1
I I I
CHEMICAL E N G I N E E R I N G R E V I E W S
I PROCESSES REVIEW
I
II FERMENTATION II
I K THIS Ninth .Annual Revie\\ on fermentation. coverage has included only those papers that are regarded as significant contributions to the subject field. T h e prriod covered by this revieiv is from Februaq- 1955 through Frbruary 1956, inclusive. General Reviews Sumcrous publications have apprarctd during the past year which havr reviewed various aspects of the fermentation industry. A special book on pilot plant techniques of submerged fermentation has been editrd by Chain (7'4) of the Istituto Superior? di Sanith. This is a n excellent volunie covering various research developments of the institute, such as aeration studies, design and operation of various laboratory and pilot plant submerged fermrntors, mechanical agitation, evaluation of antifoam agents, and genetic stirdies of Pcnici!liuni chrjsogenum. .A book bvliich has colkcced, correlated. and evaluated pertinent information relative to antiseptics? disinfectants, and fungicides, as well as chemical a n d physical methods of sterilization, has been edited by Reddish (5.4). L'arious contributions have been made by authorities ~ v h o arc experts in their respectivr fields of antiniicrohic research. in interesting book on a symposium of perspectivcs and horizons in microbiology has been edited by LVaksiiian ( ~ 7 ~ 1 )T. h e thirteen scientific papers deal with selected aspects of broad subjects, such as the microbe as a L\.holr. metapoirtic integrations of lysogenic bacteria, cenetics of niicroorganisnir. nutrition and enzymatic studies of mutants, metabolism of bacteria and molds; biological fixation of nitrogen, microorganisms and steroid transformations, unsolved problems of immunology, virus reproduction, challenging problems of antibiotics, and the relation of microorganisms to plants. A book on microbiology has been written by Gray 13.4). He discusses biology, culturc, and control of microorganisms; host parasite relationship of cclls; disease and immunity; viruses, bacteria, yeasts, molds, and fungi; algae and protozoa; tha microbiology of soil and inland waterways; the sea and applied microbiology.
into operation in S o i w i i h r r 195.5, .A popular rapoi t on t!ic achit prior to construction of outside \valls or of fermentation chemistry and biopermanent control panels. i4ftt-r prochemical engineering has been publonged negotiations, the German firm of lished by Chas. Ffizrr & Co. (2'4). Braunschweigishe hlashinenbau-Anstalt A highly interesting book on the life of (BMA) (73B) has received a n order from bacteria has bcc-n ivrirten hy Thimann the Brazilian Sugar and Alcohol In(6.1). T h e author spent sonif 15 ycars stitute to build a n alcohol distillcry ii, in writing this hook covering the morBrazil. Cost is cstimated a t $650,000: phology and general physiology of complrtion is scheduled for latc 1 [ 5 6 . bactrria, the role of rnicroorganisnis in the nitrogen cyclc: thc metaholisni of Chas. Pfizer & Co., Iric,. (776'1. announced the formation of a Jaimnes!,carbohydrates, and gro\vth and synAmerican company for bayic nianiithesis of bacteria. T h e careful study of facturc of antibiotics. l'hr, concet.11. this book should he a pleasurr to every chemist, biochrmist: or engineer. .-IPfizcr-Taito Co., Ltd.: xvil! iint1r~i~t;rLc~ expansion of present producrion hrilili(.s highly interesting survey of the future of microbiology in relation to industrial a t Kobe, Japan, and prrparc for hasic chemistry was thc subject of a paper by manufacture of oxytetracyclinr, -scs of \vhole culturtts of plasinotiia indicated that relatiuel!. largr amount5 of riboflavin c a n be synthesized in such culturcs. Vitamin B,, Complex. Tluriiig the p s t year, Tudd a n d othrrs 1.30E)reportrd the striictiirc of t h c \-itaimin 151, molecule. \\'it11 cxcelition of the possible location of one or t\vo of rlie double bonds, the structure is positively identified. LVork is now undcr way to synthesize the vitaniin. Other papers concerned \Titi1 the structure of vitamin Bl: were published by Hodgkin and associates (77E) and by Bonnett and associates (ZE). Williams (32E) has edited a symposium on the biochemistry of vitamin Bl:. T h e isolation and chemistry of vitamin Biz, methods of measurement, adsorption, and excretion
INDUSTRIAL AND ENGINEERING CHEMISTRY
of vitamin B I in ~ man: function of Bj?in animal metabolism and in microorganisms are revie~ved. Kuehl and others (76E, 77E) have reported on 3,3-diincthy1-2,5-dioxopyrrolidine-4-propionamide, ~~-3:.?-diniethyl7 , s - dioxo - 4 - hydroxypyrrolidinc - 4!,ropionic. acid lacton:-, and 1)~-3>.3tiimrthyl-2.5 dioxopyrrolidinc-l-propiiinic acid as ne\v drgradation products of vitamin R12. Robinson and o t l i ~ r s ! 39E) lia-.x. rrl)ortrd on degradation (if I'actor 111 to '-hydros!.heiizimidazolc a n d derivatives, and biosynt!irsis of 13'actor 111. Factor I11 \vas isolated irorr! fermented selvage. Stevens, \Yolnak, and Zirin (.?!/I\the incubation of progesterone w i l t 1 Colleotrichuni antirrh2nz and the 1 5 J 1iydro.u:. rpimer by the action of Phjco-
Table 111.
Y'rari>]uinintion
Microbiological Transformations of Steroids Product Organisvi Cortisone Prednisone Fusarium solani Corynebacterium simplex Bacillus sphaericus Didymella lycopersici H\ di ocortisone Prednisolone [ C. simplex 9n-Fluoroh~drocortisone l - D e h y d r o - D o l - f l u o r o h ~ ~ ~ ~ c ~ r t i ~B. o nsphaericus e D . lycopersici Corticosterone l-Dehydl.ocorricos[~roI~[. Calonectria decora 1-Dehydro-rompound \ Compound S,I C. simplex B . bphaericus 1 -Del-iyclro-IlOC: DOC' D.i ~ c v p e rici i Su bstratc
'
1.2-l>rtndi oqenation
Prosesterone 1 1 -Dehydrocorticosterone 11B-Hydroxyprogesterone
1 -Dehyclrop:.og.esteione
li.--Pre~nadiene-21-01-3.1 1,211-tri-
C. decora B sphaericur
D.1 vcopersici B . sphaei icur
one i'~:-Pregnadiene-l18-~1-3.20-dionc
Androstenedione 11-Dehydro-l7a-methylcorticosterone 6-Dehydro-DOC 11-Dehydroprogester.one 170-Ethynyltestosterone 17a-Methyltestosterone
~',i-.'indl.ostadiene-3. 1'-&one l'a-~Iethyl-~',l-pregnaciiene-ll-
Progesterone 19-Norproqesterone 11-Ketoprogesterone 11a-Hydroxyproqesterone 11 0-Hvdroxvproge.ter.^ one 6 5-Hydroxyprogesterone 140-Hydroxyprogesterone
DOC 13-Sou-DOC: 11-Dehydrocorticosterone 1la-Hydroxy-DOC
1 la-Hydroxyprogester-
1 1a,l -ol-I~ihydros\progestel.one
one 110-Hy-droxyprogesterone
11 9,l'a-Dihydroxyprogesrerone
Androstenedione DOC
16a-Hydroxy-DOC
Strepomyces roseochromogmus Didymella codakii
15a-Hydroxylation
Progesterone DOC Compound S
150-Hydroxyprogesterone 15a-Hydroxy-DOC 150-Hydroxy-compound S
Colleotrichum antirrhirii Gibberella baccatab Hormodendrum viride
(77G. 37G1 (2G)
15p-Hydroxylation
Progesterone DOC Compound S
150-Hydroxyprogesterone 158-Hydroxy-DOC 158-Hydroxy-compound S
Phycomyces blakesleeanus Lenites abientinah Spicaria sp.
