Purification and Properties of a New L-Sorbose Dehydrogenase Accelerative Protein from Bacillus megaterium Bred by Ion-Beam Implantation

Bacillus megaterium BM302 bred by ion-beam implantation produces L-sorbose dehydrogenase accelerative protein （SAP） to accelerate the activity of L-sorbose dehydrogenase （SDH） of Gluconobacter oxydans in the 2-keto-L-gulonic acid （2KLG） fermentation from L-sorbose by the mixed culture of B. megaterium BM302 and G. oxydans. The SAP purified by three chromatographic steps gave 35-fold purification with a yield of 13% and a specific activity of 5.21 units/mg protein. The molecular weight of the purified SAP was about 58 kDa. The SDH accelerative activity of SAP at pH 7 and 50℃ was the highest. Additionally, it retained 60% activity at a pH range of 6.5 ～ 10 and was stable at 20℃ ～ 60℃. After 0.32-unit SAP was added to the single cultured G. oxydans strains, the SDH activity was apparently accelerated and the 2KLG yield of GO29, GO112, GO and GI13 was enhanced 2.1, 3.3, 3.5 and 2.9 folds respectively over that of the strains without the addition of SAP.     

Study on the Effect of Mutated Bacillus megaterium in Two－Stage Fermentation of Vitamin C

Bacillus megaterium as a companion strain in two-stage fermentation of vitamin C could secrete some active substances to spur growth of Gluconobacter oxydans to produce 2-KLG. In the fermenting system where Gluconobacter oxydans was combined with GB82-a mutated strain of B. megaterium by ion implantation, the amount of 2-KLG harvested was larger than that produced by the original B. megaterium BP52[1] being substituted for GB82. In this paper, we studied the effect of the active substances secreted by GB82 to enhance the capability of Gluconobacter oxydans to produce 2-KLG. The supernate of GB82 sampled at different cultivation times all had much more activity to spur Gluconobacter oxydans to yield 2-KLG than that of the original B. megaterium, which might be due to the genetic changes in the active components caused by ion implantation. Furthermore, the active substances of GB82‘s supernate would lose a part of its activity in extreme environments, which is typical of some proteins.     

Mutagenic Effects of BM302：GO112 Induced by Low-Energy Ion Beam Implantation

Mutant strains of GO112 and BM302 with a high 2-keto-L-gulonic acid （2KLG） transformation rate induced by ion beam implantation were separately and combinatorially compared with the original strains GO29 and BM80 to study the mutagenic effects of ion beam implantation. Both the sole GOl12 and mixed BM302：GOl12 demonstrated improved SNDH activity and 2KLG yield compared to the original strains. The mutant combinations of BM302：GOl12 showed a longer stationary phase and higher biomass than BM80：GO29. The mutant BM302 exhibited a stronger capacity to maintain a stable pH environment at mixed fermentation with Gluconobacter oxydans （G. oxydans） for 2KLG transformation and facilitated the growth of G. oxydans compared with the original strain BM80. The promotive capacity to L-sorbosone dehydrogenase （L-SNDH） from the supernate of BM302 was 1.6-fold higher than that of BM80. Genes encoded SNDH in GO29 and GOl12 were amplified and sequenced, and mutations including three transitions （CG →TA, CG →TA, GC → AT） and one transversion （AT→ TA） were confirmed from GO29 to GOl12. The corresponding amino acid was changed as Leu →Phe, Arg → Gln and Asn → Lys.     

The Mutation Breeding and Mutagenic Effect of Air Plasma on Penicillium Chrysogenum

Low temperature air plasma was used as the mutation tool for penicillin-producing strain Penicillium chrysogenum. The discharge conditions were RF power of 360 W, temperature of 40℃ in a sealed chamber, and pressure of 10 Pa to 30 Pa. The result showed that the kinetics of the survival rate followed a typical saddle-shaped curve. Based on a statistic analysis, at the treating duration of 10 min, the positive mutation rate was as high as 37.5% while the negative mutation rate was low. The colonial morphology changed obviously when the plasma treating duration reached or exceeded 45 min. After both primary and secondary screening, a mutant designated as aPc051310 with high productivity of penicillin was obtained, and a strong mutagenic effect on P. chrysogenum was observed in the process. It was proved that after five generations, the mutant aPc051310 still exhibits a high productivity. All the results prove that the plasma mutation method could be developed as a convenient and effective tool to breed high-yield strains in the fermentation industry, while expanding the plasm application at the same time.     

