紫外诱变选育米曲霉高产蛋白酶菌株

以从自然发酵黄豆酱中筛选的5株野生米曲霉为供试菌株,以这些菌株产蛋白酶(酸、中、碱)、淀粉酶(α-淀粉酶、糖化酶)活力大小为评价标准,筛选出米曲霉K61作为诱变出发菌株。采用紫外诱变对米曲霉K61菌株进行改造,最终筛选出一株蛋白酶活力高且遗传性能稳定的突变株Y29。将米曲霉Y29菌株应用于黄豆酱的生产中,并与目前工业生产中广泛应用的米曲霉沪酿3.042菌株进行比较。性能实验结果表明米曲霉Y29菌株的蛋白酶(酸、中、碱)活力明显高于米曲霉沪酿3.042菌株,但α-淀粉酶、糖化酶、生长速度和孢子数这4个指标两者的差异并不显著;制酱品质试验结果表明,米曲霉Y29菌株的酱香更浓郁一些,氨基酸态氮含量达到0.77g/100mL,高于米曲霉沪酿3.042菌株,其它指标均符合国家标准GB2718-1996。     

中性蛋白酶高产菌株的筛选及产酶酶系分析

目的:满足水产中对中性蛋白酶的需求。方法:以实验室保藏的米曲霉ZW为出发菌株,经Co60定向诱变,通过透明圈法初筛、摇瓶发酵复筛,筛选中性蛋白酶活力高的菌株,并对其进行产酶酶系分析。结果:筛选到的米曲霉ZW-06产中性蛋白酶酶活可达15000U/g干曲,比诱变前酶活提高了74%,是目前国内报道的固体发酵产中性蛋白酶活力最高的菌株;经过10代传代之后,酶活力仍保持稳定。通过对米曲霉ZW-06进行产酶酶系分析,发现发酵产物中除了有较高的中性蛋白酶酶活,还有较高的木聚糖酶和酸性纤维素酶酶活,酶活分别达到49879U/g干曲和21099U/g干曲。结论:米曲霉ZW-06在饲料工业中有很大     

米曲霉在产脱氨酶过程中的超微结构动态

腺苷酸脱氨酶(AMP deaminase,EC 3.5.4.6)催化5′-腺苷酸脱氨生成5′-肌苷酸。核酸经5′-磷酸二酯酶降解后,用AMP催化其中5′-腺苷酸使其生成5′-脱苷酸。产脱氨酶高活力米曲霉菌株(Aspergillus oryzae)AS3.800,在制作麸麯过程中,当米曲霉分生孢子颜色由浅黄变为绿色时,酶活力下降。现对培养1.5、2、2.5及3天的麸麯进行了电镜观察与脱氨酶     

人工神经网络在米曲霉菌体固定化研究中的应用

在液体培养基中,将米曲霉3042菌体培养成直径为1～2mm、菌壁上含较高氨基酰化酶浓度的菌丝球,以甲醛为交联剂,明胶为酶活性保护剂对米曲霉菌丝球进行固定化研究。在正交实验L₁₆(4⁵)的基础上,选用结构为410-15-1的BP(Back propagation)人工神经网络,对固定化工艺条件进行优化和预测,得到了优化的固定化条件。实验测定在此条件下制备的固定化米曲霉菌体比酶活为1500u,比酶活保留率达到83%,说明人工神经网络可以用于米曲霉菌体固定化工艺条件的优化。     

米曲霉在产脱氨酶过程中的超微结构动态

腺苷酸脱氨酶(AMP deaminase,EC 3.5.4.6)催化5′一腺苷酸脱氨生成5′一肌苷酸。核酸经5′-磷酸二酯酶降解后,用AMP催化其中5′一腺苷酸使其生成5′一脱苷酸。产脱氨酶高活力米曲霉菌株(Aspergillus oryzae)AS3.800,在制作麸麯过程中,当米曲霉分生孢子颜色由浅黄变为绿色时,酶活力下降。现对培养1.5、2、2.5及3天的麸麯进行了电镜观察与脱氨酶活性测定,希望能找到米曲霉菌体发育过程与产酶之间的相关性。     

大豆异黄酮糖苷酶产生菌培养条件优化及转化

将经过筛选的产大豆异黄酮糖苷水解酶的菌株米曲霉3042经过单因子及正交试验,确立了产酶的最适培养基配方为：玉米芯＋麸皮4%,（NH4）2SO40.1%,水扬苷0.01%,KH2PO40.1%,Vc 0.1%,MgSO40.1%.产酶的最佳培养条件为：发酵培养基起始pH 6.0,发酵温度27℃,摇床转速160r/min,发酵时间84 h时酶活力最高.粗提取的大豆异黄酮经发酵液转化后,其结合态含量降低,游离态含量增加.     

