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Production of Lysine Amino Acid
- 2. INTRODUCTION AN OVERVIEW OF AMINO ACIDS AND LYSINE
- 3. • AMINO ACIDS ARE THE MONOMERIC SUBUNITS BUILDING BLOCKS OF PROTEINS. ALL PROTEINS IN OUR WORLD ARE MADE FROM THE UBIQUITOUS SET OF 20 AMINO ACIDS • AMINO ACIDS ARE ORGANIC COMPOUNDS, THEY HAVE TWO FUNCTIONAL GROUPS, A CARBOXYL GROUP (-COOH) AND AN AMINE GROUP (-NH2) BONDED TO THE SAME CARBON ATOM (THE α CARBON) • THE AMINO GROUP IS BASIC IN NATURE AND THE CARBOXYL GROUP IS ACIDIC IN NATURE • THEY ALSO CONTAIN A HYDROGEN ATOM AND A SIDE CHAIN (R GROUP) WHICH IS SPECIFIC TO EACH AMINO ACID. • THE KEY ELEMENTS OF AN AMINO ACID ARE CARBON (C) , HYDROGEN (H) , OXYGEN (O) , AND NITROGEN (N). • ACCORDING TO THE REQUIREMENTS OF THE AMINO ACIDS IN DIET , THEY ARE DIVIDED INTO TWO CLASSES - ESSENTIAL AMINO ACIDS AND NON ESSENTIAL AMINO ACIDS. WHAT IS AN AMINO ACID ? 3
- 4. WHAT IS LYSINE ?? 4 H I H2N—C—COOH I CH2 I CH2 I CH2 I CH2 I NH2 LYSINE (Lys, K) • L- LYSINE (2,6-DIAMINOHEXANOIC ACID) IS AN ESSENTIAL AMINO ACID FOR THE NUTRITION OF HUMANS AND OTHER ANIMALS. OTHER NAMES OF LYSINE ARE Lys , K AND HAVING MOLECULAR FORMULA (C6H14N2O2) • IT IS USED FOR SUPPLEMENTING THE CEREAL PROTEINS LACKING THIS AMINO ACID. • THIS AMINO ACID IS USED MEDICALLY AS A NUTRIENT IN FORM OF SUPPLEMENTS AND MEDICINES • LYSINE IS PRESENT IN TWO FORMS , L - FORM AND D - FORM
- 5. HISTORY… 1889 • LYSINE FIRST ISOLATED FROM CASEIN 1956 • LYSINE WAS COMMERCIALLY INTRODUCED AS FEED 1978 • THE FIRST PRODUCTION OF FERMENTED L-LYSINE WAS PRODUCED BY A JAPNESE COMPANY KYOWA HAKKO KOGYO 5
- 6. PRODUCTION OF LYSINE OVERVIEW OF LYSINE PRODUCTION NATURALLY AND INDUSTRIALLY 6
- 7. • PRODUCTION OF LYSINE NATURALLY , TAKES PLACES BY TWO BIOSYNTHETIC PATHWAYS 1. THE DAP (DIAMINOPIMELIC ACID) PATHWAY FOUND IN BACTERIA (e.g. Corynebactrium glutamicum), ALGAE , HIGHER PLANTS AND OOMYCOTA 2. THE AAA ( α – AMINO – ADIPATE) PATHWAY FOUND IN FUNGI AND EUGLENOIDS 7 NATURALLY (LYSINE BIOSYNTHESIS)
- 8. 8
- 9. I U F D O INBOUND LOGISTICS 9 UP STREAM PROCESSING • CULTURE OPTIMIZATION • MEDIA FORMULATION FERMENTATION DOWN STREAM PROCESSING OUTBOUND LOGIDTICS • RAW MATERIALS • MICROBIAL CULTURE • MEDIA • MEDIA UTILIZATION & LYSINE PRODUCTION • SEPERATION • EXTRACTION • PURIFICATION • PACKAGING OF L- LYSINE
- 11. • FERMENTATION MEDIA CARBON SOURCE CANE MOLASSES NITROGEN SOURCE CORN STEEP LIQUOR / SOYABEAN MEAL MINERAL AND SALTS KH2PO4 / K2HPO4 / CaCO3 TRACE ELEMENTS CORN STEEP LIQUOR ANTI FOAMING AGENT PEG-2000, SILICONE BASED OIL. • INSTRUMENTS 11 RAW MATERIALS
- 12. • IT IS A GRAM POSITIVE BACTERIUM • IT IS ROD SHAPED BACTERIUM • THEY LIVES IN CHAINS OR IN CLUSTERS • THE BACTERIUM IS USUALLY PRESENT IN SOIL • IT IS NON MOTILE • THE BACTERIUM IS A NON SPORE FORMER • THE BACTERIUM IS A NON PATHOGENIC CULTURE ( Corynebacterium glutamicum ATCC 21526) 12
