood plasma substitutes. They are partly degraded
`by controlled acid hydrolysis and fractionated to give products of suitable
`size, c.g., molecular weights of about 75,000. Only about 10 per cent
`of the original dcxtran is obtained as material suitable for clinical use.
`An extensive search is now in progress for microorganisms that will pro-
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 382
`Petitioner Microsoft Corporation - EX. 1032, p. 382
`
`

`

`)lfi'l‘ABUlJfihl or manooaoamsns
`
`37"
`
`dnee more suitahle polysaeehariiies than those obtained from L.
`1‘ r'Tofr l cs .
`
`inesca-
`
`l’olysacclnn'illes of this type are produced from sucrose
`{d} Lt"i'ttHS‘.
`by several microorganisms, c.g.. Bacillus- sabtih's: Yields of
`lcvan up
`to 30 per cent, based on the sucrose used, have been obtained. Enzyme
`preparations give approximately the same yields, and the reaction seems
`to follow the same equation as for dextrans, but the polysaccharidc and
`free sugar are reversed. Thus:
`
`“(C|2H:12011l_>[Callmnfiln‘l'iilCnlh-gOa)
`Sucrose
`Levan
`Glued-'1'
`
`By yeast
`
`In the absence of air, ethyl alcohol and carbon dioxide account for about
`90 per cent of the. sugar fermented, as indicated by the following equation:
`
`mono, —> segnfion + see,
`
`If the
`Small amounts of acetic acid and glycerol are also produced.
`medium is kept alkaline, pH about 8.5, large quantities of acetic acid
`and glycerol are formed. The metabolism of the yeast
`is shifted so
`that a minor product, glycerol, becomes a major product. The equation
`for the fermentation may he represented as
`
`ECIJI120" + 1130-9
`2C03 + CH;,CO0H + Cli3CI'1201'I-l- 2C1120ll ' CHOU ' CI130H
`
`This theoretical distribution of products is not. realized, since more alcohol
`and less glycerol are usually formed.
`Glycerol production may also be increased by adding sulfites to the
`medium. This fixes the intermediate acetaldehyde as CI—IaCl-IOH-O-
`'SO-JXn and prevents its reduction to ethyl alcohol. A corresponding
`amount of another intermediate, dihydroxyaectone phosphate,
`is con-
`verted to glycerol. The sulfite process for production of glycerol was
`used by the Germans in World War I.
`It is still under consideration, but
`to date has not been operated successfully. The fermentation equation
`may be written as
`
`(annoys €11,011~Ci101-1-e11,01-1+ C}13CHO + (:02
`
`is obtained in practical
`Only about one-half of this yield of glycerol
`operations because some of the acetaldeliyde escapes fixation and instead
`goes to ethyl alcohol.
`is around
`Under anaerobic conditions the yield of yeast [dry weight]
`5 per cent of the sugar fermented, about one-tenth as much as is pro-
`duced under aerobic conditions.
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 383
`Petitioner Microsoft Corporation - EX. 1032, p. 383
`
`

`

`38“
`
`METABOLISM OF MICRDORGANISMS
`
`SYSTEMS OF INTERMEDIARY METABOLISM
`
`Aerobic metabolism
`
`Microorganisms that use oxygen have a glycolysis and oxidizing system
`such as animals possess and metabolize glucose via pyruvic acid and the
`citric acid cycle to carbon dioxide and water.
`llowevcr, another route,
`by which glycolysis is side-stepped and pessihly also the citric acid cycle,
`appears to function in yeast. and bacteria, and to some extent in liver.
`This route has been known for a long time but has not received much
`attention until recently.
`It has l'iecn called “the hexose monophosphatc
`shunt,” but. this is a. poor name since the route appears to be more than
`a detour around glycolysis.
`It. is a direct oxidation of glucose in which
`a number of entirely new Compounds appear as intermediates in the
`following sequence:
`
`Glucose '49-» glucose—GPO.
`
`«El—a-
`
`2-ketogluconic acid-6—P04
`
`fl!- glueonic acid-fi-PO.
`(4)
`.
`— co,
`-—" r1hulosc‘5-P0,
`
`,
`
`(a)
`
`{
`
`(til
`Cs compound (X 2) ——»
`
`tetrose
`
`t?)
`
`tl'lflse- 3-P0 4 ——""'—_""'
`
`9
`
`—* sedoheptulose-‘f-PO.
`{glucose-GPO; All“ recycled
`
`(3)
`+ triose-3-P01 H
`
`(10)
`tctrosc-4«P04 __,
`
`? triose-3-PO. + CO;
`
`The nature of the Ca compound in step 5 is still unknown; it does not
`appear to be a glycolie aldehyde. The kctopcntose, ribulose-5-P04,
`is
`in equilibrium with the aldopentose, ribosc-5-PO4, but the predominant
`form seems to be the ketose. The occurrence of a 0,: sugar, scdoheptulose,
`in the metabolism of a Ca sugar is an unexpected and noteworthy phenom-
`enon. The disposition of the tetrosc—4-PO4 (step 10} is still uncertain,
`but it appears to go to a triosc-phosphatc and a one carbon compound,
`which may be carbon dioxide. All details of the direct. oxidation pathway
`have not- yet been “‘Ul‘kf'd out, but the main outlines of the route are
`evident.
`
`Since some cells are equipped with both the glycolysis-eitric acid cycle
`mechanism and the direct oxidation system, the question naturally arises
`as to the relative importance of the two systems.
`Investigators are
`cautious about expressing an opinion, because sufficient data are not
`yet available for answering this question. However, judging from the
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 384
`Petitioner Microsoft Corporation -IEX. 1032, p. 384
`
`