Improvement of L（＋）-Lactic Acid Production of Rhizopus Oryzae by Low-Energy Ions and Analysis of Its Mechanism

The wild type strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion implantation （15 keV, 7.8×10^14 ～ 2.08 ×10^15 ions/cm^2） to find an industrial strain with a higher L（＋）-lactic acid yield, and two mutants RE3303 and RF9052 were isolated. In order to discuss the mechanism primarily, Lactate Dehydrogenase of Rhizopus oryzae was studied. While the two mutants produced L（＋）-lactic acid by 75% more than the wild strain did, their specific activity of Lactate Dehydrogenase was found to be higher than that in the wild strain. The optimum temperature of Lactate Dehydrogenase in Rhizopus oryzae RF9052 was higher. Compared to the wild strain, the Michaelis constant （Km） value of Lactate Dehydrogenase in the mutants was Changed. All these changes show that L（＋）-lactic acid production has a correlation with the specific activity of Lactate Dehydrogenase. The low-energy ions, implanted into the strain, may improve the specific activity of Lactate Dehydrogenase by influencing its gene structure and protein structure.     

Analysis of a Partial Male-Sterile Mutant of Arabidopsis thaliana Isolated from a Low-Energy Argon Ion Beam Mutagenized Pool

A screen for Arabidopsis fertility mutants, mutagenized by low-energy argon ion beam, yielded two partial male-sterile mutants tc243-1 and tc243-2 which have similar phenotypes. tc243-2 was investigated in detail. The segregation ratio of the mutant phenotypes in the M2 pools suggested that mutation behaved as single Mendelian recessive mutations, tc243 showed a series of mutant phenotypes, among which partial male-sterile was its striking mutant characteristic. Phenotype analysis indicates that there are four factors leading to male sterility, a. Floral organs normally develop inside the closed bud, but the anther filaments do not elongate sufficiently to position the locules above the stigma at anthesis, b. The anther locules do not dehisce at the time of flower opening （although limited dehiscence occurs later）, c. Pollens of mutant plants develop into several types of pollens at the trinucleated stage. as determined by staining with DAPI （4＇,6-diamidino-2-phenylindole）. which shows a variable size. shape and number of nucleus. d. The viability of pollens is lower than that of the wild type on the germination test in vivo and vitro.     

Optimization of L（＋）-Lactic Acid Production from Xylose with Rhizopus Oryzae Mutant RLC41-6 Breeding by Low-Energy Ion Implantation

In order to obtain an industrial strain with a higher L（＋）-lactic acid yield, the strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion beam implantation and the mutant strain Rhizopus oryzae RLC41-6 was obtained. An experimental finding was made in surprise that Rhizopus oryzae mutant RLC41-6 is not only an L（＋）-lactic acid producer from corn starch but also an efficient producer of L（＋）-lactic acid from xylose. Under optimal conditions, the production of L（＋）-lactic acid from 100 g/L xylose reached 77.39 g/L after 144 h fed-batch fermentation, A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment.     

Creation of High-Yield Polyhydroxyalkanoates Engineered Strains by Low Energy Ion Implantation

Polyhydroxyalkanoates （PHAs）, as a candidate for biodegradable plastic materials, can be synthesized by numerous microorganisms. However, as its production cost is high in comparison with those of chemically synthesized plastics, a lot of research has been focused on the efficient production of PHAs using different methods. In the present study, the mutation effects of PHAs production in strain pCB4 were investigated with implantation of low energy ions. It was found that under the implantation conditions of 7.8×10^14 N^＋/cm^2 at 10 keV, a high-yield PHAs strain with high genetic stability was generated from many mutants. After optimizing its fermentation conditions, the biomass, PHAs concentration and PHAs content of pCBH4 reached 2.26 g/L, 1.81 g/L, and 80.08% respectively, whereas its wild type controls were about 1.24 g/L, 0.61 g/L, and 49.20%. Moreover, the main constituent of PHAs was identified as poly-3-hydroxybutyrates （PHB） in the mutant stain and the yield of this compound was increased up to 41.33% in contrast to that of 27.78% in the wild type strain.     