- 14. MEDIA FORMULATION 14 INGREDIENTS AMOUNT (GRAM / LITRE ) GLUCOSE 100 g UREA 5 g BIOTIN 0.01 g LEUCINE 0.4 g MgSO4 2.85 g KH2PO4 / K2HPO4 0.5 g MnSO4 0.016 g CaCO3 20 g (NH4)2SO4 46 g DISTILLED WATER 1000 L MEDIA PREPRATION FOR GROWTH OF CULTURE AND INOCULLUM DEVELOPEMENT
- 15. • DONE BY GENOME SHUFFLING • RIBOSOME ENGINEERING • PROTOPLAST FUSION CULTURE OPTIMIZATION DONE TO OVEREXPRESS THE EXPRESSION OF INDIVIDUAL BIOSYNTHETIC GENE Lys C AND Dap A 15
- 16. INOCULLUM DEVELOPEMENT 17 500 ml 1 litre L . F 5 L SEED FERMENTER 50 L PILOT FERMENTER 100 L 100 ml PRE CULTURE SLANT TO PRODUCTION FERMENTER
- 17. FERMENTATION 17
- 18. CHARACTERISTICS OF THE FERMENTATION FERMENTATION PROCESS SUBMERGED FERMENTATION AEROBIC FERMENTATION MODE OF OPERATION BATCH PROCESS FED-BATCH PROCESS FERMENTER TYPE STIREDTANK FERMENTER AIRLIFT FERMENTER PROCESS PARAMETERS OPTIMUM PH 7.2 OPTIMUM TEMPERATURE (35 – 37)° C TIME 100 HRS PRODUCTION CYCLE 18
- 19. • FERMENTATION PERFORMED IN FED-BATCH MODE AND UNDER AEROBIC CONDITION • IN BATCH PHASE SEED CULTURE IS FED INTO THE FERMENTER PREVIOUSLY FILLED WITH FERMENTATION BATCH MEDIUM • WHEN GLUCOSE IS EXHAUSTED, THE BATCH FERMENTATION PHASE ENDS AND FED-BATCH FERMENTATION PHASE STARTS THE FERMENTATION PROCESS • CULTURE MEDIA PREPATED BY MIXING PROCESSED WATER, GLUCOSE & NUTRIENTS CULTURE CARBOHYDRATE SOURCE PRODUCE AMINO ACID 19
- 21. SEPERATION • CELL REMOVAL • L-LYSINE RECOVERY EXTRACTION • FURTHER REMOVAL OF IMURITIES • CONCENTRATION OF L-LYSINE PURIFICATION 21 • PURIFIED L-LYSINE READY FOR PACKAGING D S P - SCHEMATIC
- 22. SEPERATION OF L-LYSINE LIQUID L-LYSINE L-LYSINE SEPERATED FROM CELLS BY ULTRAFILTRATION 22 L-LYSINE SELECTIVELY ABSORBED BY ION-EXCHANGE CHROMATOGRAPHY L-LYSINE ELUTED FROM CHROMATOGRAPHY RESINS BY WASHING WITH AQUEOUS AMMONIA SOLUTION
- 23. 24 L – LYSINE HCL CONCENTRATED (ACIDIFIED L-LYSINE) L - LYSINE MOTHE R LIQUOR HCL LIQUID L - LYSINE MOTHER LIQUOR (CANE MOLASSES) HCL EXTRACTION OF L-LYSINE
- 24. PURIFICATION OF L-LYSINE ACIDIFIED L - LYSINE PURE L – LYSINE LIQUID OR POWDERED L- LYSINE OR CRYSTALINE L-LYSINE 24
- 26. PACKAGING OF L-LYSINE 25 KG BAG 50 g BOTTLE PURIFIED L-LYSINE 26
- 27. USES OF L-LYSINE 27 FIELD OF APPLICATION USED IN IN FOOD NUTRITIONAL SUPPLEMENTS, FOOD DECORANTS IN BEVERAGES NUTRITIONAL SPORTS DRINK IN PHARMACEUTICAL PREVENTING AND TREATING COLD SORES CAUSED BY HERPES SIMPLEX LABIALIS VIRUS IN HEALTH AND PERSONAL CARE COSMETICS, PERSONAL CARE PRODUCTS, BABY PRODUCTS, BATH PRODUCTS, EYE MAKEUP, SAVING KITS IN AGRICULTURE / ANIMAL FEED / POULTRY ANIMAL FEED AND USED IN FERTILIZERS IN OTHER INDUSTRIES AS INTERMEDIATE TO PRODUCE OTHER ORGANIC CHEMICALS