耐低温β-葡聚糖酶高产菌株的选育

通过低温驯化、紫外诱变琼脂平板上的孢子的方法,对局限曲霉进行诱变。根据刚果红与β-1,3—1,4-葡聚糖耦合而保持红色的特征,采用鉴定平板与诱变平板为同一平板的快速高效的方法粗筛耐低温β-葡聚糖酶高产霉菌。最后经过摇瓶进一步复筛,筛选出一株产酶活力比原始菌株提高4倍多的诱变株QY-00305,该菌株有较好的稳定性。     

微波诱变结合化学诱变选育纤维素酶高产菌的研究

以纤维素酶产生菌TP1202为出发菌株,通过微波诱变和硫酸二乙酯、氯化锂诱变剂进行诱变,选育到1株纤维素酶高产菌株AS5。在适宜条件下,其产生的羧甲基纤维素酶活力(CMC),滤纸酶活力(FPA)和棉花糖酶活力分别是出发菌株的107%、152.4%和140.5%。     

N~+离子注入选育高产氨基酰化酶菌株的研究

选用N~+离子注入的方法对米曲霉(Aspergillus oryzae)CICC 2339-1进行诱变育种,通过三角瓶发酵法筛选氨基酰化酶高产株。N~+离子注入选择能量为10 KeV,剂量在(1.30～4.94)×10~(15)ions/cm~2之间。根据剂量与存活率以及剂量与突变率曲线选择最佳的注入剂量。通过三角瓶发酵筛选得到突变菌株SN-110-15其酶活提高率为139.5%,诱变试验效果显著。     

重离子诱变枯草芽孢杆菌高产复合酶菌种的筛选

重离子诱变作为一种新型高效的辐照诱变方式,具有突变率高、突变谱广、突变体稳定等特点。枯草芽孢杆菌(Bacillus subtilis)因具有多种水解酶活力而广泛应用于工业生产,虽然经过多次诱变和改造,其酶活力不断提高,但其复合酶活力仍有提升空间。同时,多次同种诱变剂的使用容易导致菌种产生"抗性",酶活力提高不大。为此,本研究首次利用不同剂量¹²C⁶⁺重离子束对枯草芽孢杆菌20076进行辐照诱变。诱变后通过透明圈初筛和酶活力验证复筛方式选育出一株复合酶活力较高的菌株B. subtilis KC-1。与原始菌株相比,其发酵液滤纸酶活力、内切葡聚糖酶活力、β-葡萄糖苷酶活力、外切葡聚糖酶活力、蛋白酶活力和α-淀粉酶活力分别提高了20. 2%、79. 6%、98. 2%、7. 4%、10. 4%和27. 4%。11代遗传稳定性实验结果表明该突变株具有良好的稳定性。     

Assessment of genetic diversity and population structure of Xanthomonas oryzae pv. oryzae with a repetitive DNA element.

A repetitive DNA element cloned from Xanthomonas oryzae pv. oryzae was used to assess the population structure and genetic diversity of 98 strains of X. oryzae pv. oryzae collected between 1972 and 1988 from the Philippine Islands. Genomic DNA from X. oryzae pv. oryzae was digested with EcoRI and analyzed for restriction fragment length polymorphisms (RFLPs) with repetitive DNA element as a probe. Twenty-seven RFLP types were identified; there was no overlap of RFLP types among the six races from the Philippines. Most variability (20 RFLP types) was found in strains of races 1, 2, and 3, which were isolated from tropical lowland areas. Four RFLP types (all race 5) were found among strains isolated from cultivars grown in the temperate highlands. The genetic diversity of the total population of X. oryzae pv. oryzae was 0.93, of which 42% was due to genetic differentiation between races. The genetic diversities of strains collected in 1972 to 1976, 1977 to 1981, and 1982 to 1986, were 0.89, 0.90, and 0.92, respectively, suggesting a consistently high level of variability in the pathogen population over the past 15 years. Cluster analysis based on RFLP banding patterns showed five groupings at 85% similarity. The majority of strains from a given race were contained within one cluster, except for race 3 strains, which were distributed in three of the five clusters.     

Assessment of genetic diversity and population structure of Xanthomonas oryzae pv. oryzae with a repetitive DNA element.

