CN105385610A - Preparing method for Aspergillus oryzae protoplast - Google Patents

Abstract

The invention discloses a preparing method for Aspergillus oryzae protoplast, and relates to the technical field of methods for protoplast preparation in bioengineering. The method includes Aspergillus oryzae culture, hypha collection, enzymolysis and the like. By the adoption of the method, the yield of the protoplast can reach 1.5*10<7>/mL, the regeneration rate can reach 90%, and a foundation is laid for Aspergillus oryzae gene modification.

Description

The preparation method of aspergillus oryzae protoplastis

Technical field

The present invention relates to a kind of aspergillus oryzae method for preparing protoplast, belong to technical field of bioengineering.

Background technology

Aspergillus oryzae is a kind of aerobic fungi, belongs to Deuteromycotina, Aspergillus, and its mycelia is generally in yellow-green colour, and sporulation quantity is large and containing multinuclear, lack sexual reproduction in its growth cycle.Aspergillus oryzae is the bacterial strain that a class can produce prozyme, the enzyme produced comprises cellulase, amylase, saccharifying enzyme, proteolytic enzyme etc., therefore aspergillus oryzae brewageing, the industrial circle such as feed, food occupies very important status, very extensive comprising applying in producing at rice wine, soy sauce, commercial enzyme and medical protein.In addition, aspergillus oryzae has efficient protein excretion ability to express, and the expression level of the outstanding expression system pichia spp of its heterologous protein expression level and eukaryotic protein is suitable.It is a kind of safe microbial strains (GenerallyRecognizedasSafefoodingredients, GRAS level) that FDA Food and Drug Administrations (FDA) in 1989 and U.S. feed Gesellschaft announce aspergillus oryzae.Be produce bacterial strain with aspergillus oryzae, there is ripe fermentation and post-processing technology, the ideal object of therefore Chang Zuowei genetic expression, protein excretion research and heterologous protein expression.In actual production, the enzymatic productivity of aspergillus oryzae is often not good enough.If can carry out aspergillus oryzae artificial reconstructed alive to improve its enzyme, will enhance productivity to a certain extent, reduce costs; Utilize genetic engineering technique, build aspergillus oryzae genetic transformation system such as engineering strain and efficient exogenous protein expression systematic research and be day by day subject to heavily.

Summary of the invention

The object of the invention is the preparation method providing a kind of aspergillus oryzae protoplastis, and the inventive method protoplast yield is high, regeneration rate is high, for process provides good condition further.

Concrete technical scheme is as follows:

The preparation method of aspergillus oryzae protoplastis, step is as follows:

1) by 10 ⁷individual aspergillus oryzae spore inoculating, in liquid nutrient medium, fully shakes cultivation 20 hours in 30 DEG C; Liquid nutrient medium is G-P substratum, and its component is: peptone 1%, glucose 0.5%, potassium primary phosphate 0.5%, SODIUMNITRATE 0.1%, magnesium sulfate 0.05%, and surplus is water;

2) with lens wiping paper collecting by filtration mycelia, clean mycelia with aqua sterilisa and mycelia moisture is removed;

3) mycelia is put into enzyme liquid, enzymolysis 2-4 hour;

Described enzyme liquid preparation: often get helicase 1g and Yatalase lyase 0.8g, with 0.8mol/L sodium-chlor for solvent, adjusts pH6.0 to be settled to 100mL, mixes for subsequent use;

4) protoplastis is collected.

Step 3) enzymolysis time is 3 hours.

Aspergillus oryzae is cultivated and can first be adopted PDA solid medium to cultivate.After cultivation completes, Bechtop collects spore with physiological saline and counts.Get 10 ⁷individual aspergillus oryzae spore inoculating is in liquid nutrient medium, and substratum liquid amount is 50mL/250mL, 28 DEG C, 200 revs/min, shaking culture 20h, collects mycelia.

Beneficial effect:

Adopt the inventive method, protoplast yield can reach 1.5 × 10 ⁷individual/mL, and regeneration rate can reach 90%.Technical foundation has been established in aspergillus oryzae strain transformation prepared by the present invention.

Accompanying drawing explanation

Fig. 1 substratum is on the impact of protoplast yield

Fig. 2 enzyme liquid is on the impact of protoplast yield

Fig. 3 pH is on the impact of protoplast yield

Fig. 4 enzymolysis time is on the impact of protoplast yield

Fig. 5 permeate agent kind is on the impact of protoplast yield.

Embodiment

Embodiment 1

By 10 ⁷individual aspergillus oryzae spore inoculating is in G-P liquid nutrient medium (peptone 1%, glucose 0.5%, potassium primary phosphate 0.5%, SODIUMNITRATE 0.1%, magnesium sulfate 0.05%) in, fully shake cultivation after 20 hours in 30 DEG C, substratum is filtered with four layers of lens wiping paper, and the mycelia be trapped on lens wiping paper is rinsed with aqua sterilisa, in sterile environment, get mycelia 2g, add 10mL enzyme liquid (helicase 1%, Yatalase lyase 0.8%) in, enzymolysis time is 3h, and microscopy counts, and collects.

Enzyme liquid (helicase 1%, Yatalase lyase 0.8%) is prepared: get helicase 1g and Yatalase lyase 0.8g, with 0.8mol/L sodium-chlor for solvent, adjusts pH6.0 to be settled to 100mL, mixes for subsequent use.0.8mol/L sodium-chlor is also permeate agent.