(77G) (77G, 37G) (2G)
21 -Hydroxylation
17a-Hydroxylation
160-Hydroxylation
0
DOC: 1I-deoxycorticosterone.
1 590
01-3,11.20-trione ~',"''-Preanatrieiie-21-01-3.20-dione ~','."-Pregnatriene-3,ZO-dione 1-Dehydro-1-a-ethynyltestosterone 1-Dehvdro-1-a-methyltestosterone
118-Hydroxy-DOC 6p-Hydroxy-DOC 14a-Hydroxy-DOC
1ba-H~-droxyandrostcnedione
Compound S : 170-hydroxy-11-deoxycorticosterone
INDUSTRIAL AND ENGINEERING CHEMISTRY
:lsperyil/ur niger
Ilnctylzum dendroides
(77G') (.?7G) (71G)
FERMENTATION myces blai:s!eeanus on progesterone. Proof of the structures was adduced from the preparation of 15-ketoprogesterone. a derivative common to both stproids, and the differences in ease of esterification and chromatographic mobility exhibitcd by the h>-droxy compounds. T h e corresponding 11-deoxycorticosterone compounds were described by hkystre aiid associates (77G). T h e 15a-hydroxy compound \vas produced by the action of Gibberella baccata and the 153- by Lenites ohientina (37C). I n the same paper tlirse investigators also reported for the first time the 7cr-hydroxylation of a stcroid. This transformation \vas achieved by the incubation of 11 -deoxycorticosterone with various species of Pzzizci and Currularia. TTVO additional cultures for the 1 5-hydroxylation of steroids have b w n supplird bv the
7 ~rarr,jvrnia/i011
1.j:-Hydroxylation
publication of Bernstein and associates (ZG). They isolated l 5 B - hydroxycompound S from the fermentation of compound S ivith a Spicaria species and 15, - hydroxy - compound S from a Hvrmodendrurn riridt. fermentation of tht. same steroid. .-Inovel type of microbiological transformation \vas achieved by Bloom and Shull (3G, 23G) with certain unsaturated sreroids. They obtained the 14a,15aepoxido derivative of compound S by cxposing 14-dehydro-compound S to the activity of a variety of 14a-hydroxylating organisms (Table 11). In a similar manner: the 9D,ll$-epoxido- derivative of compound S \vas produced by incubation of 3(11 )-dehydro-compound S \vir11 Curruic;ria lunata and Cunninghameiln blakesleeana, n v o 1 1P-hydroxylating culturcs. The 9a. l la-epoxido compound
\cas not produccd Jvhen the latter substrate \vas employcd ~ v i t h 1 la-hydroxylating cultures. From their studies thrse investigators postulate that microbiological epoxidation occurs when a stcroid containing a n isolated double bond i, incubated ivith a culture capablr of introducing a n axial hydroxyl group into the steroid nucleus a t tiit. sit? oi‘ thr unsaturation. -4 ne\v class oi’ biologicall). active corticoids, representing derivatives of cortisone aiid hydrocortisonr functionally substituted in the D-ring. !vas reported b ~ , .%gnello and associates (7G). The starting steroid for this s:xric,s of chemical transformations was 140-hydroxyhydrocortisone, itself an active glucocorticoid, produced by the action of Curmiaria lunata on compound S ( Z J G ) . The genus Aspergiiius has proved to be
Table 111. Microbiological Transformations of Steroids (Continued) Product Substrate 1 j;-Hydroxc)-pro_estei one Progesterone
14n-Hydroxylation
ProgesteI one DOC
14a-Hydroxy-nOc:
1 1 wHytiroxvlation
1ba-H~dl.osyprogest~,r-
11n.lba-~ehydroxyprogesterone
14a-Hydrozyprogesteroiie
one
16a.l -n-Osidoproqrstrrone Progesterone
1l a - H y d r o x y - 1Ga,l ‘a-oxidoproqrste:-one 1 1 u-Hydros!.progestrroiie
1’~-Hydi.oi~pl.ogestt.r.-
1 1a,I ’ c r - D c h y d r o x ) - p r o ~ ~ ~ t ~ r ~ n e
one
DOC Compound S
11-Epicorticosterone
11 3-Hydroxylarion
Compound li
Hydr-ocortisonc
C.’oniothJ, ium ,p.
85- or 9:-Hvdrox)-lation
DOC
8:- or 9t-Hydroxy-DOC
Proge3te:o:ic
8:- or 3 ;-Hyd~oxypro~esterone
-25urospora crassa Curuularia pullfsceiis Strejtoniyce~arrreofacirnr
’a-Hydroxvlation
DOC
’a-Hydroxy-DOC
t’eziza and Curmlaiia sp
-t-Hydroxylation
Progesterone
7:-Hydroxyprogesterone
I’hyomyces hlaX-esieeaniis
63-Hydroxylation
Progesterone .Androstenedione
6B-Hydroxyprogesterone 6j3-Hydrosyandrostenedione
Streptomyes aureofacicni ilsp~rgillusniger Lenriies abidilla ;lip~r,oillusnidrilari s
noc
b@-Hydroxy-DOC 613,16a-Dihydroxyprogesterone
Progesterone
I1 a , l -a-Dihydroxyprogesterone
DOC
11-Epi-hydrocortisone
l’a-Hydroxyprogesterone Compound S
11~ ~ 1 4 a , l 7 a - T r i h y d r o x y p r o ~ e s terone
C u r d a r i a lunata
14a-Hydrosyhydrocortisone
Curiularia lunata
14-Dehydro-compound S
14a.15a-Epoxido-compoundS
9( 11 )-Dehydro-cornpound S
9$,1lB-Epoxido-compound S
C u r d a r i a lunata Cunninghamella blakesleeana Helicostylum pariforme M u c o r grise0cyanu.r .Vfucor parasiticus Curvularia lunata Cunninghamella blakesleeana
Progesterone
Androstenedione and testolactone
16a-Hydroxyprogesterone
1 1a ,1‘a-Hydroxylation
Epoxidation
Oxidative cleavage of side chain
11-Epihydrocortisone
Dactylzum dendroides Asjergillus sp. 1)actyliurn d e n d r o i d ~ s
Cephalosporium subuerticullaturn
b The configuration of the 15-hydroxyl group in the DOC derivatives produced by these two organisms was orginally assigned in reverse order (77G) and later revised (37G).
