Streptomycin production

streptomycin production, uses, disadvantages , medium, inoculum preparation, commercial production, harvest and recovery process, biosynthetic pathway from glucose to streptomycin, flow sheet of streptomycin production by submerged culture method, chemical structure of streptomycin,
which functional unit have antibiotic activity?

1. Mrs.K.Sudha Rameshwari,
Assistant Professor, Department of PG Biochemistry,
V.V.Vanniaperumal college for women, Virudhunagar, Tamilnadu

2. Introduction
•Streptomycinisanantibiotic
•ItwasdiscoveredbySchatz,BugieandWaksman(1944)in
oneofthesoilisolatesStreptomycesgriseus
•Mostoftheindustryusedthisparentstrainforstreptomycin
productiontodayalso.
•Mutationandselectionemployedtoincreaseyieldstothe
presentdaylevels.
•But,nowadaysalsoStreptomycinisproducedbystreptomyces
griseusandonlyafewstrainsofthisorganismhavethe
abilitytoproducereasonableyieldsoftheantibiotic
•ItisactiveagainstgramPositiveandNegativebacteriaand
againstthetuberculosisorganismMycobacteriumtuberculi

3. Uses
Ithasbeenusedtherapeuticallyinthetreatmentof
infectionscausedbyorganismsresistanttoPenicillin
Itisalsousedinthetreatmentofplantdiseases
causedbybacteria
Itisusedinthetreatmentoftuberculosiscausedby
Mycobacteriumtuberculi.

4. Disadvantage
Inman,prolongedstreptomycintreatment
athighdosagecanproduceneurotoxic
reactions
particularlyproblems inbalance
maintenanceandpartialhearingloss
Chemicalreductionofstreptomycinto
dihydrostreptomycindecreasesthis
neurotoxicity.

5. Somemicroorganismsgainresistance
relativelyeasilytostreptomycinand
streptomycintherapy.
So,itiscarriedoutinconjuctionwithpara
aminosalicyclicacidorisoniazid
(isonicotinicacidhydrazide)todecrease
thisresistancebuildupinsensitive
microorganisms

6. Chemical structure
•Streptomycinanddihydrostreptomycinarebasic
compounds
•Theyareusuallypreparedassalts
•Streptomycinisavailableasthehydrochloride
C21H39N2O12.3HCLasacrystallinehydrochlroide
doublesaltwithcalciumchlorideorasthe
phosphateorsulfate
•Dihydrostreptomycinasthehydrochlorideor
sulfate

7. Dependingonthestrainofthisorganism
beingusedorontheproductionofmedium,
smallamountsofmannosidostreptomycin
orhydroxystreptomycinareaccumulatedin
additiontostreptomycin
Somemannosidostreptomycinisproduced
earlyinthefermentation,butthisantibiotic
islargelyenzymaticallydegradedby
Streptomycesgriseustosterptyomycinatthe
timeofharvest
Themannosidostreptomycinisnotdesired
becauseofitslowantibioticactivity

8. Theuseofprecursorsdoesnotincreaseyieldsof
streptomycin
Mostofthecarbonofthestreptomycinmolecule
hasshowntooriginatefromglucoseandnotfrom
themorecomplexcarboncompoundsofthe
medium,althoughsomeofthecarbonmolecule
originatefromcarbondioxide
Thecarbonylfunctiononthestreptosemoietyis
involvedintheantibioticactivityofstreptomycin.
Mostchemicaladditionstothecarbonylgroup
destroytheantibioticactivity.

9. Chemical Structure of Streptomycin
Streptomycinischaracterisedchemicallyasan
aminoglycosideantibiotic.
Itconsistsofthreecomponentslinkedglycosidically
(byetherbonds):
(i)Streptidine(inositolwithtwoguanidogroups),
(ii)Streptose(methylpentose),and
(iii)Streptoscamine(N-methyl-L-glycosamine)asshown
inFig.1
Bothguanidogroupsofstreptidineareessentialforthe
antibioticactivityandremovalofonegroupreduces
antibioticactivityupto90%.
Dihydrostreptomycinisproducedbyreductionof
carbonylgrouponthestreptosemoiety

10. Fig1. Chemical structure of Streptomycin

11. Mechanism of Action of Streptomycin:
Streptomycin, like other aminoglycosidicantibiotics (e.g.,
gentamycin, neomycin, kanamycin, tobramycin), inhibits protein
synthesis in bacterial cells by binding to the 30S subunit of
ribosomes.
Streptomycin is aprotein synthesis inhibitor. It binds to the small
16S rRNAof the 30S subunit of the bacterial ribosome, interfering
with the binding offormyl-methionyltRNAto the 30S subunit.
This leads to codonmisreading, eventual inhibition of protein
synthesis and ultimately death of microbial cells through
mechanisms that are still not understood. Speculation on this
mechanism indicates that the binding of the molecule to the 30S
subunit interferes with 50S subunit association with
themRNAstrand. This results in an unstable ribosomal-mRNA
complex, leading to aframe shift mutation and defective protein
synthesis; leading to cell death. The mechanism of inhibition of
protein synthesis by streptomycin is schematically shown in Fig. 2

