Decomposition of myceliar matrix and extraction of chitosan from Gongronella butleri USDB 0201 and Absidia coerulea ATCC 14076

Free chitosan, 2 g/100g mycelia from Gongronella butleri and 6.5 g/100g mycelia from Absidia coerulea were isolated by 1M NaOH at 45 degrees C for 13 h and 0.35 M acetic acid at 95 degrees C for 5 h. Both myceliar matrixes did not break down under these conditions. However, myceliar matrix could be decomposed by treatment with 11 M NaOH at 45 degrees C for 13 h and 0.35 M acetic acid at 95 degrees C for 5 h and then extracted the total chitosan, 8-9 g/100g mycelia from both fungi. According to these results, G. butleri has higher amount of complexed chitosan and A. coerulea has higher amount in free chitosan.     

原生质体紫外诱变技术选育1,3-丙二醇高产菌株

以肺炎克雷伯氏杆菌(Klebsiella pneumoniae)为研究对象,应用原生质体紫外诱变技术提高其对甘油及1,3-丙二醇的耐受性,获得1,3-丙二醇高产菌.在原生质体制备过程中,运用滤膜去除酶解后细胞悬液中的正常菌体,简化菌体酶解过程,提高再生率及形成率.经过原生质体诱变后,以耐受高浓度甘油和1,3-丙二醇及高产酸能力为筛选方向,最终筛选到了3株高产菌株(Kp-1、Kp-4和Kp-5).在补料发酵实验中,上述诱变菌产1,3-丙二醇能力分别为70.24 、65.21和75.51 g/L,比野生菌株WT(55.78 g/L)分别提高了25.92%、16.91%和35.37%.     

白腐菌紫外诱变选育高产漆酶突变株及其产酶条件研究

以白腐菌为出发菌株,利用紫外线(UV)进行诱变,筛选高产漆酶突变菌株。通过测定致死率绘制出发菌株的致死曲线,采用PDA-RBBR平板变色法进行初筛,ABTS检测酶活对突变株进行摇瓶复筛。结果表明:利用15 w紫外灯在照射距离为30 cm,照射时间为120 s,致死率为72.1%的条件下进行诱变处理,获得一株高产菌株,其酶活提高79.54%,经过5代传代培养,未见酶活下降,具有较好的遗传稳定性,进一步研究了初始pH值,接种量和培养基装液量等对诱变菌株产酶的影响,结果表明在最佳的培养条件pH值6.0,15%的装液量于28℃下,酶活达214.9 U/L。     

紫外诱变选育恩拉霉素高产菌株

本研究采用紫外诱变育种技术对一株产恩拉霉素抗真菌链霉菌(Streptomyces fungicidicus)F110进行了诱变处理,经链霉素抗性、利福霉素B抗性以及双重抗性筛选,共获得了132株抗生素抗性突变株,其中26株突变菌株的恩拉霉素产量与出发菌株相比均有明显提高。摇瓶发酵条件下,突变株SR93的恩拉霉素产量最高可达2 400μg/m L,与出发菌株相比提高了38%。传代结果表明:该突变株产素水平稳定,因此具备较好的开发及工业应用价值。     

UV-induced recessive lethals in uvs strains of Neurospora which are deficient in UV mutagenesis

The frequencies of spontaneous and UV-induced recessive lethal mutations were compared for UV-sensitive and wild-type heterokaryons of Neurospora crassa. These heterokaryons were homokaryotic either for one of two alleles of uvs-3, or for uvs-6 or uvs+. For uvs-3, which is known to have mutator effects, spontaneous recessive lethals were found to be 4-6 times more frequent than observed in uvs+. After correction for clonal distribution of spontaneous mutants, an observed 2-fold increase for uvs-6 was not statistically significant and may have been due to chance occurrence of a few large clones of mutants. Treatment with low doses of UV (50-200 J/m2) produced very similar overall rates of increase for recessive lethals in uvs and uvs+ heterokaryons. This means, that in contrast to results obtained when mutation to ad-3 was measured, both uvs-3 alleles showed highly significant increases for recessive lethals when treated with UV. It is proposed that certain types of UV damage may be processed into recessive lethal mutations by an alternate mechanism from that responsible for viable mutations.     

