Aspergillus and method for producing pneumocandin B0 by using aspergillus

A technology of pulmonary candidin and bacterial strains, which is applied in the field of microbial engineering, can solve the problems of increasing production costs, achieve high fermentation units, reduce the difficulty of purification, and achieve the effects of good genetic stability

Active Publication Date: 2017-09-26
HISUN PHARMACEUTICAL (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] WO00 / 08197 discloses the fermentation of bacterial strain ATCC74030 to prepare pulmonary candidin B 0 When using 125g / L fructose as a carbon source, the method of reducing impurities by adding amino acids and trace elements, wherein adding 15g / L proline can reduce pulmonary candidin C 0 decreased to 4.4%, but pulmonary candidin E 0 Increased to 2.7%, adding the trace element zinc makes pulmonary candidain B 0 Serine analogs and B 5 content were reduced by 1%, but pulmonary candidae 0 Pulmonary Candidain B 0 Potency is reduced by 50%, adding trace element cobalt can make pulmonary candidin E 0 content decreased from 2.2% to 1.8%, but pulmonary candidin B 0 Serine analogs and B 5 Pulmonary Candidain B 0 The potency is reduced by 25%, and amino acids and trace elements are added during the fermentation process, which increases the production cost

Method used

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  • Aspergillus and method for producing pneumocandin B0 by using aspergillus
  • Aspergillus and method for producing pneumocandin B0 by using aspergillus
  • Aspergillus and method for producing pneumocandin B0 by using aspergillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 strain source

[0040] The bacterial strain HS-2158 of the present invention is an original bacterial strain isolated from Xin'an River in Zhejiang, and a mutant bacterial strain obtained by mutagenesis.

[0041] After the original strain was cultured in PDA slant medium at 25°C for 13 days, the mycelium was scraped off with an inoculation shovel under aseptic conditions, and the mycelium was ground in a ground test tube and suspended in sterile water to obtain a bacterial suspension. solution for NTG (nitrosoguanidine) mutagenesis treatment.

[0042] Weigh 5 mg of NTG crystals, dissolve in 5 ml of sterile Tris buffer (pH 8.0), pipette 3 ml of NTG solution into 2 ml of bacterial suspension, and place on a rotary or reciprocating shaker in a medium at 25°C Shake for 30 minutes. The remaining specific steps are as follows:

[0043] (1) Preparation and cultivation of mycelia

[0044] PDA solid medium, sterilized at 121°C for 30 minutes, cooled to 50-60°C and ...

Embodiment 2

[0050] Embodiment 2 strain identification

[0051] Experiments were carried out with reference to relevant contents in books such as "General Mycology", "Handbook of Common Bacterial System Identification", "Guidelines for Molecular Cloning Experiments" and "Chinese Pharmacopoeia" (2005 edition XIH).

[0052] 1. Culture characteristics: Use 10 kinds of culture medium: ISP1, ISP2, ISP3, ISP4, ISP5, Chase, PDA, calcium malate, nutrition and Gaoshi No. 1 medium, culture at 28°C for 7 to 10 days, and observe the mycelium color and pigmentation. The culture characteristics of the strains are shown in Table 3.

[0053] Table 3 Culture characteristics of bacterial strain HS-2158 (CGMCC NO.13367) on 10 kinds of media

[0054] culture medium

growing situation

Substrate hyphae

aerial hyphae

Soluble pigment

ISP1

2

olive green

White

none

ISP2

4

olive green

yellow-green

none

ISP3

4

olive green

yellow-g...

Embodiment 3

[0081] Example 3 Strain HS-2158 (CGMCC NO.13367) When the main carbon source of the fermentation medium is sorbitol and maltodextrin

[0082] Research on the fermentation process of

[0083] (1) PDA solid medium, sterilized at 121°C for 30 minutes, cooled to 50-60°C, poured into a plate, inserted into 0.1ml of bacterial suspension and cultured for 13 days, the mycelia matured.

[0084] (2) The formulation and culture conditions of the seed medium are the same as those in step (2) in Example 1, and the culture period is 87 hours.

[0085] (3) Pulmonary Candactin B 0 Preparation and cultivation of fermentation medium.

[0086] Fermentation medium formula I is equal to step (3) in embodiment 1;

[0087] Fermentation medium formula II (g / L): sorbitol 50.0, maltodextrin 50.0, glucose 5.0, soybean cake powder 20.0, peptone 5.0, cottonseed cake powder 30.0, potassium dihydrogen phosphate 5.0, ammonium sulfate 3.0, calcium chloride 4.0 , Zinc sulfate 1.0g, L-proline 15.0, pH7.0; ...

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Abstract

The invention discloses a novel aspergillus strain and application thereof. The strain is named as Glarea lozoyensis HS-2158, and has the preservation code being CGMCC NO.13367. The strain HS-2158 provided by the invention is a high-yield strain of the pneumocandin B0; the generated byproducts such as pneumocandin C0, E0, B5, B0 serine analogues account for low percentage in the pneumocandin B0; the extraction and separation difficulty of the pneumocandin B0 is reduced, so that the strain HS-2158 has good application values.

Description

technical field [0001] The invention relates to the technical field of microbial engineering, in particular to a kind of aspergillus and its preparation of pulmonary candidin B 0 (Pneumocandin B 0 ) in the application. Background technique [0002] Caspofungin (Caspofungin) was developed by Merck, and its trade name is Cancidas. Its structural formula is shown in the following formula 1. It was first launched in the United States in 2001 and has been widely used in more than 70 countries. At present, the product has become the ace drug in the systemic antifungal drug market. [0003] [0004] Caspofungin is an inhibitor of glucan synthase (an enzyme not found in mammalian cells that is required by many pathogenic fungi to synthesize a major part of the cell wall). The enzyme can catalyze the transfer of glucan groups in uridine diphosphate to generate β-1,3-D-glucan. β-1,3-D-glucan synthase is necessary for the growth of fungi. Inhibition of this enzyme can lead to ab...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P21/02C12R1/645
CPCC07K7/56C12N1/14C12N1/145C12R2001/645
Inventor 李娜江沛郑玲辉
Owner HISUN PHARMACEUTICAL (HANGZHOU) CO LTD
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