Cyclosporin A-producing strain of two different protoplast preparation and regeneration of
Author: Lin Cheng Yuan-Rong Zheng Min Zheng
【Abstract】 The cyclosporin A-producing strain in two
different - white bassiana and Fusarium solani as the starting strain, carried out protoplast preparation and regeneration conditions of the study and were achieved in two different kinds of strains protoplast regeneration.
Key words Cyclosporine A white bassiana protoplasts of
Formation and regeneration of protoplasts from two
ABSTRACT Protocols for formation and regeneration protoplasts for two different fungus species, Beauveria nivea and Fusarium solani, were described. High yield of protoplasts
was obtained by using a combined enzyme system containing lywallzyme and snailase. The effects of factors such as osmotic stabilizers, cultivation time , temperature, pH, incubation time on the formation and regeneration were studied.
KEY WORDS Cyclosporin A; Beauveria nivea; Fusarium solani; Protoplast
Cyclosporin A (cyclosporin A) is composed of 11 amino acid cyclic peptide, has served as efficient
immunosuppressants in human organ transplantation is widely applied . Sandoz company of Switzerland in 1976, first reported by the semi-porous known genus Trichoderma
(Tolypocladium inflatum) [later renamed as the white bassiana Beauvcria nivea)] and gloss column cinerea (Cylindrocarpon lucidum) produced by cyclosporin A [ 2]. Since then scientists
from various countries has been reported upon by the 13 kinds of other producing bacteria, such as the Fusarium fungus (Fusarium solani) and the invasion of new red Kan globosum (Neocosmospora varinfecta) etc. in cyclosporin A. Fujian Institute of Microbiology was first reported in 1983 the soil was screened from our production of cyclosporin A strain of Fusarium solani 4 11, is the first in the successful
development of cyclosporin A [3,4]. According to Ains worth of fungal classification of Fusarium solani and internationally commonly used white cyclosporin A-producing strains belonging
to Trichosporon Class Beauveria bassiana (Hyphomycetes) silk Mycelia (Hyphomycetales) and tumor-seat Mycelia
(Tuberculariales ). Both producing strains not only in morphology, cultural characteristics and physiological characteristics are different, but also in industrial
production processes and the results have different characteristics. Production of Fusarium solani with cyclosporin A low-cost fermentation medium and fermentation period is short (5d) the advantages, but cyclosporin A low
titer and component content; white bassiana production of cyclosporin A titer with cyclosporin A and components of high content of advantages, but the high cost of fermentation medium and fermentation cycle is long (10 ~ 15d). Kim et al  the use of snow-white bassiana for intraspecific fusion obtained dependence L Val and L Leu fusion of strains of
cyclosporin A titer of up to 8920 mg / L, but the fermentation medium price of the required expensive, the use of amino acids in the medium of up to 36g / L, fermentation cycle, as long as 10 ~ 13d. We envision two different cyclosporin A-producing
strain of the bacteria to protoplast fusion between head, trying to filter out the collection of the advantages of integration of two different sub-strains used in production,
ensure higher productivity cyclosporin A and economic benefits of the new strain. Reported in this paper with white bassiana and Fusarium solani as the starting strain, respectively conditions of protoplast formation and regeneration research.
1 Materials and methods
Fusarium solani S 42 150 2 and the white
bassiana X 441 50 provided by the Fujian Institute of Microbiology.
PDA liquid medium Weigh 20g peeled potatoes, cut into
small lumps, add appropriate boiled 30min. With 4-layer gauze
filter, abandoned to the residues in the filtrate by adding 2g of glucose, fixed volume to 100ml.
PDA slant agar 100ml PDA agar 2g liquid culture for preparation of radical addition.
PDA regeneration medium PDA liquid medium, yeast dipping sauce (paste) 0.3g, KCl 3.9g, agar 2g, constant volume to 100ml.
