CN103320328A

CN103320328A - Aspergillus niger and application thereof

Info

Publication number: CN103320328A
Application number: CN2012100811125A
Authority: CN
Inventors: 赵萍; 王雅; 陆届瑾
Original assignee: Lanzhou University of Technology
Current assignee: Lanzhou University of Technology
Priority date: 2012-03-23
Filing date: 2012-03-23
Publication date: 2013-09-25

Abstract

The invention provides aspergillus niger and an application thereof. The aspergillus niger has an ability of decomposition of cellulose and lignin. Microscope morphological characteristics of the aspergillus niger are that: a conidia head is radial in shape, the diameter is about 200 [mu]m, and a conidiophore stem is about 2 mm; a top sac is nearly spherical in shape, the diameter of the top sac is 20 to 30 [mu]m, and a conidia is spherical in shape and black brown; an optimum temperature of a lipase of the aspergillus niger is 40 DEG C, and has good stability at a temperature within 35 DEG C; and the lipase of the aspergillus nigerin has good stability in a pH value range of 5 to 9.

Description

A kind of aspergillus niger and purposes

Technical field

The invention belongs to biological technical field, be specifically related to the purposes that a plant height is imitated lipolytic active bacterium and this bacterium.

Background technology

Lipase is one of important industrial enzyme preparation kind, can catalysis solution fat, the reaction such as transesterify, ester be synthetic, be widely used in the industry such as grease processing, food, medicine, daily use chemicals.The lipase of different sources has different catalysis characteristics and catalysis activity.Fat application of enzymes 1) foodstuffs industry utilizes the shorter lipid acid of chain that discharges after the enzyme effect, increases and improve local flavor and the fragrance of food; Utilize lipase-catalyzed alcoholysis and esterification to produce various essence esters, make modifier; With there being the narrow spectrum microbial lipase of 1,3-to improve the assortment of edible oil nutritive value and increase edible oil, preparation is for high-grade greases such as cocoa esters; In addition, lipase can also be used for going turbid slagging-off, improving Bread Quality of drinks, improves foaming etc. the aspect of albumen.2) detergent industry Novo company at first pushes the washing composition of fatty enzyme to world market, as the synergistic agent of washing composition, but this enzyme wipe oil under 15～60 ℃, condition more than the pH9.0.3) the synthetic fine chemistry product of lipase-catalyzed in organic solvent, the research and development that catalyze and synthesize the series product such as low-molecular-weight carboxylic acid ester's series essence ester, terpene alcohol ester, middle longer chain fatty acid short chain alcohol ester and longer chain fatty acid long-chain fat alcohol ester with lipase in organic solvent make remarkable progress, and product can be included into GRAS (being commonly considered as safe) category, regards natural " green " substitute as.At present the synthetic ester product of enzyme process is from the laboratory study suitability for industrialized production of marching toward, become the another new production technique of replenishing and replacing part chemosynthesis product, the products such as a lot of esterified prod such as biofuel, dimension A cetylate, iso-octyl palmitate have surpassed chemical synthesis in the advantage aspect economy and the environmental protection two in production reality.4) high efficiency, highly-solid selectively and the mild reaction conditions of the optical resolution enzymic catalytic reaction of racemic mixture are so that the enzyme catalysis Split Method can be used for the chipal compounds that some is difficult to split with general law.Lipase is lytic enzyme, to substrate can catalysis asymmetric hydrolysis or asymmetric esterification widely.Lipase all has preferably stereoselectivity to chipal compounds such as alcohol, carboxylic acid, ester, acid amides and amine etc., so lipase is widely used for the resolution of racemic alcohol and carboxylic acid.The main path that splits is stereo selective hydrolysis, esterification or transesterification reaction.Be active ester with vinyl ester, the chemistry with Pseudomonas Lipases catalysis cyanalcohol in anhydrous methylene chloride splits, and gets S type alcohol, through the synthetic beta-adrenaline inhibitor of chemical conversion.In addition; lipase also is used for the fractionation of ester ring secondary alcohol; the fractionation of pyrethroid intermediate; the fractionations of weedicide anisic acid base naproxen, Bu Luofen etc., the large-scale production of the lipase with transesterification or esterification function that wherein is used for organic synthesis is significant for the synthetic fine chemicals of enzyme catalysis and chipal compounds.Esterification is the high-temperature acid base catalysis mostly at present, exists by product poisonous, severe reaction conditions, and energy consumption is high, the problems such as environmental pollution.

