CN1260349C - Streptomyces nanningensis - Google Patents

Abstract

The present invention relates to a microbe with the function of inhibiting acetylcholinesterase, which relates to the field of microbes. The present invention is characterized in that the microbe is named as Streptomycesnanningensis YIM33098 and is preserved in a preservation organization designated by the State Intellectual Property Bureau on Feb. 14th, 2003; the name of the preservation organization is the China Center for Typical Culture Collection, and the preservation number is M203019. The microbe has the activity of inhibiting acetylcholinesterase; the inhibiting efficiency of a fermentation liquid of the microbe on acetylcholinesterase is 100%, IC50 is equal to 4.52*10<6>mug/ml, and the effect is stable and durable; the microbe has a great potentiality of developing novel, efficient, nontoxic and natural drugs.

Description

The Nanning streptomycete

Technical field

The present invention relates to microbial technology field, particularly relate to a kind of inhibiting microorganism of enzyme acetylcholine that has.

Background technology

Neurophysiology research discloses, (Acetylcholinesterase, the vagusstoff (Ach) that is discharged when AchE) the hydrolysis central nervous system is unified fiber behind the parasympathetic ganglion and main neural preganglionic fiber impulsion is to guarantee the coordinating and unifying of nervous excitation and inhibition for acetylcholinesterase.Ach is the essential neurotransmitter of memory formation and the physiological foundation of long-term memory.Ach keeps the brain waking state by the reticular formation of brain stem ascending reticular activating system, increases the decomposition that Ach synthesizes or reduce Ach, can influence learning and memory function.It is human that the memory function regression descends parallel with maincenter choline system function with the age increase.The activity of acetylcholine esterase inhibition can be improved man memory.

Summary of the invention

The purpose of this invention is to provide the microorganism that a kind of its fermented liquid has the acetylcholine esterase inhibition effect--Nanning streptomycete.

Streptomycete called after Nanning, Nanning of the present invention streptomycete Streptomyces nanningensis YIM33098, now be deposited in the preservation of specified depositary institution of State Intellectual Property Office, depositary institution's title: Chinese typical culture collection center, preservation date is on February 14th, 2003, and deposit number is CCTCC NO:M203019.

Nanning of the present invention strepto-fungus strain separates from the pedotheque that gather in China Nanning and obtains.Now be deposited in the preservation of depositary institution of Patent Office of State Intellectual Property Office, depositary institution's title: Chinese typical culture collection center, preservation date are on February 14th, 2003, and deposit number is M203019.

Streptomycete of the present invention is well-grown on most of substratum, and it is yellowish-white to olive-gray that aerial hyphae is, and substrate mycelium is yellowish-white to beige.Spore chain is dredged spirrillum.The spore rod-short, spore surface is smooth.Gelatine liquefication, starch hydrolysis are positive, and milk solidifies, peptonizes, nitrate reductase is negative, and does not produce hydrogen sulfide, does not form melanochrome.Can utilize glucose, seminose, ribose, inositol, Histidine.Cell walls contains L-DAP and glycine, and full cell hydrolyzate does not contain characteristic sugar.

Culture condition:

(1) slant culture condition: substratum: glucose 4g; Yeast extract paste 4g; Malt extract 5g; Vitamin complex 3.75mg/L; Agar 15g; Distilled water water 1000ml, pH7.2; Culture temperature is 28 ℃, and incubation time was 1 week.

(2) seed culture condition: insert the seed liquor shake-flask culture 36 hours from inclined-plane picking part mycelium.Seed culture medium: dextrin 120g; Soyflour 40g; Yeast extract paste 2g; Tryptophane 0.5g; Beta-alanine 5g; Sal epsom 0.5g; Ammonium phosphate 0.2g; Distilled water 1000ml, pH7.2, culture temperature is 28 ℃.

(3) fermentation culture conditions: receive in the seed culture medium shake-flask culture 6 days by 10% inoculum size.Substratum: soyflour 10g; Glucose 10g; Peptone 3g; Sodium-chlor 2.5g; Lime carbonate 2g; Distilled water 1000ml, pH7.2, culture temperature is 28 ℃.Obtain fermented liquid.It is as follows to do the acetylcholine esterase inhibition activity test with this fermented liquid:

1. principle

Cholinesterase in the blood (acetylcholinesterase) catalysis acetylcholine hydrolyzation.Act on certain hour under the condition of certain temperature and certain pH, the vagusstoff amount of hydrolysis is relevant with the activity of enzyme, after adding a certain amount of vagusstoff, measure the vagusstoff amount that remaining vagusstoff amount can calculate hydrolysis, thereby learn activity of cholinesterase.