Cellulase (cellulase) Biotechnology Co., Ltd., Xiamen, Beijing, Thailand; snail enzyme (snailase) Biotechnology Co.,
Ltd., Xiamen, Beijing, Thailand; lywallzyme (lywallzyme) Institute of Microbiology, Guangdong Province; dithiothreitol (DDT), Xiamen, Beijing, Thailand Bio-Technology Co., Ltd..
Osmotic stabilizer, respectively 0.6mol / L of KCl, NaCl,
MgSO4 * 7H2O, D mannitol, D sorbitol and sucrose
1.4 Preparation of Protoplasts
Were picked one part white Beauveria bassiana and Fusarium solani agar slant cultures were inoculated on PDA liquid medium, the amount of 40ml/250ml shake flask bottles, 26 ?, 200r/min culture after a certain time into a number of diameter for 0.3mm glass beads, oscillation 20min. Sand heart G3 glass filter funnel. Take 0.2g wet pellet by adding 1ml 0.01mol / L of DDT, blending, put it aside for 20min after the
10000r/min centrifugation 10min, abandoned to the supernatant, the mycelium suspended in 2ml sterile water, sediment, 10000r/min Centrifuge 10min. Abandoned to go to Qing Ye, add 1ml of different enzyme solution, mixing, placed in 28 ?
water bath pot per hour through a straw blowing and drawing
three times, blood cell count plate count. After digestion of additional 1ml 0.6mol / L KCl, blowing and drawing three times, with three-layer lens cleaning paper filter, the
filtrate transferred to a centrifuge tube. 3000r/min
centrifugal 7min, abandoned to the supernatant, the protoplast suspension in 1ml 0.6mol / L KCl, the blood count plate count.
1.5 Regeneration of Protoplast
Will be better prepared protoplast suspension with 0.6mol
/ L KCl solution to make the appropriate dilution of culture on PDA plate coated recycled 0.1ml; protoplast protoplast regeneration control, will the hypotonic treatment with sterile water, after coating on the regeneration medium. 26 ?
culture regeneration of 5 ~ 7d observed colonies, at the same time the number of regenerated bacterial count:
Regeneration rate = experimental group, the number of regenerated colonies - the control group the number of
protoplasts regenerated colony number × 100% reposted
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2 Results and discussion
2.1 The composition of enzyme solution on protoplast formation of
Using lywallzyme, snail enzymes and cellulase enzyme solution composed of 9 groups were age of 24h hydrolysis of
white fungus Beauveria bassiana and Fusarium solani hyphae to 0.6mol / L of KCl as a stabilizer (pH6. 0, reaction temperature 28 ?, reaction time 4h), the results in Table 1.
From Table 1 shows that lywallzyme bacterial digestion
with snail enzyme mixture, the formation of protoplast volume up. Preparation of different types of fungal protoplasts and generate the conditions necessary for the amount of difference between the large, white bassiana protoplast need to form a
lower enzyme concentration (0.5% lywallzyme 0.5% snail enzyme) to form protoplasts more, to reach 8.0 × 107 Ge / ml (Figure
1A). Fusarium solani at a higher concentration of enzyme solution (1% lywallzyme 1% snail enzyme) in the formation of 5.3 × 105A/ ml protoplast (Figure 1B). The use of complex enzyme broken, the effect is better than a single enzyme.
Alone, or snail cellulase enzyme was found in the mycelium production of protoplasts, while alone lywallzyme hydrolysis of cell wall structure can be generated protoplasts, and under
the action of snail enzyme-assisted protoplast volume to reach a maximum.