Mutagenesis of T richoderma V iride by Ultraviolet and Plasma

Considering the importance of a microbial strain capable of increased cellulase production, a mutant strain UP4 of T richoderma viride was developed by ultraviolet (UV) and plasma mutation. The mutant produced a 21.0 IU/mL FPase which was 98.1% higher than that of the parent strain T richoderma viride ZY-1. In addition, the effect of ultraviolet and plasma mutagenesis was not merely simple superimposition of single ultraviolet mutation and single plasma mutation. Meanwhile, there appeared a capsule around some of the spores after the ultraviolet and plasma treatment, namely, the spore surface of the strain became fuzzy after ultraviolet or ultraviolet and plasma mutagenesis.     

Screening of Bioflocculant-Producing Strain by Ion Implantation and Flocculating Characteristics of Bioflocculants

A bioflocculant-producing mutator strain, NIM-192, was screened out through nitrogen ion implanting into F J-7 strain. The results showed that NIM-192 had good genetic stability and high flocculating activity, and the flocculating rate increased by 34.26% than that of the original. Sucrose, complex nitrogen source contained yeast extract, urea and pH 7.0～ 9.0 were chosen as the best carbon source, nitrogen source and initial solution pH for bioflocculant production, respectively. The bioflocculant kept high and stable flocculating activity at alkalinous reaction mixture with a pH beyond 7.0, while the flocculating activity was remarkably reduced when the reaction pH was lower than 7.0. Addition of many cations could obviously increase the flocculating rate, among which Ca^2＋ demonstrated the best effect. The bioflocculant had very strong acid-base stability and thermo-stability.The flocculating rate kept over 86% when pH of the bioflocculant was in a range of 3.0 ～ 12.0, and the change of flocculating activity was not great when heated at 100℃ for 60 rain.     

Improvement of Vitamin K2 Production by Escherichia sp.with Nitrogen Ion Beam Implantation Induction

Low-energy ion implantation as a novel mutagen has been increasingly applied in the microbial mutagenesis for its higher mutation frequency and wider mutation spectra.In this work,N~+ ion beam implantation was used to enhance Escherichia sp.in vitamin K_2 yield.Optimization of process parameters under submerged fermentation was carried out to improve the vitamin K_2yield of mutant FM5-632.The results indicate that an excellent mutant FM5-632 with a yield of 123.2±1.6 μg/L,that is four times that of the original strain,was achieved by eight successive implantations under the conditions of 15 keV and 60×2.6×l0~(13)ions/cm~2.A further optimization increased the yield of the mutant by 39.7%,i.e.172.1±1.2 μg/L which occurred in the mutant cultivated in the optimal fermentation culture medium composed of(per liter):15.31 g glycerol,10 g peptone,2.89 g yeast extract,5 g K2HPO_4,1 g NaCl,0.5 g MgSO_4·7H_2O and 0.04 g cedar wood oil,incubated at 33℃,pH 7.0 and 180 rpm for 120 h.     

