A New Industrial Enzyme Preparation

INTRODUCTION OF GS – CHITOSANASE — A NEW INDUSTRIAL ENZYME PREPARATION

  GS-CHITOSANASE is a new type of industial enzyme preparation developed by the company. The enzyme preparation is mainly used for making high quality chitosan oligosaccharides (amino- oligosaccharins) produced by the method of specific enzymolysis degradation.

  Unique feature

1. The strains producing the enzyme preparation is a Bacillus subtilis GF07 that does not produce toxin and is not pathogenic to humans, as a result it is safe to use.

2.The main active ingredient of GS-CHITOSANASE is chitosanase（EC 3.2.1.132）that is a enzyme with specificity endo-hydrolysis activity against chitosan and the exo-hydrolase activity of exo-β- glucosaminidase （EC 3.2.1.165）and β-N- acetyl-hexosaminidase（EC 3.2.1.52）has not yet detected in the enzyme preparation. As a result, GS-CHIYOSANASE is very suitable for the preparation of chitooligosaccharides.

3. The main enzymolysis products of GS-CHITOSANASE against chitosan are the chitosan oligosaccharides with degree of polymerization from 2 to 10.In which the relative yield of oligomer with DP (5-10) can be as high as 40-80%.

  Use

1 GS-CHITOSANASE is very suitable for contral of the enzymolysis on chitosan to make some product of chitooligosaccharide with special polymerization degree and narrow molecular weight distribution.

2 GS-CHITOSANAE is also used to produce of water-soluble low-molecular-weight chitosan.

  Examples of application

Example 1

Under normal circumstances ，the chitooligosaccharide ,obtained from chitosan hydrolysis more fully by GS-CHITOSANSE, is a product of average molecular weight 1000, dispersity 1.06 and polymerization degree distribution from 3 to 12.

① The test result of high-performance GPC of the product

HPGPC spectrum （determinated by  College of Resource and Environmental Science, Wuhan University  Chitin Research and Development Centre of Wuhan University）

   Data of HPGPC

Weight-average molecular weight                          1005.6

Number-average molecular weight                          942.3

Molecular weight distribution of the product                 1.0670

  ②The test result of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry of the product

  Fig1  MALDI-TOF-MS （determinated by Beijing MS centre ,Institute of Chemistry Chinese Academy of Sciences ）

 Fig 2  Date analysis of MALDI-TOF-MS（the digital at the top of ion peak of chitosan oligomer in the spectrum show the polymerization degree of the oligomer .）

Fig 3 The polymerization degree distribution and relative content of the chitooligosaccharides in the product

 Fig 4 The relative content of homogeneous chitosan-oligosaccharides in the product

 Example 2

The product, obtained from GS-CHITOSANSE limited hydrolysis against chitosan and grading separation according to molecular weight size of the oligomer, may be chitooligosaccharides of average molecular weight 2500, dispersity 1.15 and polymerization degree distribution from 6 to 9.

① The test result of high-performance GPC of the product

HPGPC spectrum （determinated by  College of Resource and Environmental Science, Wuhan University  Chitin Research and Development Centre of Wuhan University）

 Data of HPGPC

Weight-average molecular weight                          2352.8

Number-average molecular weight                         2041.6

Molecular weight distribution of the product                 1.15240

  ②The test result of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry of the product

Fig1  MALDI-TOF-MS （determinated by College of Life Science  Hunan Normal University ）

 Fig 2  Date analysis of MALDI-TOF-MS（the digital at the top of ion peak of oligomer in the spectrum show the polymerization degree of the oligomer .）

Fig 3 The polymerization degree distribution and relative content of the chitooligosaccharides in the product

 Example 3

An domestic enterprise small-lot produced chitooligosaccharides by using GS-CHITOSANASE, the testing results of matrix-assisted laser desorption ionization / time-of-flight mass spectrometry shows that all oligomer is uniform chitosan oligosaccharides with polymerization degree distribution from 2 to 9 , the relative content of chitohexaose is high 20% in the product.

Pig The polymerization degree distribution and relative content of the chitosan oligosaccharides in the product

Standard

* The measurement of GS-CHITOSANAE activity is based on the reducing amount of the chitosan substrate in unit time. One unit of chitosanase in the enzyme preparation is defined as the amount of enzyme required to transform 1mg of chitosan into water-soluble components  in one min at 45℃，pH 5.5.