12. Fig 2: Schematic representation of protein
synthesis inhibition streptomycin

13. Production –medium
Two types of medium were used
1.Woodruff and Mc. Daniel (1954)
1% soyabeanmeal
1% glucose
0.5% sodium chloride
2.Hockenhull (1963)
2.5% glucose
4% soyabeanmeal
0.5% distillers dried soluble
0.25% sodium chloride
pH -7.3 to 7.5 before sterilization

14. Inoculumpreparation
Highyieldingmutatedstrainsofstreptomyces
griseusaregeneticallyunstable,afacttobe
consideredinmaintenanceofstockcultures.
Becauseofthisconsideration,sporesofthe
organismusuallyaremaintainsoilstocksorare
lyophilizedinacarriersuchassterileskimmilk.
Sporesfromthesestockculturesarethen
transferredtoasporulationmediumtosporulated
growthtoinitiateliquidculture buildupof
mycelialinoculuminflasksorinoculumtanks

15. Factors to be consider during production
Streptomycin yields in production fermentors
respond strongly to agitation and aeration. The
optimum temperature in the range of 25oC to 30 o
C ,probably closer to 28o C
The optimum pH for streptomycin production
occurs between pH 7 & 8, high production occur in
the range of pH7.6 to 8
The fermentation production approximately 5-6
days and provides streptomycin yields probably
exceeds of 1200 micrograms per mililiter

16. Contaminants
Contaminants decrease the antibiotic yield
Actinophage

17. Biosynthesis of Streptomycin
Streptomycinisdirectlyderivedfromglucose.Thoughthe
enzymesinvolvedinthesynthesisofN-methyl
glucosaminearenotyetknown,itisexpectedthatabout28
enzymestakepartintheconversionofglucoseinto
streptomycinasprécisedinFig.3
Fig 3. Biosynthetic pathway from D-glucose to Streptomycin

18. Commercial streptomycin fermentation passes through
three phases:
I phase:
lastsapproximately24hours
Withrapidgrowthofthemycelium
Proteolyticactivityofstreptomycesgriseusreleases
ammoniatothemediumfromthesoyabeanmeal
Carbonnutrientsofthemediumutilizedforthegrowth
Glucoseofthemediumisutilizedslowlyduringthis
period
Onlyslightstreptomycinproductionoccurs
Duringthisperiod,thepHofthemediumrisesfrom
approximately6.7or6.8to7.5orslightlyhigher
STREPTOMYCIN PRODUCTION

19. II phase
Streptomycin is produced at high rate
Lasts approximately 24 hours to 6 or7 days of
incubation
Almost, no mycelium growth, weight of the mycelium
remains constant
The ammonia is utilized and the pH remains fairly
constant in a range of approximately about 7.6-8
Glucose and oxygen are required in a large quantity.

20. III phase
Sugarhasbeendepletedfromthemedium
Streptomycinproductionceases
Myceliumundergoesautolysis,releasing
ammoniaandthepHvaluerises
Thefermentation,howeverusuallyisharvested
beforecelllysis.

21. Harvest and Recovery of streptomycin
Aftercompletionoffermentationthemyceliumis
separatedfromthebrothbyfiltration.Streptomycinis
recoveredbyseveralmethods.Thechoiceofprocedure
dependingontheindustrialconcern.
Inoneprocedure,
Thestreptomycinisadsorbedfromthebrothontothe
activatedcarbonandthenelutedfromthecarbonwith
diluteacid.Theelutedstreptomycinisprecipitatedby
acetone,filteredanddriedbeforefurtherpurification.

22. Inanalternativeprocedure,
Thefermentationbrothisacidified,filtered
andneutralized.Itisthenpassedthrougha
columncontainingacationexchangeresinto
adsorbthestreptomycinfromthebroth.
Thecolumnisthenwashedwithwaterandthe
antibioticiselutedwithhydrochloricacidor
cyclohexanolorphosphoricacid.Itisthen
concentratedatabout60°Cundervacuum
almosttodryness.

23. The streptomycin is then dissolved in methanol
and filtered and acetone is added to the filtrate to
precipitate the antibiotic. The precipitate is again
washed with acetone and vacuum dried. It is purified
further by dissolving in methanol. The streptomycin in
pure form is extracted as calcium chloride complex.
Byproduct Vitamin B
12
Vitamin B
12is produced as a byproduct which will not
affect adversely the yield of streptomycin.

24. Fig 4.Flow chart of Streptomycin production by
submerged fermentation

25. REFERENCES
Industrial Microbiology -Casida
Industrial Microbiology –A.H.Patel
http://www.biologydiscussion.com/medical-
microbiology/streptomycin-discovery-structure-and-mechanism-
antibiotics/55919