2.2 bacteria age on protoplast formation of
Age is the preparation of protoplasts of bacteria during the important factor. With 0.6mol / L of KCl as a stabilizer
(pH6.0, reaction temperature 28 ?), 4h after the observation,
the results shown in Figure 2. 28h culture at 26 ? for 16h
white bassiana or culture of mycelium of Fusarium solani protoplasts prepared largest amount, indicating that the
period of the cell wall structure is suitable for preparation of protoplasts. Table 1 the composition of the enzyme solution on protoplast formation of a few weeks ago of the mycelium cell structure of the degrading enzyme is not sensitive to cell wall and protoplast membrane difficult to separate, resulting in the release of the difficulties protoplasts . In the logarithmic growth phase of young hyphae easy digestion, this time hyphae of endogenous secretion of large amount of lywallzyme are conducive to the release of
2.3 osmotic stabilizer on protoplast formation of
Osmotic stabilizer on the nature and concentration is to maintain and control an important factor in the number of protoplasts. Respectively, 0.6mol / L stabilizer D
sorbitol, sucrose, D mannitol, NaCl, KCl and MgSO4 * 7H2O Preparation of protoplasts results show that white bassiana in 0.6mol / L NaCl the number of protoplasts prepared under the most of Fusarium solani in 0.6mol / L NaCl, and MgSO4 * 7H2O
as a stabilizer when the largest number of protoplasts (Table 2).
2.4 The temperature on protoplast formation of
Enzyme reaction kinetics based on the principle of Protoplast enzyme solution temperature and pH can directly affect the enzymatic reaction rate and the degree of
protoplasts. In the enzyme under different temperatures and found that two kinds of hyphae digestion 4h, Fusarium solani
protoplasts at 32 ? to form the largest number, while the
white bassiana in the range of 28 ~ 36 ? can be obtained with
more protoplast volume (Figure 3). Table 2 permeability pressure stabilizer on protoplast formation of
2.5 pH on protoplast formation of
Comparison of enzyme solution in pH5.0, 5.5,6.0,6.5,7.0 of 0.2mol / L sodium citrate - sodium hydrogen phosphate
buffer, where the role of protoplasts, pH6.0 ~ 7.0 The results show that within the context of two kinds of bacteria The protoplast had no significant effect (Figure 4).
2.6 hydrolysis time on protoplast formation of
Preparation of protoplast digestion time is another important factor. Enzyme lack of time, protoplast can not get all of them. But the enzyme for too long, protoplast membrane stability was reduced, resulting in broken protoplasts . White Beauveria bassiana 32 ? in the liquid under the action
of enzymes, 3h, was the highest number of protoplasts. The Fusarium solani after 4h to reach a maximum number of protoplasts (Figure 5).
2.7 Protoplast Regeneration
Protoplast fusion breeding of the most important aspect
is the need for re-producing cells in protoplast before
breeding in ordinary medium. Respectively 0.6mol/LD
sorbitol, sucrose, D mannitol, NaCl, KCl and MgSO4 * 7H2O
Preparation of PDA as a stabilizing agent regeneration medium,
26 ? culture 5d, was regenerated colonies (Table 3).
Different strains of the large difference between the regenerative capacity of protoplast regeneration medium, the stability of the agents on the regeneration rate impact is relatively large. White Beauveria bassiana In a KCl medium as a stabilizer in the regeneration rate of 4.7%, while in order to sorbitol as the stabilizer of the medium, almost no growth. Snow-white white-Jiang higher rate of bacterial protoplast, but its low rate, perhaps due to the lack of the majority of
protoplasts or loss of the nucleus can not be recycled in some organelles . Preparation of Fusarium solani low regeneration rate, with KCl as a stabilizer in the medium, its
recycling rate as high as 26%. Fusarium solani protoplasts
unable to sorbitol and mannitol as a stabilizer in the regeneration medium. Table 3 protoplast regeneration rate of the cell wall of filamentous fungi because of the composition of more complex, so to get a lot higher protoplast viability,
select the appropriate conditions for preparation and regeneration is very important. Through the above experiments identified the snow-white white-chiang bacteria and Fusarium
solani in protoplast preparation and regeneration conditions for the next step to carry out protoplast fusion between head and lay a good foundation for the study, relevant research work in progress.
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