Decoloration of chitosan by UV irradiation

Decoloration of chitosan by UV irradiation, which was used to replace a bleaching step during chitosan preparation, was evaluated under four separate treatments (effect of irradiation time, chitosan/water ratio, stirring speed, and UV light source). The optimal decoloration condition was defined as that producing white chitosan with higher viscosity. Decoloration of chitosan could be achieved effectively using a UV-C light by stirring unbleached chitosan in water (1:8, w/v) for 5 min at 120 rpm. UV irradiation applied under the optimal conditions could be used to produce chitosan with desirable white color (L^* = 76.95, a^* = −0.37, and b^* = 14.04) and high viscosity (1301.7 mPa s at 0.5% w/v in 1.0% v/v acetic acid).     

Elevated yield of monacolin K in Monascus purpureus by fungal elicitor and mutagenesis of UV and LiCl

In China, Monascus spp., a traditional fungus used in fermentation, is used as a natural food additive. Monascus spp. can produce a secondary metabolite, monacolin K namely, which is proven to be a cholesterol-lowering and hypotensive agent. Hence, recently, many researchers have begun focusing on how to increase the production of monacolin K by Monascus purpureus. In the present study, we investigated the effect of the fungal elicitor and the mutagenesis of UV & LiCl on the amount of monacolin K produced by Monascus purpureus. The fugal elicitor, Sporobolomyces huaxiensis, was isolated from tea leaves and its filtrate was added into the culture filtrate of Monascus purpureus during growth to induct the production of monacolin K. The results showed that the highest amount of monacolin K produced by the liquid fermentation was 446.92 mg/mL, which was produced after the fungal elicitor was added to the culture filtrate of Monascus purpureus on the day 4; this amount was approximately 6 times greater than that of the control culture filtrate, whereas the highest amount of monacolin K produced by the mutated strain was 3 times greater than the control culture after the irradiation of UV light in the presence of 1.0 % LiCl in the medium.     

The two-step model of bacterial UV mutagenesis

Recent results are discussed which have led to a two-step model for UV mutagenesis in excision-deficient Escherichia coli. After exposure to UV, the replication fork is assumed to continue until immediately before certain photoproducts where it stops and leaves a gap which cannot be dealt with by recombination repair. In the first (misincorporation) step, bases (a proportion of which are ‘wrong’) are postulated to be inserted opposite the photoproduct under the direct influence of the recA gene product. These misincorporated bases can be revealed as mutations by delayed photoreversal in umuD, C and lexA (ind⁻) bacteria. Their level is determined by the particular allele of recA that is present (recA441 > recA⁺ > recA430) and their rate of formation by the amount of recA protein in the cell and the degree of enrichment of the medium. No other protein needs to be synthesized for this step to occur. The second (bypass) step requires induced levels of the products of the umuD and C genes which are postulated to facilitate continued DNA synthesis on the priming end opposite the photoproduct. In principle, further errors could be made at this stage which might appear as ‘hitch-hiking’ rather than ‘targeted’ mutations.     

Recombinant carbazole-degrading strains for enhanced petroleum processing

Biotechnological upgrading of fossil fuels is of increasing interest as remaining stocks of petroleum show increasing levels of contaminants such as heavy metals, sulfur and nitrogen-containing heteroaromatic compounds. Carbazole is of particular interest as a major petroleum component known to reduce refining yields through catalyst poisoning. In this study, the biotransformation of carbazole was successfully demonstrated in a liquid two-phase system, when solubilized in either 1-methylnaphthalene or in diesel fuel. The effects of solvent toxicity were investigated by expressing the carbazole-transformation genes from MB1332, a rifampicin-resistant derivative of Pseudomonas sp. LD2, in a solvent-resistant heterologous host, P. putida Idaho [1]. This solvent-resistant strain successfully degraded carbazole solubilized in 1-methylnaphthalene and in the presence of 10 vol% xylenes similar to the non-recombinant strain Pseudomonas sp. LD2. Identification of a suitable recombinant host, however, was essential for further investigations of partial pathway transformations. Recombinant P. putida Idaho expressing only the initial dioxygenase enzymes transformed carbazole to an intermediate well retained in the oil phase. Partial carbazole transformation converts carbazole to non-aromatic species; their effect is unknown on refinery catalyst poisoning, but would allow almost complete retention of carbon content and fuel value. Electronic Publication     