Pigment analysis of a color-leaf mutant in Wandering Jew （Tradescantiafluminensis） irradiated by carbon ions

Many mutants of plant induced by heavy ion beam irradiation have been reported in recent years,but leaf anthocyan mutants induced by ion irradiation in evergreen were rarely found.In this study,a color-leaf mutant with purple leaves,stems and petals was isolated from clones of Wandering Jew irradiated by 95.8 MeV/u carbon ion beam.The concentration and histological distribution of leaf pigment were surveyed in wild type and mutant.In mutant,contents of total chlorophylls (Chl),chlorophyll a (Chl a),chlorophyll b (Chl b) and carotenoids (Car) decreased significantly,while concentration of the anthocyanins was 6.2-fold higher than that of wild type.Further composition analysis of anthocyanins by electrospray ionization mass spectrometry (ESI-MS) indicated that the purple pigmentation of leaves in mutant was caused by accumulation of petunidin anthocyanin.Microscopic examination showed that most petunidin anthocyanin accumulated in the lower epidermis,and little in vascular parenchyma of mutant,while there was no pigment in wild type.Meanwhile,in spongy parenchyma of mutant we observed little Chl,which the wild type abounds in.In conclusion,the color-leaf mutant of Wandering Jew induced by irradiation of carbon ions was improved in ornamental value,and it could be contribute to variation in level,component and distribution of foliar pigment.The possible mutation mechanisms were discussed.     

Effect of Ar^＋ Implantation and Maize Genome DNA on Autotetraploid Rice

The effect of Ar^＋ beam implantation and maize genome DNA on autotetraploid rice is studied. Better mutation types and higher mutation rates were discovered in M2 of T3 with ion implantation and immersion in maize genome DNA. In the five agronomic categories investigated, the mutation rate of the seed setting rate was 9.1%, and the total mutation rate was 14.8% in the T3. However, the total mutation rate was 2.1% with the treatment of only ion implantation and 1.3% with the treatment of only immersion in maize genome DNA. Mutant FA36（4） was discovered in M1 with ion beam implantation and immersion in maize genome DNA. Its RuBPCase activity, PEPCase activity and seed setting rate were 32%, 153%, and 36.79%, respectively, higher than its parent IR36（4）. Rapid analysis of polymorphicDNA （RAPD） analysis of three M2 plants of FA36（4） （FMI, FM2, FM3） and two controls （purple maize and IR36（4）） were also conducted with 40 random primers. S5-3 was RAPD fragment amplified with a template of purple maize, FM2 and FM3 genome DNA using primer S5. There was no S5-3 in the RAPD pattern of IR36（4） or FMI.     

A high-throughput screening method for breeding Aspergillus niger with 12C6+ ion beam-improved cellulase

In this study,a high-throughput screening method was established through the 24-square deep-well microliter plate(MTP) fermentation and micro-plate detection for large-scale screening of the mutants.It was suitable for screening a large number of mutants and improving the breeding efficiency after heavy-ion beam irradiation.Seventeen strains showed higher cellulase activity compared with the initial strain after the screening of plate and MTP fermentation.The filter paper activity and β-glucosidase activity of Aspergillus niger H11201 had increased 38.74 and 63.23%separately compared with A.niger H11 by shaking flask fermentation,and it was genetically stable after being passaged to nine generations.The results indicate that the high-throughput screening method can be used for the quick breeding of A.niger with high cellulase activity.     

Preliminary Studies on Base Substitutions and Repair of DNA Mismatch Damage Stimulated by Low Energy N＋ Ion Beam Implantation in Escherichia coli

Ever since the low energy N+ ion beam has been accepted that the mutation effects of ionizing radiation are attributed mainly to direct or indirect damage to DNA. Evidences based on naked DNA irradiation in support of a mutation spectrum appears to be consistent, but direct proof of such results in vivo are limited. Using mutS, dam and/or dcm defective Eschericha coli imitator strains, an preliminary experimental system on induction of in vivo mutation spectra of low energy N+ ion beam has been established in this study. It was observed that the mutation rates of rifampicin resistance induced by N+ implantation were quite high, ranging from 9.2×10-8 to 4.9 ×10~5 at the dosage of 5.2 ×1014 ions/cm2. Strains all had more than 90-fold higher mutation rate than its spontaneous mutation rate determined by this method. It reveals that base substitutions involve in induction of mutation of low energy nitrogen ion beam implantation. The mutation rates of mutator strains were nearly 500-fold (GM2929), 400-fol     

Study on Mutagenic Breeding of Bacillus Subtilis and Properties of Its Antifungal Substances