Summary of the invention

The purpose of this invention is to provide a kind of aspergillus niger and uses thereof.

The present invention is a kind of aspergillus niger (Aspergillus niger) Yz15 No.110411-3CGMCC No.5705, and it has the ability of decomposition of cellulose, xylogen.

The reaction conditions gentleness that enzyme process is synthetic, energy consumption is low, and the catalyzed reaction high specificity is difficult for producing by product, and is environmentally friendly, is Green Chemistry industrial expansion direction.

Lipase (Lipase EC 3113) is a class lytic enzyme, but catalyzing oil hydrolysis by using produces triglyceride, monoglyceride, lipid acid and glycerine.Also have the stereoisomerism specificity of chemo-selective and height, more at the report in the fields such as chiral drug production, biofuel, manufacturing in recent years, and the domestic report that has no of this bacterial strain.

Description of drawings

Fig. 1 is Yz15PDA substratum form, and Fig. 2 is the Yz15 spore shape, and Fig. 3 is conidium form after the cotton blue dyeing of Yz15, and Fig. 4 is the phylogenetic tree that bacterial strain Yz15Aspergillus sp makes up by 18S rDNA sequence and Neighbor Joining method,

Embodiment

The present invention is near dining room, dining room near the soil and water sample that are rich in greasy dirt the water drain, be separated to the higher bacterial strain of a strain strain yielding lipase activity, by the research to this bacterial strain purifying and evaluation and zymologic property, by morphology and Molecular Identification, this bacterial strain Yz15 and Aspergillus niger strain CBS 513.88clone F-wpysw4 (EU710739.1) are in same branch, and sequence homology is 100%.Judge that according to morphology bacterial strain Yz15 conforms to the aspergillus niger morphological specificity of Aspergillus in conjunction with 18SrDNA sequential analysis demonstration.Yz15 is accredited as aspergillus niger Aspergillus niger, is numbered Yz15No.110411-3.Its substratum morphological specificity: colony growth is rapid on the PDA substratum, cultivates the culture dish that can cover with whole diameter 9cm in 5 days for 28 ℃, and quality is smooth, granular, first faint yellow, gradual change brown, after become Vandyke brown to black, without transudate, the bacterium colony reverse side is in various degree orange-yellow.Colony growthing slow on the grease substratum.Its microscope morphological specificity: conidial head radially, diameter is about 200 μ m.The conidiophore stem is 2mm approximately; The top capsule is subsphaeroidal, diameter 20～30 μ m, and conidium is spherical, brown.

Yz15 lipase optimum temperuture is 40 ℃.Temperature has satisfactory stability below 35 ℃, enzyme fast-descending alive when surpassing 40 ℃.At 45 ℃, measure behind the 30min the residual vigor of lipase only initial reaction about 30%.Yz15 lipase optimal pH is that 8.0, pH scope all has good activity in the work of 5.0～9.0 scope endoenzymes.From the reaction 3 stages demonstrate enzyme work having good stability in pH 5.0～9.0 scopes.Ca ²⁺, Mg ²⁺Yz15 lipase is shown preferably activation, and Cu ²⁺And Fe ²⁺Yz15 lipase is shown obvious restraining effect; In the reaction system take propyl carbinol as organic solvent, have satisfactory stability, and have with this understanding certain esterification activity.

The present invention is a kind of aspergillus niger (Aspergillus niger) Yz15No.110411-3CGMCC No.5705, and it has the ability of decomposition of cellulose, xylogen.

Table 1 is depicted as temperature to the impact (U/mL) of Yz15 bacterial strain lipase activity and stability.

Table 1

Table 2 is that pH is on the impact (U/mL) of Yz15 lipase activity and stability.

Table 3 is that metal ion is on the impact (U/mL) of Yz15 lipase.

Control group	Mg ²⁺	Cu ²⁺	Fe ²⁺	K ⁺	Ca ²⁺
						15.11	19.66	10.67	8.41	14.78	22.53

Table 4 is rough determination (U/mL) of esterification activity.

	Propyl carbinol, citric acid are substrate	Butanic acid, glycerine are substrate
			Yz15 lipase	1.38	0.30

Fig. 8

Below further launch aspergillus niger of the present invention and produce and measure.

1. materials and methods

1.1 material

1.1.1 sample collecting

Near sampling position: the soil of long-term long-pending oil, near soil and the water sample the refinery, totally 25 samples sewage of dining room sewage pipe, the dining room.

1.1.2 substratum

Enrichment medium (PDA substratum): sucrose (or glucose) 20g, agar 18g, potato 200g, 1000mL.