`

`31 B'l‘ABOLlS-.\[ or meueoacamsns
`
`381
`
`number of papers that. are appearing, some information on this problem
`should soon be. forthcoming.
`involve phosphor-yie-
`A second type of direct oxidation that docs not
`tieu operates in the metabolism of glucose by certain aerobic bacteria,
`e.g., Psendomonos aeruginoso. Oxidation of glucose.
`leads to glueonic
`acid, 2-ketoglueonic acid, pyruvic acid, and the formation of large amounts
`of a-kctoglutaric acid. Yields of this kcto acid up to 0.55 mole per
`mole of glucose have been obtained. This provides a convenient method
`for the preparation of a-ketoglutarie acid.
`
`ANAEROB IC METABOLISM
`
`Yeas:
`
`Hexosc diphosphatc and pyruvie acid, so prominent as intermediary
`products in animal 111etabolism, were first. observed in yeast. The steps
`in glycolysis are the same as far as pyruvic acid in both animal and
`yeast metabolism.
`In yeast fermentation the whole process is anaerobic;
`the pyruvie acid is decarboxylated to aectaldehyde, and this is then
`reduced to ethyl alcohol. The hydrogen necessary for
`the last step
`comes from the dehydregenation (oxidation) of pbosphoglyceraldchyde
`to phesphoglyeeric acid via DPN-Hg as carrier.
`If this reduction is
`blocked, for example, by fixing the aeetaldehyde with sulfite, the hydrogen
`is used to reduce dihydroxyacctone to glycerol. Glycerol production
`always occurs to a slight extent {3—5 per cent of the glucose), but with
`the main route of fermentation blocked, the yeast makes the side line
`a main route. The alternative pathway is a very neat and convenient
`device for continuing metabolism under adverse conditions.
`If alcoholic fermentation is studied with labeled glucose, it is found
`that carbon 1 appears in the methyl group, 2 in the carbine] group of
`ethyl alcohol, and carbon 3 in the carbon dioxide. This accords with
`the Embden-Meyerhef scheme of intermediary metabolism.
`(See Chap.
`13.)
`
`Bacteria
`
`The se-eallcd “mixed" lactic fermentation shows some unexpected
`departures from the alcoholic fermentation of yeast. Carbon 1 of glucose
`appears in the carbon dioxide. Carbons 2 and 3 are found in the methyl
`and carbine]
`(or carboxyi) groups, respectively, of ethyl alcohol
`{or
`acetic acid}. Carbon 4 comes out in the carboxyl group of the lactic
`acid. The two halves of the glucose molecule are metabolized differently.
`Various intermediary compounds have been found. The first. series of
`
`Petitioner Microsoft Corporation - Ex. 1032, p. 385
`Petitioner Microsoft Corporation - EX. 1032, p. 385
`
`

`

`332
`
`M ETABOIJSM 0F MICROORCANISMS
`
`compounds appears to be the same as in the oxidative pathway of aerobic
`organisms:
`
`Glucose —* glucose-G-phoSphate --+ gluconic acid-G-phosphate
`
`_’ 2-kctoglueonie acid-Gphosphate —"{ _ribulose-B-phosphate
`
`CO»
`
`---—--3\-
`
`{C2 compound —* ethyl alcohol or acetic acid
`
`triose-3-111105pliate —** lactic acid (+ HaPOJ
`
`The fermentation of glucose becomes a pentose fermentation after the
`loss of carbon 1. The latter part of this form