DEVELOPMENT OF ECOFRIENDLY TECHNOLOGIES

1

• DEVELOPMENT OF ECO-FRIENDLY TECHNOLOGIES
• APPLICATION OF FUNGAL ALKALINE PROTEASE IN
  LEATHER INDUSTRY
• Dr. Vasanti V.Deshpande
• NCL-CLRI COLABORATIVE EFFORT

2

• WHY DEVELOPE ECOFRIENDLY TECHNOLOGIES
• ENVIRONMENTAL POLLUTION
• A Serious Problem
• ENVIRONMENT
• Land, Air, Water
• MAN POLLUTES HIS OWN ENVIRONMENT
• CHEMICAL INDUSTRIES.
• Cause Significant Pollution
• DEVELOPMENT OF TECHNOLOGIES
• Environmentally safe
• Minimum health hazards
• NATIONAL BOARD FOR POLLUTION CONTROL
• Protection of Environment
• Use of Biotechnological Approaches

3

• LEATHER INDUSTRY
• LEATHER
• Export commodity
• Foreign Exchange earner
• Thrust sector
• ENVIRONMENTAL POLLUTION
• Caused by Tannery Effluents
• CLOSURE OF SEVERAL TANNERIES
• DEVELOPMENT OF CLEANER TECHNOLOGIES
• Demand of the present day

4

• BEAM HOUSE OPERATIONS
• PRETANNING PROCESSES FOR LEATHER MANUFACTURE.
• RAW HIDES / SKINS
• ? Soaking ? Bating
• ? Liming ? Pickling
• ? Dehairing ? Degreasing
• ? Chromium tanning/Finishing
• TOXIC CHEMICALS USED
• ? Lime ? Sodium Sulphide
• ? Caustic soda, salts etc
• CLEANER TECHNOLOGIES FOR
• ? Leather Treatment
• ? Effluent Treatment

5

• LEATHER PROTEINS
• COLLAGEN
• A principal protein constituent of hides and
  skins
• ASSOCIATED PROTEINS
• Globular Albumins, Globulins, mucoids
• Fibrous Elastin, Keratin, Reticulin
• REMOVAL OF NON-COLLAGENOUS CONSTITUENTS
• By pretanning operations
• COLLAGEN CONTENT
• Decides the Quality characteristics of
  leather

6

• ENZYMATIC PROCESS FOR LEATHER TREATMENT
• ADVANTAGES OF USING ENZYMES
• ? Specificity ? Absence of Undesirable Products
  
• ? Activity under mild conditions
• ? Products of improved quality
• ? Reduction of Hazardous Polluting chemicals
• ? Biodegradable
• ALKALINE PROTEASE
• ? Potential Role in Pretanning Processes
• MICROBES
• Ideal Source of Proteases
• Rapid Propagation
• Limited space for cultivation
• Easy Genetic Manipulation

7

• NCLS FUNGAL ALKALINE PROTEASE
• Special features
• High yield (30Ku/ml)
• Short fermentation cycle (48h)
• Lower temperature optimum
• Fungal origin
• Ease of downstream processing
• Evaluation trials conducted at CLRI
• Positive Results
• Potential for Application in Leather
• Processing

8

• TRANSLATION OF LABORATORY PROCESS INTO INDUSTRIAL
  REALITY
• PILOT SCALE PRODUCTION OF ENZYME
• PROCESS FOR EVALUATION IN LEATHER PROCESSING
• INCREASING PRODUCTION OF ENZYME
• Conventional Mutagenesis
• R-DNA Technology

9

• BIOTECHNOLOGICAL DEVELOPMENT OF ALKALINE PROTEASE
• Bench to Industrial Process
• Multidisciplinary Task
• NCL Pune
• Expertise in
• ? Microbiology ? Biochemistry
• ? Molecular Biology
• Ready Access to
• ? Biochemical Engineering
• ? Process Development
• CLRI Chennai
• ? Pioneer in leather technology
• ? Infrastructural facilities for evaluation
  of protease for leather application
• ? Good rapport with tanneries

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• SCALING UP OF ENZYME PRODUCTION
• SHAKE FLASKS LARGE FERMENTORS
• Optimization of fermentation parameters
• pH 6-7
• Temp 280C
• Medium components C/N sources, Inducer
• Production in 14L Fermentor
• Dissolved oxygen 60-100
• Agitation 350-450 rpm
• Aeration 3.5-4 lpm
• Period 40h

11

• PRODUCTION IN 150L FERMENTOR
• TWO STAGE INOCULUM
• DO 60-100
• Agitation 100-10 200 rpm
• Aeration 3-4-5 lpm
• Time 22h
• Final pH - 8.2
• Maximum activity 22 Ku/ml
• DOWN STREAM PROCESSING
• Microfiltration - Gravity Filtration
• Concentration - Ammonium Sulphate
• Precipitation