Construction of Lactobacillus plantarum strain with enhanced L-lysine yield

AIM: To enhance L-lysine secretion in Lactobacillus plantarum. METHODS AND RESULTS: An S-2-aminoethyl-L-cystein (AEC)-resistant mutant of L. plantarum was isolated, and it produced L-lysine at considerably higher level than the parent strain. Aspartokinase in the mutant has been desensitized to feedback inhibition by L-lysine. The nucleotide sequence analysis of thrA2 that codes for aspartokinase in the mutant predicted a substitution of glutamine to histidine at position 421. L-Lysine-insensitive aspartokinase, together with aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase genes, was cloned from L. plantarum DNA to a shuttle vector, pRN14, and the genes were then transformed individually into the AEC-resistant mutant and the parent strain. The overexpression of the genes led to the increase in the activity of enzymes they encode in vitro. However, only the strain overexpressing aspartokinase or dihydrodipicolinate synthase produced more L-lysine. CONCLUSIONS: The desensitization of aspartokinase to L-lysine in L. plantarum led to the overproduction of L-lysine. The overexpression of L-lysine-insensitive aspartokinase or dihydrodipicolinate synthase enhanced L-lysine secretion in L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the L-lysine-overproducing strain of L. plantarum in food or feed fermentation may increase the L-lysine content of fermented products.     

热诱变筛选林可霉素高产菌株的探索

为了提高林肯霉菌产林可霉素的能力 ,对热诱变的方法进行了初步的探索 ,找出了比较合适的加热温度、时间和菌龄 ,并且杀死率也符合选种要求 ,此法正突变率高 ,简便易行     

紫外线诱变选育高产PHB解聚酶的菌株

以降解聚-β羟基丁酸酯(PHB)的青霉(Penieillium sp．)DS9713a为出发菌株,通过紫外线(UV)诱变分生孢子,采用透明圈初筛和摇瓶复筛,获得酶活高于原始菌株的突变株5株,其中DS9713a-CS01突变株的PHB解聚酶活力高于对照97．42％,并对其酶学性质进行了初步研究。     

Complexes of glucose oxidase with chitosan and dextran possessing enhanced stability

Abstract

In this study, the different mole ratios of glucose oxidase/chitosan/dextran–aldehyde and glucose oxidase/chitosan/dextran–sulfate complexes were synthesized. The modification of glucose oxidase by non-covalent complexation with dextran and chitosan in different molar ratios was studied in order to increase the enzyme activity. The enzyme/polymer complexes obtained were investigated by UV spectrophotometer and dynamic light scattering. Activity determination of synthesized complexes and free enzyme were performed at a temperature range. The best results were obtained by C_chitosan/C_{dextran–aldehyde} = 10/1 ratio and C_chitosan/C_{dextran–sulfate} = 1/5 ratio that were used in thermal stability, shelf life, salt stress, and ethanol effect experiments. The results demonstrated that both complexes were thermally stable at 60?°C and had superior storage stability compared to the free glucose oxidase. Complexes showed higher enzymatic activity than free enzyme in the organic solvent environment using 10% ethanol. The complexes were resistant to salt stress containing 0.1?M NaCl or CaCl₂. The particle size distribution results of the triple complex evaluated the complexation of the chitosan, dextran derivative, and glucose oxidase. The average size of the triple complex in diameter was found to be 325.8?±?9.3?nm. Overall findings suggest that the complexes of glucose oxidase, chitosan, and dextran showed significant enhancement in the enzyme activity.     

Isolation of alcohol tolerant,osmotolerant and thermotolerant yeast strains and improvement of their alcohol tolerance by UV mutagenesis

In this study, the yeast strains were isolated from grapes by serial dilution technique to determine their alcohol-, sugar- and thermotolerance. 34 wild type yeast strains were isolated and alcohol-, sugar- and thermotolerance of these strains were determined. The maximum alcohol tolerance was found to be 9% (v/v) in yeast strain which is named Y2. Thermotolerance behavior of 6 strains were investigated. The strains were treated with UV light with intervals of 20, 30, 40 and 50 seconds. Selected resistant colonies were investigated for alcohol tolerance. It was found that alcohol tolerance increased from 9% (v/v) to 12% (v/v) on Y2 strain.     

Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion

The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T.␣atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 g/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in␣vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 g/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T.␣atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.This work was presented as an oral lecture in section ‘Agriculture, Soil, Forest Microbiology’ at the BioMicroWorld2005 conference.     

Mechanisms of uniformity of yield strains for trabecular bone

Variations in yield strains for trabecular bone within a specific anatomic site are only a small fraction of the substantial variations that exist for elastic modulus and strength, and yet the source of this uniformity is not known. Our goal was to investigate the underlying mechanisms by using high-resolution, materially nonlinear finite element models of 12 human femoral neck trabecular bone specimens. The finite element models, used to obtain apparent yield strains in both tension and compression, assumed that the tissue-level yield strains were the same across all specimens. Comparison of the model predictions with the experimental data therefore enabled us to isolate the combined roles of volume fraction and architecture from the role of tissue material properties. Results indicated that, for both tensile and compressive loading, natural variations in volume fraction and architecture produced a negligible coefficient of variation (less than 3%) in apparent yield strains. Analysis of tissue-level strains showed that while bending of individual trabeculae played only a minor role in the apparent elastic behavior, the combined effects of this bending and tissue-level strength asymmetry produced apparent-level failure strains in compression that were 14% lower than those at the tissue level. By contrast, tissue and apparent-level yield strains were equivalent for tensile loading. We conclude that the uniformity of apparent yield strains is primarily the result of the highly oriented architecture that minimizes bending. Most of the variation that does occur is the result of the non-uniformity of the tissue-level yield strains.     

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.     

REV7, a new gene concerned with UV mutagenesis in yeast

Summary Three allelic mutations of a new yeast gene, which we have named REV7, have been isolated by testing 313 methyl methane sulfonate sensitive mutants for UV-induced reversion of a lys2 allele. Rev7 mutants are markedly deficient with respect to UV-induced reversion of lys2, are slightly sensitive to UV and appear to be in the RAD6 epistasis group for UV survival. Rev7-1, which is probably an amber mutation, does not appear to affect sporulation in homozygous diploids. The REV7 gene is located about 12 cM distal to HIS5 on chromosome IX.     

Molecular marker-assisted selection for enhanced yield in malting barley

Brewers are reluctant to change malting barley (Hordeum vulgare ssp. vulgare L.) cultivars due to concerns of altered flavor and brewing procedures. The U.S. Pacific Northwest is capable of producing high yielding, high quality malting barley but lacks adapted cultivars with desirable malting characteristics. Our goal was to develop high yielding near isogenic lines that maintain traditional malting quality characteristics by transferring quantitative trait loci (QTL) associated with yield, via molecular marker-assisted backcrossing, from the high yielding cv. Baronesse to the North American two-row malting barley industry standard cv. Harrington. For transfer, we targeted Baronesse chromosome 2HL and 3HL fragments presumed to contain QTL that affect yield. Analysis of genotype and yield data suggests that QTL reside at two regions, one on 2HL (ABG461C-MWG699) and one on 3HL (MWG571A-MWG961). Genotype and yield data indicate that additional Baronesse genome regions are probably involved, but need to be more precisely defined. Based on yield trials conducted over 22 environments and malting analyses from 6 environments, we selected one isogenic line (00-170) that has consistently produced yields equal to Baronesse while maintaining a Harrington-like malting quality profile. We conclude there is sufficient data to warrant experiments testing whether the 2HL and 3HL Baronesse QTL would be effective in increasing the yield of other low yielding barley cultivars.     

UV mutagenesis in inhibitor depleted conidia of Aspergillus nidulans

UV treated conidia of a strain of Aspergillus nidulans (meth Gl. biAl) depleted of germination inhibitory substances have been examined for inactivation and mutation induction at groups of suppressor gene loci defining three classes of methionine revertants. An exponential decline in the colony forming ability and quadratic increase in mutation frequency (for each class of revertant) as the incident dose increased were observed. The induced mutation frequency for each class and the loss of colony forming ability of the conidia are greater in the absence than in the presence of these self-inhibitors of germination. However, the relative frequencies of the individual classes of revertants did not differ whether the inhibitory substances were present or not. Liquid holding effects leading to increases in survival and mutation have been observed, but the relative frequencies of the individual revertant classes remain unchanged.