Primary dcreening operation substratum (selection substratum): vegetables oil 1.5g, agar 3.0g, K ₂HPO ₄0.2g, MgSO ₄7H ₂O 0.01g, (NH ₄) ₂SO ₄0.2g, 150mL.

Sieve again substratum (fermentation culture): vegetables oil 3.0g, K ₂HPO ₄1.0g, MgSO ₄7H ₂O 0.2g, (NH ₄) ₂SO ₄1.5g, glucose 1.0g, 150mL.

1.1.3 main agents

NaOH, phenolphthalein, propyl carbinol, butanic acid, glycerine, citric acid, sodium stearate, potassium primary phosphate, dipotassium hydrogen phosphate, MgSO ₄7H ₂O, (NH ₄) ₂SO ₄

1.1.4 key instrument

JJ-CJ-1D Bechtop, biochemical cultivation case, BS-2F constant-temperature shaking incubator, the biological digit microscope of DMB, Motic biomicroscope, HPX-9162MBE digital display electric heating incubator, TGL-16 high speed tabletop refrigerated centrifuge.

1.2 method

1.2.1 the screening of lipase strains

1.2.1.1 the enrichment culture enrichment culture with the soil sample and water sample dilution that gather after, get 1mL and put into the triangular flask that the 250mL enrichment medium is housed, be 30 ℃ in temperature, rotating speed is to cultivate 5d on the shaking table of 150r/min, and enrichment culture liquid is continued switching 3 times.

To coat on the selective medium after the dilution of enrichment culture liquid 1.2.1.2 selectivity is cultivated, cultivation is observed periphery of bacterial colonies and grease decomposition circle whether occurred, then preserves respectively appearance and decomposes the bacterium colony that encloses.

1.2.1.3 then multiple sieve is cultivated will be 30 ℃ in temperature through the inoculation of primary dcreening operation in multiple sieve nutrient solution, rotating speed is to cultivate on the 150r/min shaking table that to measure its enzyme behind the 60h alive.

1.2.2 Assay of lipase activity

The definition of lipase activity: be under 7 the condition 30 ℃ of pH values, per minute is discharged 1 μ mol lipid acid from vegetables oil enzyme amount is defined as the enzyme U of unit that lives.

The mensuration of volumetry lipase activity: respectively add 5mL enzyme liquid after preparing 2 parts of 100mL emulsions that contain 10mL 0.01mol/L sodium stearate and 1g vegetables oil, a CaCl that before reaction, adds indicator phenolphthalein and 10mL 0.1mol/L ₂Use the 0.05mol/LNaOH titration after the concussion; Another part adds the CaCl of phenolphthalein, 10mL 0.1mol/L afterwards in reaction ₂Use the 0.05mol/LNaOH titration after the concussion, the titer that deducts the front NaOH of reaction after the reaction is the growing amount of lipid acid (A mL * 0.05mol/L).

Enzyme work=A * 0.05mol/L * 10 ³/ C * T

Wherein A is the consumption of NaOH, mL; The enzyme amount 5mL of C for adding, T is the reaction times, 10min.

Add CaCl ₂Effect be the H that increases lipid acid ⁺Ionization improves titration sensitivity.

1.2.3 identification of strains

The bacterial strain that screening is obtained carries out microscopic examination, consult the microorganism identification handbook, and pass through pcr amplification, obtain bacterial strain 18S rDNA sequence, utilize CLC DNA Workbench 6 softwares with the DN A sequence of including among the section of acquisition sequence and the GeneBank compare, and with relevant strain construction evolutionary tree.Determine the kind of bacterial strain according to morphology and molecular sequences feature.

1.2.4 zymologic property

High reactivity lipase strains to resulting separation carries out respectively the zymologic properties such as temperature, pH value, cation recognition, esterification activity being measured.

2. the screening of bacterial strain

Obtained the bacterial strain that the hydrolysis of 15 strain adularescents is enclosed through selectivity cultivation (grease substratum) screening again 2.1 pass through to isolate on the enrichment medium (PDA substratum) enzyme more much higher strain bacterial strain alive, utilize fermentation culture to extract the thick enzyme of each bacterial strain, be about the enzyme of measuring each bacterial strain under 7.0 the condition 30 ℃ of pH values and live, select the enzyme the highest bacterial strain of living.

2.2 carrying out morphological feature with reference to " fungi identification handbook ", morphological feature detects.