VOL. 48, NO. 9, P A R T II
SEPTEMBER 1956
1591
UNIT PROCESSES REVIEW quite versatile in effecting steroid transrorniations. By selection of the appropriate species and strain, i t is now possible to hydroxylate steroids at carbon atoms 11, 17, and 21. For the introduction of the 21 -hydroxyl group Zaffaroni and associates (33G) employed .I. niger (XTCC No. ?142), ivhile Dulaney and associates (7G) found several species of the genus capable of converting progesterone into 1 l a , 17n-dihydroxyprogesterone. In a study of the hydroxylation of progesterone by ,lspergiilus ochraceur, Dulaney and coworkers (9G)found the transformations to proceed stepwise to yield first i 1n-hydroxvprogesterone followed by 6 8 , l l a-dihydroxypro,vcsterone. If the culture was cultivated on a zincdeficient medium, they found that the G(3-hydroxylation did not occur. This effect was attributed to a limitation placed on protein synthesis and adaptive enzyme formation by the zinc deficiency. Karow and Petsiavas (74G) studied the effect of physical variables on the hydroxylation of progesterone and compound S by the same culture. They found the rate of hydroxylation to be greatly dependent on agitation and air rate while variation in temperature in the 28' to 37' C. range had little effect. A seniicontinuous addition of steroid appeared to be preferable to a one-shot addition. Enzyme inhibitors were successfully employed by Mann and associates (76G) in increasing the yield of 11 8-hydroxy steroids produced by Cunninghamella blakesleeana in shake flask fermentations. T h e addition of 2,4-dichlorophenol increased the hydrocortisone yield from compound S. The addition of arsenate or arsenite, on the other hand, reduced the cortisone yield but did not affect the yield of hydrocortisone. When 1 1-deoxycorticosterone was used as the substrate, actidione increased the yields of corticosterone and decreased the formation of 14a-hydroxy-1 l-deoxycorticosterone. An interesting survey of a large number of Aspergilli and Penicillia was conducted by Dulaney and colleagues (7G). They found that the ability to transform progesterone into 1la-hydroxyprogesterone and compound S into 1I-epihydrocortisone was common in the genus Aspergillus. Additional hydroxylation of 1la-hydroxvprogesterone to yield 6p,1 la-dihydroxyprogesterone also occurred, hut conversion of this substrate to 1 la,i7a-dihydroxyprogesterone was found much less frequently. In the case of Penicillia, the formation of 1 la-hydroxyprogesterone from progesterone was rare (less than 1% of the cultures tested) but another derivative, tentatively identified as 15t-hydroxy-progesterone, was produced in many cases.
1592
An unsuccessful attempt to oxygenate steroidal sapogenins microbiologically has been reported by Mininger and colleagues (78G). : I \vide variety of microorganisms NWC c.inployrd in an attempt to hydroxylate sarsasapogenin, diosgenin. and A4-tigogisnone but no transformation products were obtained. Among the newer developments in steroids which have been reported since the last revie\v are thr following: the synthesis of 1-dehydro-On-fluorohydrocortisone (70G, 72G), a potent hormone \vith both gluco- and mineralocorticoid activity; the synthrsis of racrmic aldosterone (22G), the most recent adrenal hormone:; the introduction of the steroid compound, Viadril, as a surgical anesthetic (7.5G') ; thc synthrsis of the 2methyl analogs of hydrocortisone and la-fluorohydrocortisone ( I X ) , the latter compound representing the most potent mineralocorticoid kno\vn today. Except for 1 -dehydro-3a-fluorohydrocortisone, which has also been prepared microbiologically (79G', 27G,29C). all of the above-mentioned syntheses werr chemical in nature.
Antibiotics Several gcneral rr\.ivivs on antibiotics appeared during the past year, including those by Welch (727H), Eagle and Saz ( 4 8 H ) ! Binkley ( 7 5 7 1 ) , and Buchi ( 2 4 H ) . The current status of the development of antimicrobial agents was reviewed by Hobby (6911). Other reviews more specialized in nature \rere those by LlcKeen ( S 3 H ) on the mass production of mtibiotics, by Regna (707H) on the chemistry of antibiotics of clinical importance, b y Flippin (50H) with particular reference to toxic reactions, by Craig (3811) on polypeptide antibiotics. and by Khan and Chughtai (74H) on thr production of several commercially important antibiotics. Nonsynthetic media suitable for the production of actinomycete antibiotics by the shake flask method were described by LVarren and associates (725H). These media were useful for cultivating actinomycetes obtained from an antibiotic soil screening program. Penicillin. Some general considerations in the production of penicillin are revic\red by Serzedello (777H). I n a study of the effect of temperature changes on the production of penicillin by Penicillium chrysogenurn (W 49-133), Owen and Johnson ( 9 3 H ) found that there were two optima. One, about 30' C.. was best for the mycelium-producing phase; the other, about 2OoC.,was best for the penicillin-producing phase. They reported that 507, more penicillin was produced by fermentations which started at 30' C. and were transferred to 20' C. after 42 hours, than was produced in control fermentations a t 25' C.
INDUSTRIAL AND ENGINEERING CHEMISTRY
Additional studies on the role of far in the penicillin fermentation were reported. Peterson and associates ( 0 9 H ) obtained increased penicillin yields in shake flasks by the addition of lard oil to a standard medium, but found no stimulation if a richer medium, containing higher levels of corn steep solids and lactose, was used. If less than 0.4% calcium carbonate was used, the low pH resulting from the hydrolysis of the oil by the mold lipase depressed the yield. In 50-gallon fementors the average yield of penicillin without oil was 1400 units per ml. and ivith oil 2020 units per ml. DiAccadia (44H) substituted animal and vegetable oils for lactose in a corn steep medium and obtained equal penicillin yields. In other shake flask experiments he found that the substitution of pure triolein for lactose in the s)-nthetic medium of Jarvis and Johnson (72H) resulted in yields equal to those of the lactose control. Somewhat loiver yields were obtained with arachis oil or lard oil in this medium. Other medium modifications which were found to be beneficial were the addition of thiosulfate (56H), and the use of Lactobacillus delbrueckii fermentation broth as a medium adjunct (737H). -4dditional information on the utilization of valine for penicillin biosynthesis was provided by .'irnstein and Clubb (70H), \vho added valine containing both carbon-14 and nitrogen-15 to gro1ving submerged cultures of P. chrysogenuni. They concluded from their studies that D-valine is probably not used directly for penicillin biosynthesis but that dimethylpyruvic or a related deamination product of valine might be a more direct precursor. The chemical assay of benzylpenicillin (penicillin G) in fermentation liquors is based upon the determination of its phenylacctyl grouping. This procedure is complicated by the presence of othcr phenylacetyl derivatives in the sample. .Ashton and Foster ( 7 7 H ) have devised an isotope dilution assay, based on the use of carbon-14 as a tracer in benL!lpenicillin, xvhich circumvents this difficulty. T h e standard error by their procedure did not exceed i1%. I n 1948 Behrens and associates ( 1 3 H ) reported the biosynthesis of a number of new penicillins by addition of the appropriate prccursor to Penicillium noluturn fermentations. One of these new pencillins was phenoxymethyl penicillin which recently has been reinvestigated because of its unusual acid stability. This penicillin, called penicillin V, has been the subject of a series of reports (7H, 7 2 6 8 ) in which its pharmacological and clinical properties are compared with those of penicillin G. In oral therapy the new penicillin appears to offer some improvement over penicillin
FERMENTATION G, since higher penicillin blood levels are obtained brith the same dosage. Cephalosporin S:an antibiotic \vliich previously \vas identified as a penicillin containing a D-e-aminoadipic acid side chain, has been found to be identical {rith synnematin B ( 7 H ) . Streptomycin. Some characteristics of S. grispiir strains resistant to phage \ r e r e described by Van Alstyne and associates (727Hj. They found that these strains retained indefinitely t!icir resistance to\rard the phage \vith ivhich they Icere dc\~.loped!but that they \Yere not resistant to unrelated phages. ‘The phage appeared to be adsorbed on tlie resistant strains aiid to be rekased Ivithout multiplication. ‘The release of the phage \vas brought about eithrr b y aging, or by t r e a t m ~ n twith an ion cxchange resin. hlixturt.5 of soybean meal and distiller‘s soliiblcs are useful additions to the streptoiliyci:1 medium according to a patent issutd 10 h l c n a n i r l ( S I H j . Such mistui.rs appeared to serve satisfactorily ;is the source of the complex nitrogenous organic matr,rial necessary for streptonip in production.