2. measuring method

A) reagent

I. phosphate buffered saline buffer is got Sodium phosphate dibasic (3 grades of product) 16.72g and potassium primary phosphate (3 grades) 2.72g, and the adding distil water dissolving is diluted to 1000ml, and its pH is 7.2, can be stored in the refrigerator when outdoor temperature is higher.

II. matrix takes by weighing the 1.2716g Ovisot, and adding distil water is made into 0.07mol/L vagusstoff stock solution to 100ml, is stored in the refrigerator, face with preceding again with this solution dilution to 0.007mol/L.

III.1mol/L oxammonium hydrochloride (NH ₂OHHCl) solution takes by weighing oxammonium hydrochloride 13.9g, and adding distil water is diluted to 200ml, and (more than 30 ℃) can be stored in the refrigerator when room temperature was too high.

IV.3.5mol/L sodium hydroxide solution weighing sodium hydroxide (3 grades) 14.0g, adding distil water is diluted to 100ml.

V.1:2 to measure proportion be 1.18 concentrated hydrochloric acid (3 grades) 100ml to salt aldehyde solution, adding distil water 200ml.

The VI.10% liquor ferri trichloridi takes by weighing iron trichloride (3 grades) 10.0g, about adding distil water 10ml, adds concentrated hydrochloric acid 0.84ml then, and after the dissolving of heating, adding distil water is to 100ml again.

B) operation steps

^*Sample hose

I. get 0.98ml phosphate buffered saline buffer and 0.02ml whole blood in vitro, put preheating 3-5min in 37 ℃ of water-baths.

II. correctly add 1.0ml0.007mol/L vagusstoff solution rapidly by the alcohol extractive sample number into spectrum order that adds this fermented liquid, put in 37 ℃ of warm water baths.

Add alkaline hydroxylamine solution (mixing facing) 4.0ml rapidly by same order immediately behind the III reaction 30min with preceding 20min equal-volume by 1mol/L hydroxylamine solution and 3.5mol/L sodium hydroxide solution, and the 2min that fully vibrates.

IV continues to add 1: 2 hydrochloric acid soln 2.0ml, fully behind the about 2min of vibration, adds 10% liquor ferri trichloridi 2.0ml again, fully vibration.

V filters above-mentioned solution (10ml altogether) with common filter paper, and the proteic filtrate of elimination is contained in the cuvette with green color filter (530nm wavelength).Carry out colorimetric with the distilled water zeroing.

^*Control tube

I control tube 1 reaction solution is replaced by 1.0ml damping fluid and 1.0ml distilled water.Other operation is identical with sample hose, and this Guan Weiwu blood does not have the reagent blank contrast of vagusstoff.

II control tube 2 reaction solutions are replaced by 0.98ml damping fluid, 0.02ml whole blood and 1.0ml distilled water.Other operation is identical with sample hose, and this pipe is the blank of blood and reagent.

III control tube 3 reaction solutions are made into by 1.0ml damping fluid and 1.0ml damping fluid and 1.0ml 0.007mol/L vagusstoff liquid.Other operation is identical with sample hose, and this pipe is the contrast of unhydrolysed full dose vagusstoff.

Above-mentioned three kinds of control tube every batch of when test all should be cofabrication with sample hose (do a pipe or two pipes get final product)

C) calculate

I. the calculating of unit of enzyme activity

The whole blood cholinesterase activity value is represented with hydrolysis μ mol/L vagusstoff/0.02ml blood 30min.

II. the calculating of enzymic activity value percentage ratio:

This bacterial strain has the effect of acetylcholine esterase inhibition, and its fermented liquid reaches 100% to the inhibiting rate of acetylcholinesterase, IC ₅₀=4.52 * 10 ⁶μ g/ml, and lasting medicine, stable, nontoxic have the great potential of therefrom developing novel, efficient, nontoxic natural drug.

Streptomycete called after of the present invention Nanning streptomycete on February 14th, 2003 in China's typical culture collection center preservation, depositary institution is called for short: CCTCC, deposit number are M203019, depositary institution address: Wuhan, Luo Jiashan, in the Wuhan University, postcode 430072.