2.3 Molecular Identification

By pcr amplification, obtained the Partial Fragment of the 18S rDNA gene of Yz15.Fragment is carried out (company finishes by the luxuriant industry order-checking of English) after the sequential analysis, utilizes CLC DNA Workbench 6 softwares that the dna sequence dna of including among this sequence and the GeneBank is compared, and with relevant strain construction evolutionary tree.

2.4 zymologic property is measured

2.4.1 the impact that temperature is lived on enzyme

After every assembly double processed contains the 100mL emulsion of 10mL 0.01mol/L sodium stearate and 1g vegetables oil, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ Water Unders of temperature difference are bathed and are added 5mL enzyme liquid under neutrallty condition, and respectively behind reaction 10min, 20min, 30min, measure its enzyme and live, repeat 2 times and survey its mean value.

2.4.2pH on enzyme impact alive

It is 5.0,6.0,7.0,8.0,9.0 phosphate buffered saline buffer that 0.2mol/L potassium primary phosphate and dipotassium hydrogen phosphate are mixed with respectively pH, each buffered soln is 30 ℃ of water-baths in temperature, and behind reaction 10min, 20min, 30min, measure its enzyme respectively behind the adding 5mL enzyme liquid and live, repeat 2 times and survey its mean value.

2.4.3 the impact that metal ion is lived on enzyme

The Mg of preparation 0.5mmol/L ²⁺, Gu ²⁺, Fe ²⁺, K ⁺, Ca ²⁺Solution and the reaction control group that do not add ion are 30 ℃ of temperature, and the reaction times is 10min, measure its enzyme and live, and repeat 2 times and survey its mean value.

2.4.4 esterification activity is measured

A. the mensuration take propyl carbinol and citric acid as the esterification activity of substrate

Preparation 30mL propyl carbinol, 10mL 0.5mol/L citric acid, 5mL enzyme liquid is put in the Erlenmeyer flask of 150mL, is 30 ℃ in temperature, and rotating speed is to react reaction times 5h in the shaking table of 60r/min.Utilize Rotary Evaporators after the reaction, pressure is removed the reaction medium propyl carbinol in 25 ℃ of conditions of 0.82Kpa temperature, the liquid of remainder is flushed in the 150mL Erlenmeyer flask with wash bottle, and to the 0.05mol/LNaOH titration of 100mL adding indicator phenolphthalein, unreacted citric acid neutralizes.Again to Erlenmeyer flask behind 35 ℃ of water-baths concussion heating 20min, rear with 0.05mol/L NaOH titration, thus measure esterification activity.

B. the mensuration take glycerine and butanic acid as the esterification activity of substrate

Preparation 10mL glycerol, 10mL 0.5mol/L butanic acid, the 20mL propyl carbinol, 5mL enzyme liquid is put in the Erlenmeyer flask of 150mL, measures glycerine and butanic acid as the esterification activity of substrate take aforesaid method.

Esterification activity=A * 0.05mol/L * 10 ³/ C * T (h)

Wherein: the enzyme amount 5mL of C for adding, T is reaction times (5h), A is the milliliter number of 30 ℃ of reaction NaOH that 5h consumes.

Claims

1. an aspergillus niger (Aspergillus niger) Yz15 No.110411-3CGMCC No.5705, it has the ability of decomposition of cellulose, xylogen.

2. aspergillus niger claimed in claim 1 (Aspergillus niger) Yz15 No.110411-3CGMCCNo.5705, its microscope morphological specificity is: conidial head radially, diameter is about 200 μ m, the conidiophore stem is 2mm approximately; The top capsule is subsphaeroidal, diameter 20～30 μ m, and conidium is spherical, chocolate.

3. aspergillus niger claimed in claim 1 (Aspergillus niger) Yz15No.110411-3CGMCCNo.5705, the optimum temperuture of its lipase is 40 ℃, has satisfactory stability at 35 ℃ with interior; The lipase of this bacterium all has satisfactory stability in the pH value in the 5-9 scope.

4. aspergillus niger claimed in claim 1 (Aspergillus niger) Yz15 No.110411-3CGMCCNo.5705, the optimal pH of its lipase is 8.0, Ca ²⁺, Mg ²⁺Lipase to this bacterium has certain activation, Cu ²⁺And Fe ²⁺Yz15 lipase is shown obvious restraining effect.

5. aspergillus niger claimed in claim 1 (Aspergillus niger) Yz15 No.110411-3CGMCCNo.5705, the lipase of this bacterium all has satisfactory stability in the reaction system take propyl carbinol as organic solvent, and all has certain esterification activity with this understanding.