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Evaluation of proteases for the Production of Chondroitin Sulfate

IP.com Disclosure Number: IPCOM000192633D
Publication Date: 2010-Jan-26
Document File: 3 page(s) / 1M

Publishing Venue

The IP.com Prior Art Database

Abstract

A series of proteases were evaluated for their ability to hydrolyze animal tissue for the production of chondroitin sulfate. Samples of bovine trachea were hydrolyzed using Protex® P, Protex® 6L, and Protex® 5L proteases. The hydrolyzed material was physically separated and detection of chondroitin sulfate disaccharides was performed using capillary electrophoresis as described by Chaplin and Kennedy, 1994. The results show that a 0.75% w/w dose of Protex P equals a 0.5% w/w dose of Protex 6L in amounts of aqueous layer generated and provides a 15% increase in chondroitin sulfate yield.

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Evaluation of proteases for the Production of Chondroitin Sulfate

Abstract

A series of proteases were evaluated for their ability to hydrolyze animal tissue for the production of chondroitin sulfate. Samples of bovine trachea were hydrolyzed using Protex® P, Protex® 6L, and Protex® 5L proteases. The hydrolyzed material was physically separated and detection of chondroitin sulfate disaccharides was performed using capillary electrophoresis as described by Chaplin and Kennedy, 1994. The results show that a 0.75% w/w dose of Protex P equals a 0.5% w/w dose of Protex 6L in amounts of aqueous layer generated and provides a 15% increase in chondroitin sulfate yield.

Introduction

The general method for production of chondroitin sulfate involves extraction of animal tissue cartilage with sodium hydroxide, followed by enzymatic treatment with proteases to remove protein.

Materials and Equipment

Equipment:

Beckman Coulter ProteomeLab PA800 Capillary Electrophoresis,

Lab-Line Incubator-Shaker,

VWR SP20 Portable pH meter

Treatment Parameters:

  • Tracheal tissue weight:  100g
  • Enzyme dose:  0.5-1% (w/w)
  • Temperature:  60°C
  • pH:  8.5
  • Agitation speed:  35 rpm
  • Collected sample volume:  25 mLs
  • Total run time:  6 hours
  • Enzymes: Protex 6L, Protex 5L, and Protex P

Method

Bovine trachea was processed through a Panasonic SuperGrinder (Model MK-G20NR) using a coarse mesh screen (7 mm). The processed bovine tracheal tissue was combined with water in a 2:1 ratio. The pH of the slurry was adjusted with 20% NaOH and the material was placed in an incubator until the desired temperature of 60°C was reached. The selected enzymes were added at time=0.  The pH of the samples was measured and readjusted at time=1 hour and time=2 hours.  Samples for measuring separation and chondroitin sulfate yield were collected at time=6 hours.  Each sample was heat inactivated at 90°C for 20 minutes. The samples for measuring separation were allowed to settle for 1 hour at room temperature.

 

Results

The purpos...