Chlortetracycline,
Oxytetracycline,
and Tetracycline. A monograph by Lhvling ( 7 7 H ) contains a brief history of the drvclopinent and chernistry of tetracycline. and more detailed informarion on thr pharmacology and therapeutic uses of thc. antibiotic. ‘The patent litigation on tetracycline iias no1v twen settled. By the terms or this sc~rrlrment, Bristol has been %ranted a nonexclusive license for lvhich i t \vi11 pa!’ ro>-alties to Pfizrr for the inaiiufaacturt- and sale of tetracycline under the, 1)roduct claims of the Pfizer patent. retroactive to .January 11. 1955. Ypjjohn and Squibb rcceil-e nonexclusive licenses covering otil!. the salc. but not the nianufac turc of tetracycline, for \\.liich Pfizcr \rill also be paid ro!-alties. In addition. Pfizrr and Bristol exchanged options for iicensc,s under their other Cnited Statrs and foreigii patent rights relating to protwses for thc. production of tetracycliiic’. .?I1 four companies will cdntiiiue to sell the antibiotic u n d r r their respecti\-e brand names in the Vnited States and elseirhere (32H). Pfizcr recently received patents on tetrac) lint- in a numbcr of foreiqn countries W l H , ,3711). Sevrral of these foreign patciirs covered the production of tetrac!-line by direct fermentation. .4ddirional information on the production of tetracycline by direct fermentation is to bc found in several publications and patents. Gourel-itch and Lein (6711) reported that increased yields of tetracycline (and substitutcd tetracyclincs) \vex obtained by the addition to the medium of any one of s-vera1 hydroxylated polyhydrobenzoic acids, such as quinic. 5-dehydroquinic. shiki-
hlistretta and hlinicri ($511) have described a countcrcurrent distribution system for the quantitative deterinination of tetracycline, o:~ytrt;.acycline, and chlortetracycline in niixtures and a method for calculating the perccmt,igc composition from the u1traviolr.t absorbence of the aqueous layer. Chloramphenicol. ~:liloramphe~iicc,!, one of the first broad spectrum antibiotics [ o be marketed, appears t o be regaining some of the ground previously lost to other broad spcctruin antibiotics. This earlier loss in salcs voluinc was occasioned by reports in 1952 of blood disorders ivhich apparently arose from the use of the antibiotic. Subsequent surveys and investigations. hoivever, proved chloramphenicol to be a valuable antibiotic when properl!. used and t h c sales of the drug have made corresponding gains since this initial slump (122H. 723H). I n a continuation of the work on the role of precursor5 in the biosynthesis of chlorarnphenicol, RobbiBis and colleagues (IO.?H) liavc tmployed coinpounds tagged with carbon-14. ‘Tiley conclude from their stiiciies ~ . i t hphtnylalanine, norleucine, lcui iiir, serinc. ;it,-throinycinis prol-idrd in the recently publishrd r n o n o ~ r r i p l iby EIerrell (071f). S ~ v e r a l pap^ on the structi!w of erytliroin!-cin have been piiblishcd. ‘Thc -13improved medium for the producearlier papcrs cstablislird tlic fat.[ that tion of chlortetracycline is described in a erythroiii>-cinwas ii y,lycosiclr con t‘iiiiing Iiatent issued to Sicdercorn (9UH). the c l i i ~ i r t l i y l a ~ n i ~ i o c l e osugar, ~ ~ y dewA n essLXntial fcaturc. is the use of calcium, amine. a n d till. nic.thoxydeoxy s1:gar. or a si~:iilar biv,ilent cation, to dctoxif!. cladiiiose. Sigal and associat-s ( / 1 /If) “staling” prodiirts in tlie n!ecliuni. ha\,? recently csta!Jished that the c ~ q l y The precipitation of ciiloltctrac).cline b y cone portion uf tlie antibiotic is a 21thcse particular (.ations n1:iy he of carbon polyii).dr(ix)- lietolactoric,. for in-portance in this c o m t c tion. ’The Lrhich t!ie nainc crythronolide \\.as prcviously notcd reduction in chlorproposed. ’Tlic. latter is c o n n e c t 4 b y t ~ ~ t r a c ~ d produccion inc by S,uurro/uriens i\-iieii potassiuin ~nonoh)-drogcn ~ ~ h o s - glycocidic linhagrs to the tivo sugar moieties. C:l;irk and ‘rat1~1-a ( 3 I”) pliatc (KZHPO$) is addcd to the inediuin found t5ar cr).throl!iycin B! 3 s(.cond lias no\v been a ~ t r i b u t e dto a changc in antibiotic i ~ r o d ~ c rbt yi Strcjtornjcer e r i l h r!ie carhoh).drate iiict,ibolisiii of the reus, yields on mild acid hydrolysis two or.qnism by D i l l a r c o and asso:.iatrs crvstallint- bascs, both diIFercnt from the (.i5H). ‘They found that a kero-acid is one obtained froin erythromycin. and formed and that rhc. avumulation of this cladinos!-. Strong acic! liydrol>sis keto-acid and the inhibitioii of antibiotic yielded desoamine and unidmtiiied production Ivere related to the coiicenproducts. tration of orthophosphate. Some studies Neomycin. T h e synrhesis o f earbonon the production of chlortetracycline by 14-labeled neomycin {vas accomplished S. aureojuciens have been reported by by Sebek (709H) by the addition of Rokos and colleag~irsi103H).