12

• ENZYME PRESERVATION AND STABILIZATION
• Preservation
• Important for ? Prolonged Shelf Life ?
  Commercial Applications
• Common Preservatives
• Unable to prevent microbial growth
• Nipacide
• Effective in preventing bacterial and fungal
  growth
• Stabilization ? Glycerol and Sorbitol
• Effective in increasing half life
• ? Enzyme more stable at pH 7.0
• ? Ammonium Sulphate conferred Maximum
  stability

13

• LEATHER PROCESSING USING NCLS ENZYME AT CLRI
• Enzyme used for
• ? Soaking ? Dehairing ? Bating
• Goat / Sheep Skins, Cow / Buffalo Hides
• ? Cut Pieces ? Full Skins
• Performance by
• ? Visual Assessment ? Histology Studies
• ? Physical Appearance
• Tests Are Carried Out at
• ? CLRI LAB ? CLRI Model Tannery
• ? Commercial Tanneries
• Multilocational tanneries

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• ENZYMATIC SOAKING PROCESS
• Soaking of Hides and skins
• Cleaning and Softening with water
• Removal of globular proteins
• Conventional Soaking
• Water containing
• Sodium Hypochlorite
• Sodium Pentachlorophenate
• Formic acid
• Enzymatic Method
• NCLs Enzyme and Wetting agent
• ADVANTAGES
• Loosening of scud,
• Opening of fibre structure
• Leather with less wrinkled grain
• Decrease in soaking time

15

• ENZYMATIC DEHAIRING PROCESS
• Dehairing of hides and skins
• Main operation in the Beamhouse
• CONVENTIONAL CHEMICAL METHOD
• Efficient ? Uses Lime and Na2S
• Leads to Pulping of hair.
• DISADVANTAGES
• Significant contributions to tannery pollution
  load
• Sulphide Toxic with obnoxious odour
• Alkaline condition- Health hazard.

16

• ENZYMATIC DEHAIRING
• Environment Friendly
• Facilitates recovery of good quality hair
• DEHAIRING WITH NCLS ENXYME
• Significant reduction in use of Na2S
• Reduced pollution
• Quality comparable with chemical process

17

• ENZYMATIC BATING PROCESS
• Objectives of Bating
• To remove non-leather forming proteins
• To allow splitting up of collagen fibres for
  penetration of tanning materials
• Conventional Method
• Use of animal dung or manure
• Disadvantages
• Unhygienic
• Difficult to control fermentation
• Enzymatic Method
• Visual Assessment Silky grain with good
  flaccidity Positive air pocket test
• Histological Studies Removal of epidermis
• opening of fibre structure

18

• COMMERCIAL TRIALS
• Commercial tanneries selected
• M/s K.A.R. Leathers (P) Ltd.
• M/s Saddique Leathers
• M/s C.K.C.M. Khadersha Bros.
• M/s Shri Ramajayam Prime Tanners
• Situated in and around Dindigul
• A leather belt in South Tamilnadu
• Dehairing Experiments
• In four tanneries
• Soaking Experiments
• In Two Tanneries
• Results Show
• NCL Protease is suitable for using In
  Pretanning Processes of Leather Manufacture

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• INCREASING PRODUCTION OF ENZYME
• USE OF CONVENTIONAL MUTAGENESIS
• Isolation of Hyperproducing Mutant
• By UV Double Mutagenesis
• Optimization of Production
• Using Factorial Design
• Comparision of parent and mutant enzyme
• Identical pH optimum
• Temperature optimum
• Temperature stability

20

• INCREASING PRODUCTION OF ENZYME
• USE OF RECOMBINANT DNA TECHNOLOGY
• Identification of Protease Gene in E. coli
• Construction Genomic Library of Fungus
• In E. coli using pGEM - 7Z f ()
• Isolation and Purification of Enzyme
• N-Terminal Sequence Analysis
• Preparation of a DNA probe
• Screening of library
• Preparation of antiprotease antibodies.
• Study of gene expression
• Cloning in yeast
• For extracellular production

21

• CONCLUSION
• Present
• Change over of Chemical Technologies
• into Enzyme Technologies
• A Slow process
• Due to high cost of enzyme production
• Near Future
• Combination of chemical and enzymatic
  processes
• Future
• May witness ecolabelled leather products

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• PARTICIPANTS
• NCL
• Investigators
• Dr. (Mrs.) V.V. Deshpande
• Dr. (Mrs.) Mala rao
• Dr. B. Seeta Rama Rao
• Dr. (Mrs.) R.Seeta Laxman
• Dr. (Mrs.) M.V. Rele
• Dr. V.V. Jogdand/Dr. R.V. Gadre
• Scientific Staff
• Dr. H. Balkrishnan
• Mrs. S. More