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Preparation method of thermostable chitosanase

A chitosanase and thermostable technology, which is applied in the field of preparation of thermostable chitosanase, can solve the problems of difficulty in large-scale production and wide application, low enzyme yield and low enzyme activity of enzyme-producing strains, etc.

Inactive Publication Date: 2018-12-14
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the generally low enzyme production and enzyme activity of enzyme-producing strains, it is difficult to scale production and wide application.

Method used

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  • Preparation method of thermostable chitosanase
  • Preparation method of thermostable chitosanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 A kind of preparation method of thermostable chitosanase, concrete steps are as follows:

[0051] 1. Cloning of chitosanase BmCsn

[0052] (1) Obtaining the partial sequence of chitosanase BmCsn

[0053] The strain with high chitosanase catalytic activity comes from the sandy soil in the coastal zone of Yantai, and the strain is identified as Bacillus mojavensis. Since the genome sequence of B. mojavensis has not been published yet, degenerate primers were designed by using the sequence alignment of other chitosanases from Bacillus, and the upstream primer BmCsn-partialF was 5'-GAAAGCGATGACATMAGCAA-3' (SEQ ID NO. 1), the downstream primer BmCsn-partialR is 5'-ACACGGGCAACHGATTCTCTCC-3' (SEQ ID NO.2), and the partial sequence BmCsn-partial (SEQ ID NO.3) of B. mojavensis chitosanase is obtained. Sequencing results showed that the partial sequence was the partial sequence of chitosanase BmCsn.

[0054] (2) Chromosome walking method to obtain the full-length ...

Embodiment 2

[0066] Embodiment 2 A kind of preparation method of thermostable chitosanase, concrete steps are as follows:

[0067] 1. Culture medium preparation

[0068] (1) Solid preservation medium: yeast extract powder 5g / L, peptone 10g / L, NaCl 5g / L, kanamycin 100mg / L, agar 20g / L, pH 7.0, autoclaved at 121°C for 20min;

[0069] (2) Liquid seed medium: yeast extract powder 1-10g / L, peptone 10g / L, NaCl 5g / L, kanamycin 100mg / L, pH 7.0, sterilized by high pressure steam at 121°C for 20min;

[0070] (3) Fermentation medium: glucose 20g / L, yeast extract powder 10, peptone 20g / L, MgSO 4 40.5, NaCl 2.5, KH 2 PO42, CaCl 20.1, pH 6.5, sterilized by high pressure steam at 121°C for 20min.

[0071] 2. Strain activation: pick the chitosanase engineering bacteria BL21-BmCsn strain obtained in Example 1 and inoculate it into a solid preservation medium by streaking and inoculating it in a constant temperature incubator at 37° C. for 14 to 20 hours;

[0072] 3. Preparation of liquid seeds: pick the...

Embodiment 3

[0075] Embodiment 3 A kind of preparation method of thermostable chitosanase, concrete steps are as follows:

[0076] (1) The acquisition of chitosanase engineered bacteria BL21-BmCsn is the same as in Example 1.

[0077] (2) The preparation of the culture medium is the same as in Example 2.

[0078] (3) The fermentation condition of chitosanase is as follows: insert liquid seed into fermentation medium by 5% inoculum size, rotating speed 200r / min, pH7.0, bacterium liquid cultivates 2-3 hour at 37 ℃, to OD 600 Add 0.3% lactose to induce at ≈0.3-0.5, cool down to 28°C after lactose induction, and ferment for 16 hours. After the end, centrifuge at 6000rpm for 10min to obtain wet bacteria containing chitosanase, and the activity of chitosanase reaches The highest value is 750U / mL.

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Abstract

The invention discloses a preparation method of thermostable chitosanase. The method comprises the following steps: cloning a chitosanase gene having a high catalytic activity and a lasting normal-temperature enzyme activity, constructing a chitosanase engineering bacterium having a high catalytic activity and a lasting normal-temperature enzyme activity, and preparing the chitosanase BmCsn havinga high catalytic activity and a lasting normal-temperature enzyme activity. The chitosanase has an obvious stability, and the enzyme activity of the chitosanase undergoing heat insulation at 28 DEG Cfor 7 d is only 5% less than the initial activity. The stability of the chitosanase is higher than the stability of reported general products. After the fermentation conditions of the chitosanase areoptimized, so the protein content reaches up to 4.5 g / L. After the chitosanase undergoes high-density fermentation, the enzyme activity reaches up to 834 U / mL fermentation solution.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering and relates to a preparation method of thermostable chitosanase. Background technique [0002] Oligochitosan is the only basic amino oligosaccharide found in nature, and it is a kind of animal cellulose. Due to its good solubility, strong anion exchange capacity, and high biological activity, chitosan oligosaccharides have been widely used in the fields of medicine, food, agriculture, and chemical industry. At present, there are mainly physical, chemical and biological methods for the preparation of chitosan oligosaccharides. The physical method is simple and easy to operate, but it is difficult to control the production of chitosan oligosaccharides with a specific molecular weight, and the waste of raw materials is serious. The chemical method reacts quickly, but the reaction conditions are harsh, the cost is high, and the pollution to the environment cannot be ignored. Relatively ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70
CPCC12N9/2402C12N15/70C12Y302/01132
Inventor 张娟冯志彬段春利
Owner LUDONG UNIVERSITY
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