mic, or j-deh:.droshikirnic acid. Compounds of this type have been shown to be precursors in the synthesis of aromatic rings. Minieri and associates (6-iH) obtained tt-tracyline by culturing Streptomjces aureofaciens on a lo\v chloride medium. thcrcby greatly restricting the chlorine available for the synthesis of chlortetracycline. the antibiotic normally produced in largest quantity by this organism. ‘They achieved a low chloride ion concentration either by using a syiithetic mediuin or by using natural materials deionized by processing irith ion exchange resins. Kolland and Sensi (7011H) also obtained nearly exclusive tetracycline pimduction by limiting the chloride concentration in media inoculated \vith a chlortetracycline-producing strain of Streptomyces. Another method of limiting the production of the chlorinated antibiotic \viis reported by Goure\-itch and associates (62”). They found that the addition of bromide to the 5‘. uurmJacii’ns feermrntations resulted in a markedly greater production of tetracycline aiid a concomittant dccrease in the amount of chlortetracycline. l‘i neiv antibiotic: bromtetracycline, apparently is produced Fvhen sufficient bromide is introduced into a low-chloride medium. The production and isolation of this antibiotic arc described by Doerschuk and colleagues ( ViN) and by Sensi and coivorkers ( I /Of€). T\\,o papers by BorenszEajn and ll-olf ( 7 6 N : 7 i H ) describe the laborarory and pilot plant production of oxytetracycline by a mutant of . C ! r i j ~ t o r n y r s rimorus. Ultraviolet irradiation and nitrogen mustards were usrd to obtain higher !.idding mutants. T h e production of ox>.tetracycliiie by t\ro other Streptotnjces species! S. )Iatens?s and S. armillatus,
VOL. 48, NO. 9, P A R T I1
SEPTEMBER 1956
1593
UNIT PROCESSES REVIEW uniformly labeled glucose carbon-14 to the medium of Streptomycer Jradiae. He found 19.5% of the total carbon-14 was incorporated into the neomycin molecule. Seomycins B and C have been purified b y carbon chromatography by Ford and associates (57H). Xlethanolysis studies on these antibiotics indicated that c.quimolar amounts of neamine a n d methyl neobiosaminides are produced and suggest a n empirical formula oi C23H16N6012 for both neornycins. Elementary analyses and potentiometric titrations were in somewhat better ag-reement with a C&4&6013 formula. A paper chromatographic method for the rapid differentiation of the neomycin group of antibiotics has been described by Saito and Schaffner (705”). I n this technique the filter paper is buffered with 1.0,M sodium sulfate? p H 2.4, and a methanol-aqueous sodium chloride solution is employed as the solvent system. Seomycins A? B, and C, framycetin, and catenulin were separated. Pan and Dutcher ( 9 6 H ) effected a separation of neomycins B and C by first acetylating the antibiotics and then using a butanol-pyridine-water paper chromatographic system. Other Antibiotics. Each )-ear; as additional new antibiotics are described, the task of correlating and integrating all of the information becomes increasingly more difficult. T h e same antibiotic may be knoivn by several trade names and, in certain instances: by several generic names as ivell. For this reason, it was decided to summarize the information on most of the nekv antibiotics in tabular form, as is presented in Table IV. Several of the antibiotics listed in this table and some of the newer antibiotics mentioned in the previous revieiv are also discussed beloiv. Cycloserine, a broad spectrum antibiotic first described by Commercial Solvents investigators (6-H. 6 8 H ) , has been shown to bk identical with Mcrck’s oxam)-cin (25H, 65H)and Pfizer’s P.4-94 ( 7 73H). Chemically the antibiotic is D-4-amino-3-isoxazolidinone. T h e companies involved have recently agreed on cycloserine as the generic name. I t is produced by both Streptomjces garyphaius and Streptomycr lavendulae according to the Merck and Pfizer publications. N o description of the Commercial Solvents culture \vas provided. A synthesis of the antibiotic has been reported by Stammer and colleagues (77627). Cycloserine has been termed a n “antibiotic paradox” (86H): as it was found to be inactive against tuberculosis in animals; when the standard methods for screening of antitubercular drugs were employed, but lvas active clinically against tuberculosis. -4 series of papers on the clinical a n d pharmacological
1 594
properties of cycloserine appears in Antibiotics Annual ( 6 H ) . A report by Sutton and Stanfield (718H) on the reversal of cycloserine inhibition by mycobactin is of interest, as it may help to clarify the mode of action of the antibiotic. Mycobactin is produced by Mycobacterium phlei a n d is a gro\vth factor for certain other mycobacteria, including the strain causing tuberculosis in humans. A situation analogous to that described above for cycloserine has also turned up for another antibiotic. In this case, cathomycin, first described by 1Ierck (73H, 72dH). streptonivicin, a n Cpjohn antibiotic (79H, 775H): and Pfizer‘s PA-93 have all bcen found to bc idcnrical (728H). T h e generic name novohiocin has now been sclccted for the antibiotic ( 5 H ) . Three different Strtp/,ton~jct~ species, S. spheroides, S. niz’eus? and S. griseus \\-ere reported to produce novobiocin (Table I\.). T ~ c oseries of papers which deal with the various properties of the antibiotic have been published (4H>h”). A new antifungal antibiotic, filipin, has been described by Amrnann and associates ( 3 H ) . I t is produced by Streptomyces j l i p i n e n s i s , a new species isolated from Philippine soil. Filipin is reported to inhibit a \vide variety of fungi including parasites of plants and animals and to give protection against fungi attacking both seeds and foliage. I t has been characterized as a conjugated diene with the empirical formula CS0HjoO10 by \\’hitfield and collragues (729H). Filipin and fungiochromin. another recently reported fungicide ( / _ ‘ o H ) ~ appear to belong to the samc family of polyene antifungals. In addition to cephalosporin N; Seivton and .Abraham (8SH SLUlj have also isolated anothcr related hydrophilic antibiotic, cephalosporin C, from the same Cephdosporjuni broth. I t resembles Cephalosporin S in yiclding D-a-aminoadipic acid on hydrol\-sis but differs from I i in not yiclding penicillaniine. Besides these hydrophilic antibiotics, the same organism also producis a family of solvent-extracrablc antibiotics called cephalosporin P. T h e actinoni)-cin g r o ~ i pof antibiotics has been s l i o ~ to , ~ be a group of closcly related compounds containing a quinoid chrorcophore linked to a peptide chain. Thc various actinomycins reportcd in the literature are A, B: C? D: I, J: and S. The)- arc believed to differ as a result of the variations in the number, arrangement, and kinds of amino acids in the peptide rr-oiety of the molecule. Goss and associates ( 5 Q H ) have reported that the actinomycin B complex. produced in the early stages of gro\vth by Streptomyces ( S o . 3720), was convrrtcd to the actinomycin I complex Tvhen the incubation ivas continued for a longer
INDUSTRIAL AND ENGINEERING CHEMISTRY