Bacillus subtitles JA isolated by our laboratory produced a large amount of antifungal substances, which had strong inhibitory activity against various plant pathogenic fungi,such as Rhizoctonia solani, l?usarium graminearum and so on. Ion beam implantation as a new mutagenic methods was applied in our studay. After B. subtitles JA was implanted by N^ ions,a strain designated as B. subtitles JA-026 was screened and obtained, which had a higher ability to produce those antifungal substances. A series of experiments indicated that the antifungal substances were thermostable and partially sensitive to proteinases K and tryproteinase. When the fermentating broth was fractionated with ammonium sulphate of a final saturation of 70%,the precipitate-enhanced inhibitory activity while the supernatant lost this activity. It appeared that the antifungal substances were likely to be protein.     

A Mutant of Bacillus Subtilis with High-Producing Surfactin by Ion Beam Implantation

In order to generate a mutant of Bacillus subtilis with enhanced surface activity through low energy nitrogen ion beam implantation, the effects of energy and dose of ions implanted were studied. The morphological changes in the bacteria were observed by scanning electron microscope （SEM）. The optimum condition of ions implantation, 20 keV of energy and 2.6 × 10^15N^＋/cm^2 in dose, was determined. A mutant, B.s-E-8 was obtained, whose surface activity of 50-fold and 100-fold diluted cell-free Landy medium was as 5.6-fold and 17.4-fold as the wild strain. The microbial growth and biosurfactant production of both the mutant and the wild strain were compared. After purified by ultrafiltration and SOURCE 15PHE, the biosurfactant was determined to be a complex of surfactin family through analysis of electrospray ionization mass spectrum （ESI/MS） and there was an interesting finding that after the ion beam implantation the intensities of the components were different from the wild type strain.     

Properties of radicals created by γ—ray irradiation of silk

The propoerties of radicals fromγ-ray irradiated silk fabrics were studied by electron spin resonance method(ESR).The ESR spectra of silk fabrics irradiated in N2 showed a doublet at room temperature,The doublet became a singlet at g=2.0057 after placing the sample in air for 24 hours.This can be explained by formation of peroxide radicals.The radical concentration of the irradiated silk fabric and the decay rate of radicals are significantly affected by irradiation conditions.which include the absorbed dose.atmosphere,and water content of the silk fabric samples.However,no dose rate effect on the radical concentration was observed.The results are of help in our practice of property modification of silk products by radiation graft copolymerization.     

Mutation induction of Pleurotus ferulae by low-energy N^＋ ion implantation and characters of the selected mutant

In order to obtain Pleurotus ferulae with high temperature tolerance,mycelium mono-cells of wild type strain ACK was treated by nitrogen ion(5～30 keV,1.5×1015～1.5×1016cm-12)implantation,and mutant CGMCC1762 was selected through auxotrophy screening method,which was Lys.VB6 auxotrophy stress with high temperature.We found that during riper period the surface layer mycelium of the mutant was not aging neither grew tegument even above 30℃.The mycelium endurable temperature of the mutant was increased 70℃ compared with that of the wild type strain.The fruiting bodies growth temperature of the mutant was 16-20℃ in daytime and was 6～12℃ at night.The highest growth temperature of fruiting bodies of the mutant Was increased by 5℃ than that of original strain.Through three generation investigation,we found that the mutant CGMCC1762 was stable with high temperature tolerance.     

Properties of radicals created by γ-ray irradiation of silk fabrics

The properties of radicals from γ-ray irradiated silk fabrics were studied by electron spin resonance method (ESR). The ESR spectra of silk fabrics irradiated in N2 showed a doublet at room temperature. The doublet became a singlet at g=2.0057 after placing the sample in air for 24 hours. This can be explained by formation of peroxide radicals. The radical concentration of the irradiated silk fabric and the decay rate of radicals are significantly affected by irradiation conditions, which include the absorbed dose, atmosphere, and water content of the silk fabric samples. However, no dose rate effect on the radical concentration was observed. The results are of help in our practice of property modification of silk products by radiation graft copolymerization.