Description of drawings

Fig. 1 is the electromicroscopic photograph of Nanning of the present invention streptomycete on yeast extract paste-malt extract substratum.

Fig. 2 is the cultural characteristic of Nanning of the present invention streptomycete.

Fig. 3 is the physiological and biochemical property of Nanning of the present invention streptomycete.

Fig. 4 utilizes situation for the carbon nitrogen source of Nanning of the present invention streptomycete.

Fig. 5 is that Nanning of the present invention streptomycete and part correlation bacterium are in the 16SrDNA of GenBank database sequence and accession number.

Fig. 6 is Nanning of the present invention streptomycete and the part correlation bacterial strain phylogenetic tree according to 16S rDNA sequence construct.

Embodiment

Embodiment:

The pedotheque of gathering from China Nanning is cultivated by following condition in the laboratory, can obtain Nanning of the present invention streptomycete.

(1) strain culturing condition:

1. slant culture condition: substratum: glucose 4g; Yeast extract paste 4g; Malt extract 5g: vitamin complex 3.75mg/L; Agar 15g; Distilled water water 1000ml, pH7.2; Culture temperature is 28 ℃, and incubation time was 1 week.

2. seed culture condition: insert the seed liquor shake-flask culture 36 hours from inclined-plane picking part mycelium.Seed culture medium: dextrin 120g; Soyflour 40g; Yeast extract paste 2g; Tryptophane 0.5g; Beta-alanine 5g; Sal epsom 0.5g; Ammonium phosphate 0.2g; Distilled water 1000ml, pH7.2, culture temperature is 28 ℃.

Cultivate by following fermentation culture conditions, can obtain fermented liquid: receive in the seed culture medium shake-flask culture 6 days by 10% inoculum size.Substratum: soyflour 10g; Glucose 10g; Peptone 3g; Sodium-chlor 2.5g; Lime carbonate 2g; Distilled water 1000ml, pH7.2, culture temperature is 28 ℃.

The 16S rDNA partial sequence of streptomycete of the present invention is analyzed as follows:

GCGGCGTGCTTAACACATGCAAGTCGAACGATGAAACCGCTTCGGTGGTGGATTAGTGGCGAA

CGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTA

ATACCGGATAATACCGGGGAAGGCATCTTCTCTGGTTGAAAGCTCCGGCGGTGCAGGATGAGC

CCGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCT

GAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGT

GGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTT

CGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGC

CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTG

GGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGG

GTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGG

TGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGAC

GCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC

GTTGGGAACTAGGTGTGGGTGACATTCCACGTCATCCGTGCCGCAGCTAACGCATTAAGTTCCC

CGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG

GCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGG

AAAGCATTAGAGATAGTGCCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGC

TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGC

ACGCCCTTCGGGGTGGTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGG

GGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTAC

AATGAGCTGCGATACCGNGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGG

TCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAA

TACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCG

GTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGGACGAAGTCG

TAACAAGGTAGC

Claims (2)

1. Nanning streptomycete, it is characterized in that called after Nanning streptomycete Streptomycesnanninggensis YIM 33098, in the preservation of specified depositary institution of State Intellectual Property Office, preservation date is on February 14th, 2003, depositary institution's title: Chinese typical culture collection center, deposit number is CCTCCNO:M203019.

2. according to a kind of Nanning streptomycete of claim 1, it is as follows that the 16S rDNA that it is characterized in that this bacterial classification partly gives row:

GCGGCGTGCTTAACACATGCAAGTCGAACGATGAAACCGCTTCGGTGGTGGATTAGTGGCGAA

CGGGTGAGIAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTA

ATACCGGATAATACCGGGGAAGGCATCTTCTCTGGTTGAAAGCTCCGGCGGTGCAGGATGAGC

CCGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCT

GAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGT

GGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTT

CGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGC

CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTG

GGCGTAAAGAGCTCGIAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGG

GTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGG

TGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGAC

GCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC

GTTGGGAACTAGGTGTGGGTGACATTCCACGTCATCCGTGCCGCAGCTAACGCATTAAGTTCCC

CGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG

GCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGG

AAAGCATTAGAGATAGTGCCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGC

TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGC

ACGCCCTTCGGGGTGGTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGG

GGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTAC

AATGAGCTGCGATACCGNGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGG

TCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAA

TACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCG

GTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGGACGAAGTCG

TAACAAGGTAGC。

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