period. T h e structure question, a t least for the actinomycin C complex, has been clarified considerably by the brilliant studies of Brockmann and his colleagues. In the most recent of these publications (27H), actinomycins C1. C1, and CDWere shown to have a common chromophore-viz.: 3 - amino - 1,8dirneth>-l - 2 - phenoxazone - 4,5 - dicarboxylic acid-and to diffyr only in the constitution of the peptide-lactone side chains attached to thr t\vo carboxyl groups of thr chromophorr. The amino acid scquence, proceeding from the phenoxazone ring, was r-threonine, either o-allo-isoleucine or D-valine, Lproline, sarcosine: and .V-mc.th>-l-L-valine. Thus, actinomycin C; contained valine in both side chains, C3 alloisoleucine in both: and C, valine in one and allo-isoleucine in the other. A drscription of the pilot plant preparation and purification of the actinomycins produced by Streptomyces (KO. S67) was given by Delcambe ( d 2 H ) . These actinomycins resembled the C1: C : . and C3 types of Brockmann. I n studies concerned with inosinic acid biosynthesis Hartman and associates (6BH) found that L-azascrine exerted a powerful inhibition action on the de novo synthesis of purines. Specifically, the antibiotic \vas found to inhibit the conversion of ( ~ - ~ ~ - f o r m y l ) - g l ~ ~ i i i amide to (a-.V-formyl)-glycinamidine ( 7 9 H ) . Gots and colleagues (60H) reported that. L-azaserine acts as an inducing agent for the development of E. coli phage. The structure of acetopyrrothine (Thio!utin) and aureothricin were shown to be acetamido and propionamido drrivatiws. respectively: of 3-amino-5-methylpyrrolin-4-ono-(4.3-d)-1,2-dithiolr by Celmer and Solomops ( 2 8 H ) . Progress in the structurc studies of several other antibiotics has also been reported. King and Craig (75H.76H) found that tyrocidine B contained ten amino acids linked together in a cyclic peptide and that the sequence of amino acids was the samc as in tyrocidine A except L-tryptophan replaced 1.-phenylalanine. T%e .\trucrure of gramicidin J, another polypeptidr antibiotic: was found to be siriiilar to that of gramicidin S by Orani and Saito (92”). They proposed a , lic heptapcptide as a minimum unit and determined the sequence of amino acids in this unit. T h e structure of the amino sugar, mycaminose, obtained from the acid hydrolysis of carbomycin has been published by Hochstein and Regna (70H). Another sugar, mycarose. had previously been isolated and identified. Txco compounds, xanthomycinic acids I and 11, were obtained as oxidation products of xanthomycin -4 by Peterson and colleagues ( Q 8 H ) . T h e capabilities of a single culture in
FERMENTATION carrying out several fermentation processes was illustrated in the publication of Perlman and associates ( 9 7 H ) . They demonstrated that a streptomycete produced a basic antibiotic and vitamin B,? (up to 0.8 y per ml.) and also converted progestercne inro 1 6 a-hydroxyprogesterone in 30 to 405; yield. Xlthough the medium composition did nor affrct the oxidation of progesterone, i t did influence both the antibiotic and vitamin production, and the best medium for the production of one did not give the best vields of the other. Fermentation a s a Unit Process
T h e previous review of the literature by Beesch and Shull ( 3 4 \vhich apprared in this series, gave a comprehensive picture on industrial fermentation practicr. T h e reader is advised to consult this paper for a detailed discussion of the problems involved. Numerous factors normally make u p a fcrinentation process. T h e niicroorganism, the medium, rrace materials. steril-
ization. and aseptic techniques, fermentation equipment, the actual fermentation process, recovery of fermentation products, and disposal of fermentation wastes \cere listrd in our previous reviejv. T\vo additional factors which assume more significance each year are also given some consideration. They are cost evaluations and the application of statistics to laboratory and plant data. The Microorganism. T h e selection of a microorganism and its maintenance of viability and productivity are essential to industrial production. T h e question of stability of the organism apparently has two schools of thought. O n e believes that the culture should be completely stable, ivhile the other believes that the best yields can be obtained by use of a so-called unstable strain. Lt'hile the use of a n unstable strain imposes additional requirements of manpoiver. time: and headachcs for laboratory and plant personnel, it may more than repay the cost by delivering exceptionally high yields of a product-a
Table IV. Some Produced by Antibiotic Streptomyces sp. .\ctiduins dctinomyces subtropieus Albomycin Amphomycin Strefitornyces canus Amphotericins X & B Streptomycrs sp. Streptomyces eurythermus .L\ngolamycin Antibiotic 8 9 9 Streptomyces aminophilus Antibiotic 1968 Streptomyces sp. Antibiotic X465 Antibiotic Substance Escherichia coli Antibiotic Substance E. coli Bacillus pumilis Antibiotic Substance Antibiotic Substance Streptomyces (No. TVC 3628) Streptomyes hawaiiensis Bryamycin Psalliota campestris Canipestrin Streptomyces splieorides Cathomycinb Cereine Sfreptornyces (KO. A 419) Chrysomycin Pseudomonas antimycetica Comirin St rtp t om-yces Jradiae Dextromycin Eulicin Streptomyces sp. Filipin Streptomyces jlipinensis Geomycin Streptomyces xanthophaeus liegacine lfonilin Streptomyces sp. hiycolutein Streptomyces sp. Streptomyces sp. Mycothricin Streptomyces narbonemis Narbomycin Streptomyces garyphalus Oxamycin,& Streptomyces griseus PA-93 PA-9 4,J Streptomyces laaendulae Streptomyces sp, P.4-114.4 & B Pumilin Bacillus pumilis Ramnacin Streptomyces sp. Rubidin Streptomyces sp. Soframycin S.laiendulae Spiramycin Streptomyces am bofmiens Streptomyces lydicus Streptolvdigin Streptonivicinb Streptomyces niceus Tetain Bacillus pumilis Thiostrepton Streptomyces sp. Trickothecin Tricothccium roseum Usno Reindrer lichrn Valinomvcin Streptomyces fulrsissimus Vancomycin Streptomyces orientalis a Oxamycin, PA-94, and cycloserine are identical ( 8 3 H ) . b Cathomycin, PA-93, and streptonivicin are identical ( l07H).
consideration which is important i n this age of intense comprtirion. I n the biosynthesis of antibiotics by submerged culture, there is a limit to the yield that any strain is capable of giving. Antibiotic manufacturers therefore have come to regard fermentation and strain improvement as an essential part of their production program. T h e presrnt linou.ledge of the genetics involved in thr biosynthesis of antibiotics is meager and research i n this field is in its infancy. The comparatively recent discovery of the part parasexual proccsscs could play in the breeding of neIv strains has disclosed many possibiiitirs of development. I t is evident that a new rra in industrial microbiological research is in siqhr. Dickinson ( 9 J ) has recently given :In excellent report on strain-iniplovt,mrnt research in the ;intibiotic industry. T h e genetics of Penicillium c h T ) s o g t w m . particularly on hcterokaryosis, and on segregation, and recombination from a heterozygous diploid was the subject of reports b!- Scrmonti (56.1: 57.J).