Counted from microscopy, as enzymolysis 3h, the output of protoplastis can reach 1.5 × 10 ⁷individual/mL, and regeneration rate can reach 90%.

Embodiment 2

By 10 ⁷individual aspergillus oryzae spore is inoculated in murphy juice substratum respectively, czapek's solution, G-P substratum (peptone 1%, glucose 0.5%, potassium primary phosphate 0.5%, SODIUMNITRATE 0.1%, magnesium sulfate 0.05%), YPD substratum (glucose 2%, peptone 2%, yeast leaching powder 1%) in, each substratum three is parallel, cultivation is fully shaken after 20 hours in 30 DEG C, substratum is filtered with four layers of lens wiping paper, and the mycelia be trapped on lens wiping paper is rinsed with aqua sterilisa, mycelia 2g is got in sterile environment, , put into the enzyme liquid (helicase 1%) making permeate agent with 1mol/ sorbyl alcohol, result as shown in Figure 1.

As shown in Figure 1, the growth of different substratum on mycelia has impact, and when substratum is nutritious, mycelial growth is very fast, and cell walls is thicker, thus affects hydrolysis result.From Fig. 1 data, when with G-P substratum, the output of protoplastis is higher, can reach 6.0 × 10 ⁵individual/mL, and relatively less with protoplastis during other substratum.

Embodiment 3

To be taken in G-P liquid nutrient medium 30 DEG C fully concussion cultivate the aspergillus oryzae mycelia 2g of 20h, make permeate agent with 1mol/ sorbyl alcohol, get the mycelia cultivating 20h, put into the enzyme liquid making permeate agent and solvent with 1M sorbyl alcohol, do three parallel, enzymolysis 3h.

Enzyme liquid: 1M sorbyl alcohol is solvent, is made into the mixed enzyme solution of 1% helicase and 0,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%Yatalase lyase respectively.Result as shown in Figure 2.

The preparation of amount on protoplastis adding Yatalase lyase as shown in Figure 2 in 1% helicase has impact, and when the amount adding Yatalase lyase is 0.8%, the quality of protoplastis reaches 4.2 × 10 ⁶individual/mL, and when adding amount < 0.8% or the > 0.8% of Yatalase lyase, protoplastis quantity is relatively less.

Embodiment 4

Be taken at the aspergillus oryzae mycelia 2g cultivating 20h in G-P liquid nutrient medium, make permeate agent with 1mol/ sorbyl alcohol, regulate pH to be 5.0,5.5,6.0,6.5,7.0,7.5 with HCl and NaOH, each pH tri-is parallel, and enzymatic hydrolysis condition is as embodiment 1, and result as shown in Figure 2.

The preparation of pH on protoplastis has impact as shown in Figure 3, and pH affects the activity of enzyme, and as pH6.0, the quality of protoplastis can reach 6.4 × 10 ⁶individual/mL, and during pH < 5.5 or > 6.5, protoplastis quantity reduces relatively.

Embodiment 5

Be taken at the aspergillus oryzae mycelia 2g cultivating 20h in G-P liquid nutrient medium, make permeate agent with 1mol/ sorbyl alcohol, pH6.0, enzymolysis time is respectively 1h, and 2h, 3h, 4h, 5h each time three is parallel, and all the other conditions are as embodiment 1, and result as shown in Figure 3.

As shown in Figure 4, on protoplast yield impact significantly, when enzymolysis time is at 2 ~ 4h, protoplast yield can reach 3.4 × 10 to enzymolysis time ⁶individual/mL, when enzymolysis time is 3h, the quantity of protoplastis can reach 7.5 × 10 ⁶individual/mL, and other enzymolysis time protoplastiss are relatively less.

Embodiment 6

Choose 0.8mol/L sorbyl alcohol, Repone K, sodium-chlor, Adlerika is as homeo-osmosis agent, and each stablizer three is parallel, gets the aspergillus oryzae mycelia 2g cultivating 20h, 30 DEG C of water enzyme digestion 3h, and all the other conditions are as embodiment 1, and microscopy counts.

Protoplastis easily absorbs water and rises brokenly in hypotonic solution, so select specific hypertonic solution as homeo-osmosis agent to maintain protoplastis form, as shown in Figure 5, with sodium-chlor as homeo-osmosis agent, the output of protoplastis is relatively high, and quantity can reach 9.0 × 10 ⁶individual/mL.

Claims (2)

1. the preparation method of aspergillus oryzae protoplastis, is characterized in that, step is as follows:

1) by 10 ⁷individual aspergillus oryzae spore inoculating, in liquid nutrient medium, fully shakes cultivation 20 hours in 30 DEG C; Liquid nutrient medium is G-P substratum, and its component is: peptone 1%, glucose 0.5%, potassium primary phosphate 0.5%, SODIUMNITRATE 0.1%, magnesium sulfate 0.05%, and surplus is water;

2) with lens wiping paper collecting by filtration mycelia, clean mycelia with aqua sterilisa and mycelia moisture is removed;

3) mycelia is put into enzyme liquid, enzymolysis 2-4 hour;

Described enzyme liquid preparation: often get helicase 1g and Yatalase lyase 0.8g, with 0.8mol/L sodium-chlor for solvent, adjusts pH6.0 to be settled to 100mL, mixes for subsequent use;

4) protoplastis is collected.

2. method according to claims 1, is characterized in that: step 3) enzymolysis time is 3 hours.