Recent Antibiotics Principally A c h e against Gram-positive bacteria Gram-positi\re & negative bacteria Gram-positive bacteria Fungi Gram-positive bacteria Gram-positive bacteria Fungi Act inom)-cetes Lfycobacterium tuberculosis Bacteria Gram-positive bacteria Gram-positive Sr negati\-e bacteria Gram-positive bacteria Gram-positive Br negativr bacteria Gram-positive bacteria
Bacteriophages Fungi Gram-positive Sr negative bacteria Fungi Fungi Gram-negative bacteria Candida sp. Fungi Bscteria and fungi Gram-positive bacteria Gram-positive & nqative bacteria Gram-positive bacteria Gram-positive Sr negative bacrei i,i Gram-positive bacteria Gram-positive bacteria Gram-positive bacteria Gram-positive bacteria Gram-positive Br negatix bacteri'i Gram-positive bacteria Gram-positive bacteria Gram-positive bacteria
Gram-positive bacteria Fungi .llycobacterium tubercuIosi3 Gram-positive bacteria
VOL. 48, NO. 9, P A R T II
(35" (40H. 65H, (179Hi (173H1 (27H~ (73H) (2H1 1 72H I (112H, ( 7OOH 1 ( 4 7 ~ ) (7OH, 1 7 i H ) (77H) (95H)
(53H) (33H i (20H. 2.3H) (80H'
SEPTEMBER 1956
1595
UNIT PROCESSES REVIEW Parasexual processes leading to a grnetic recombination other than via sexual reproduction for Stret/tonzjces coelicoior was reported b y Serrnonti and Sermonti (584. The formation of new recornbinants (stable green strains) from hetcrokaryons bcnveen albino and yello\v-type mutant strains in Aspevgdius sujne was investigated by Isitani (3I J ) . Iguchi (32J>.33/) studied tht. rriorphological chai1gt.s in induced mutants. Some 202 mutants were produced by ultraviolet, x-ray, and nitrogen inustard treati~icntof A . sojcie. More rhan ?Oyoof t!ic mutants had a decreased enzyme ac tivit)-. Studies by Glvatkins arid Gottlirb production of nlutants of S. z~t.nezuelar. Spore suspcnsions Jvere exposed io 25,000 to 300,000 y of x-rays at 50 y pcr second; 3G0,OOO y killed spores. Onl!. about 0.5 vivors failed to prodi amoiints of c~hloramphenicol i n liquid shaken culture. Xbout 17;) were loxv producers. LIutation resulted most frequently in loss of sporulation: loss of hro\z.n melaninlike pignient j and irregularity of the colony accompanied by flatness, reduction in size, and soft consistcncv! indicating mycelial fragmentation. After four to six srrial transfers, 3'; of mutants lacking brown pigment regained the pignient, one out of eleven bactcrialike niutants reverted to the parent while 44yGof the nonsporulating mutants regained sporulation. .A symposium on radiation rffects on crlls and bacteria was revie\v)- Hartsell ( 2 4 J ) . Xltliougii tht,rc. ; i w s u i i i c disadvantagvs to the its(' ul illis inethod. including incrvastd ~xissibilities of containinatcd cult~irvsc t u r t o laid t;: stcrilization of oil flasks. and the need of upright s t o r q c racks. i l i t s advantages of simplicity and successful preservation for periods of mort. thari >.rars of cultures not prcst-i,vt,d h y l!-op!iilization! havc r n c o i i r a g d \vidcspead use of this technicjue. 'The nxiintrnancc of protozoa and alqac \vas ricscrihcd by Hutner and Pi-ovasoli i ? 7 J ) . \vho pointc'd out that it is a good lxacticc. not io use chemically de1inr.d invdia f o i - iilainrrnancr because not onl!. nrc' siniIJ1t. media grossly unphysiological: b a t also contamination ma!. be rasily. ovcrlookvd. Sharp and \\.hcatc.r (5Q.J)made a coinp r i s o n of pti!.siological ar:d sero charactcristici of 41 strains of Lnct heforr freezc-dr!-inq and aftrr reczcd r y i i : ~and stot.;rpr for 6 months. 'Their results sho\vccl rhat frr~cze-clr).in~ did not iiiatt.rially alter thr charactrLristics. Briggs and associatcs (4J ) also studied the preservation of Lact.i!iczci~liby drying. 'They paid particular at to thr, survival rate. 'The nisin influt,ncing siirvival rate MYIS tiit: si.ispending medium ; best results bring ohtaincd in horse srruin \ikans!iaya, I;. A I . , 7 ; u d j h s / . .Ifil:rcbiol.> .l/:ad h'auk S.S.S. R . 3, 3 3 (1954). ( 3 E ) 1;s-aris, J.?J . '4qr. Sci. 46, 329 ( 1 (61;) Fiigonbara, '1. (to Scientilic Kesearch Inst., Ltd.), Japan. P'itenr 4,192 (.ruiy 10, 1954). (7E) Ganglily, S . , Ganguli, T.C.? Rov. 3. C., Sci. Ind. Resiurcii (IidIa 1 13B, 847 (1954). Gray. \\-, D., Oiiio J . Si.5 5 , 212. (1 955). Harris, L. J., .Valure 176, 384 (1955 i . Hashirnoto, Yoshiro, J . T'ilaininol. 1, 4 3 (1954). Hodqkin, D. C., others, .Tuture 176, 32; (1955). Imai, K . , J . Agr. Chm.Soc. ( J a p a n ) 7, 567 (1955). Ibid., p. 570 Ibid., p. 575
(1955). 15G) Chas. Pfizer & Co., Inc.. Brit. Patent 740,838 (SOL,'. 23, 1955). (6'2) Chem. Eng. 1Veri.s 33, 3670 (1955). ( 7 G ) Dulaney, E. I.., LIc.Aleer, LV. .I., others, A j p l . Alicrobio!. 3, 336 (1955). ( 8 G ) Ibid.? p. 372. (1355). (10G) Fried, J., Florry, K . , others, J. A n . Chem. SOC.77, 4182 (1955). ( I l G ) Fried, J., 'I'horna, R.\',,Stone. 1,. ( t o iothers, I'bid., 77, 6401 (1955). Karow, E. O., Petsiavas, D. N.. Division of Agricultural and Food Chemistry, 128th Meeting, .ICs, XIinneapolis, 1955. I .i;r. Resedrch Service, miineoqraphed Circ. Scr. .4IC 372 (1954 ). (?I7; Sqiiire, J . K.: Bull, J. E'.: rjtiicrs. '.i)ctrnn: Its Properties aqd Cse in lledicine," Black\vell, c;thers,
Antibiotics
Stsroids ( l G ) Agnello.
E. J., Bloom, 13. ST., Laubach, G. D., J . A m . Chem. SOC.7 7 , 4684 (1955). (2G) Bernstein, S., Fel dman, L. I., others, Chemistry e Industly 1956, D
r .
(31G) \Ycrtstein, .\.. Ibid., 11, 465 (19.55). (32G! il'oodford. I:. F.,Research 8, 412 (1955). (33G) Zaifaroiii, .\., Chir!pillo, C. C., ot!:ers, Q b e r i e n t i a 11, 1 1 9 (1955).
1- 1- 1
- '
Bloom, B. hi., Shull, G. Xi., J.A7n.
(1H) Abraham, E. P., Newton, G. G. F., others, .\hture 176, 5.51 (1955). (2H) Xhtnad, K , , Islam, hI. l?., Ibid., 176,646 (1955!. (3Hj Ammann, A , , Gottlieb, D., others, Phytoputho/ogj45, 559 (1955). ( 4 H ) Antibiotics & Chemotherapy 6 , 135170 (1956).
VOL. 48, NO. 9, P A R T II
SEPTEMBER 1956
1601
UNIT PROCESSES REVIEW (5H) Zbid.,,p:142. (6H) “Antibiotics Annual,” pp. 136-72, Medical EncvcloDedia. N e w York, 1956. (7H) Zbid., pp. 473-840. (8H) Zbid.. pp. 909-29. (9H) Araki, T., Miyake, -4.,others, A n n . Rrpts. Takeda Research L a b . 1 3 , l (1954). (10H) Arnstein, H . R. V., Clubb, hf. E., Biochem. J . 60. xxxiv (1955). Ashton, G. C.; Foster, hi. C., Analjst80, 123 (1955). Bannerjee, A. K., Sen, G. P., Sandi: P.. “Antibiotics Xnnual,” p. 640, Medical Encylopedia, S e w York, 1956. Behrens, 0. K., Corse, J., others, J . Bioi. Chem. 175, 793 (1948). Bhate, D. S., AVature 175, 816 I
f 19-56).
( l 5 H ) Binkle\r,’S. B., A n n . Reu. Biochpm. 24, 59’, (1955). ( I6H) Borensztajn, D., M’olf, J., .Wed. DoSu iadcralna i .Mikrobiol. 7. 125 (1955). (17H) Zbid., p. 135. (18H) Bose, S. R., ,Vatme 175, 468 fl9ii) \ - - - - /
Bristol Laboratories, Brit. Patent 736,325 (Sept. 7, 1955). Brockmann, H., .4bstracts, 14th International Congress Pure and ADDlied Chemistrv. D. 122. Z;;ich. 1955. (21H) Brockmann, H., Bohnsack, G., others, Angew. Chem. 68, 70 (1956). (22H) Brockmann. H.. Xiusso, H.. .Vaturzuissenchaften 41, 451 (1954). 123Hi Brockmann. H.. Schmidt-Kastner. G., Chem.’Ber.’88, 57 (1955). ’ (24H) Buchi. J., D r u t . A j o t h . Ztg. 94, 390 and 418 (1954). ( X H i Buhr, R. P.. Putter. I., others, J . A m . Chem. Soc. 7 7 , 2344 (1955!. (26H) Burton, H. S.. Chemistry & Zndustrj 1955, 442. (27H) Celmer, IV, D.. Sobin, B. .4., “Antibiotics Annual:” p. 437, hledical Encyclopedia, New York. 1956. (28H) Celmer, IV. D., Solomons, I. A., J . A m . Chem. SOL. 77, 2861 (1955). (29H) Charney. J., Slachlowitz, R . A , , others, “Antibiotics Annual,” p., 228, Medical Encyclopedia, S e i v York, 1956. 130H) Chem. En?.. *Vews33,4922(1955). (31H) Ibid.? p. 4956. (32H) Zbid.. 34, 1698 (1956). (33H) C h m . TVmk 78, 34 (Jan. 7 , 1956). 134H) Clark. R. K.. Tatkera. M., Antibioticr C? Chemotherajj 5, 206 (193). (35H) Corbaz, R., EttlinSer, I,., others, Helrm. Chini. Acta 38, 935 (1955). (36H) Zbid., p. 1207. (37H) Courtillet. D. Sf.,Sindico, S. P., Ann insf. Pasteur 8 7 , 580 (1954). (38H) Craig, L. C., Reports, Third International Congress of Biochemistry. p. 21, Brussels, 1955. (39H) Cron, hf, J., Whitehead, D. F., others, Antibiotics 3 Chemotherajy 6 , 63 (1956). (40H) Cuckler, A . C., Frost, B. S l . , others, Ibid., 5 , 191 (1955). (41H) DeBoer, C., Dietz, A., others, “Antibiotics Annual,” p. 886, Medical Encyclopedia, New York. 1956. (42H) Delcambe, LA., Zndustrie chim. belge 19,1283 (1954). , I
1602
I
(43H) DeSomer, P., VanDijck. P., An/;biotics & Chemotherapy 5 , 632 (1955). (44H) DiAccadia, F. D., Abstracts, Third International Congress of Biochemistry, p. 150, Brussels, 1955. (45H) Dihlarco, A, Borctti, G., others, Abstracts, Third International Congress of Biochemistry, Brussels, 1955. (46H) Doerschuk, A. P., Bitler, B. A., Petty, SI. 4. (to American Cyanamid Co.). Can. Patent 517,090 (Oct. 4, 1955). (47Hj Dowling, H. F., ”Tetracycline,” Medical Encyclopedia, New York, 1955. (48H) Eagle, H.. Saz? .4.K.. Ann. Rer,. Microhiol. 9. 173 11955). (49H) ElShawi, X,,’\T;irqosko: A. J., others, Bacterid. P m c . 1955, 29. (50H) Flippin, H. F.?Ani. J . Pharm. 127, 192 (1955). (51H) Ford. .T. H . . Berev. . . hl. E.. others, J . .4m. C h m Soc. 7 7 , 5811 ( 1 9 5~, 5). \
(52H) Forsyth, \V. G. C , , Biochem. J . 59, 500 (1955). (53Hj Freeman, G. G., J . Gen. .IlicrobioZ. 12,213 (1955). (54H) Fiiiii. S.. Hitomi. H.. others, A n n . kefits. TaXcda Rrsrarch L a b . 14, 8 (1955). (55H) Fuller, A. T.. .Vaturp 175, 7 2 2 f l-9.-i S- ),. \
(56H’i Glaso Laboratories, Brit. Patent 730,185 (Ilfay 18,1955). (57H! Gold, \V., Stout. H. A . others, “Antibiotics Annual,” p. 579, Medical Encyclopedia, New York, 1956. (58H) Goldberq, hi. LV.! Sternbach, L. H., others. Abstracts. 14th International Congress Pure and Applied Chemistry, p. 123, Zurich: 1955. (59H) Goss, IV. .-I,,Katz, E.. IVaksman, S. :I,.Proc. .\‘atl. Acud. Sci. U S . 42, 10 (1956). Gots, J. S.,Bird, T. J.. l i u d d , S., Biochem. Biophyc Acta 17, 419 (1955). Gourevitch. A , , Lein. J. (to Bristol Laboratories), U. S. Patent 2.712.517 iJulv 5, 1955). Gourevitch, A,; lfis’iek.’hL., Lein, J., Antibiotics 3 Chemotherapy, 5,448 (195.5). Hahn, F.. Abstracts, ‘Third International Congress of Biochemistry. p. 92, Brussrls, 195.5. Harned, K. L.. Hidp, P. H.. LaBabv, E. K.. Antibiotics 3 Chemothciab~5. 204 (1955)
Buchanan. J. 5 1 . J . A m . ?‘hem. Soc. 7 7 , 501 (1955 I. 167H ) Herrell. LV. E., ‘.Crvthromvcin,” Sledical Encyclopedia, ’ New York, 1955. (68H) Hidv, P. H.: H o d q . E. B.. others, J . Am. Chem. Soc. 7 7 . 2345 (1955). (69H) Hobby, G. L., z4nli. .Y. Y. h a d . Sci. 31, 181 (1955). (?OH) Hochstein, F. A , , Regna, P. P., J . Am. Chem. SOC. 7 7 , 3353 (1955). ( 7 l H ) Imhausen & Co., G.m.b.H., Brit. Patent 728,734 (.April 27, 1955). (72H) Jarvis, F. G., Johnson, X I . J., J . A m . Chem. SOC. 69, 3010 (1947).
INDUSTRIAL AND ENGINEERING CHEMISTRY
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