A repetitive DNA element cloned from Xanthomonas oryzae pv. oryzae was used to assess the population structure and genetic diversity of 98 strains of X. oryzae pv. oryzae collected between 1972 and 1988 from the Philippine Islands. Genomic DNA from X. oryzae pv. oryzae was digested with EcoRI and analyzed for restriction fragment length polymorphisms (RFLPs) with repetitive DNA element as a probe. Twenty-seven RFLP types were identified; there was no overlap of RFLP types among the six races from the Philippines. Most variability (20 RFLP types) was found in strains of races 1, 2, and 3, which were isolated from tropical lowland areas. Four RFLP types (all race 5) were found among strains isolated from cultivars grown in the temperate highlands. The genetic diversity of the total population of X. oryzae pv. oryzae was 0.93, of which 42% was due to genetic differentiation between races. The genetic diversities of strains collected in 1972 to 1976, 1977 to 1981, and 1982 to 1986, were 0.89, 0.90, and 0.92, respectively, suggesting a consistently high level of variability in the pathogen population over the past 15 years. Cluster analysis based on RFLP banding patterns showed five groupings at 85% similarity. The majority of strains from a given race were contained within one cluster, except for race 3 strains, which were distributed in three of the five clusters.     

Aflatoxin genes and the aflatoxigenic potential of Koji moulds

Sixty-four Aspergillus isolates, 54 of which originated from food fermentations, and 18 Aspergillus reference strains were identified and screened for the presence of aflatoxin genes aflR and omt-1. Among the Koji moulds, not only A. oryzae but also A. flavus strains were found. Furthermore, 27% of A. oryzae and 93% of A. flavus strains lacked either aflR or both aflR- and omt-1. A selection of 29 strains was also checked for the presence of pksA and nor-1. This revealed large deletions in the aflatoxin gene cluster of some strains. The hybridisation patterns also suggested a polarity in the deletion events, originating in the vicinity of pksA and extending towards omt-1. Other strains exhibited BamHI restriction fragment length polymorphisms (RFLPs) for either aflR or for aflR and omt-1. All aflR and/or omt-1 deletion strains turned out to be unable to produce aflatoxin. The RFLP-carrying strains either produced only traces of aflatoxin or none at all. In 73% of the A. oryzae strains, no apparent deletions were detected with the aflR and omt-1 probes. Nevertheless, after incubation in aflatoxin-inducing media, no aflatoxin B1 production could be detected in those A. oryzae strains.     

Genetic Diversity of Xanthomonas oryzae pv. oryzae in Asia

Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.     

BALB/c antiarsonate idiotypes: Gene complementation necessary for expression

The expression of two idiotype (id) families (5AF6 and 3C6) associated with the BALB/c p-azophenylarsonate-specific antibody response was examined in 11 mouse strains. Eight strains produced some of one or the other of these two id families with the mean percent expression in the anti-Ar responses of id(+) strains ranging from 8 to 43% for the 5AF6 and from 2 to 10% for the 3C6 idiotype. Four strains of mice (C58, AKR, PL, and RF) thought to have Lyt-3.1-linked V_L repertoire differences from other mouse strains (Lyt-3.2) were tested for their capacity to contribute to 5AF6 and 3C6 id expression. The RE strain was capable of producing 5AF6 id and small amounts of 3C6 id. Tests of Lyt-3.1 congenic strains C.AKR (AKR Lyt-3.1 on a BALB/c background) and C.C58 (C58 Lyt-3.1 on a BALB/c background) showed that C.AKR could produce 5AF6 id while C.C58 could not. 3C6 id expression was present but depressed in C.C58 mice compared with the high 3C6 id expression in C.AKR. Breeding studies mating C.C58 (bearing the required Igh-C^a-linked V _H genes) to other 5AF6(–) strains showed that gene complementation could result in 5AF6 expression in F₁ offspring. 5AF6(–) strains capable of complementation included CBA/J, C57BL/6J, AKR/J, and PL/J. C58/J (from which C.C58 were derived) was the only tested strain that failed to complement for 5AF6 id expression. Additional matings between C58/J[5AF6(–)] and CBA/J[5AF6 (–)] showed F1 offspring could produce 5AF6 id, indicating that C58/J can contribute functional V _H genes necessary for 5AF6 id expression. Depressed expression of 5AF6 and 3C6 id was noted in mice where the C58/J-derived Lyt-3.1 genotype was present. The possibility that the depression of 5AF6 and 3C6 id expression derived from C58/J mice was due to regulatory influences rather than a lack of the V _L structural genes is discussed.Abbreviations used in this paper Ar p-azophenylarsonate - DNP dinitrophenol - H heavy - HIS hyperimmune suppressed - id idiotype - Ig immunoglobulin - IEF isoelectric focusing - k kappa - L light - PC phosphorylcholine - S.D. standard deviation     

Production of fungal biomass protein using microfungi from winery wastewater treatment

This study was carried out to investigate the production of fungal biomass protein (FBP) in treatment of winery wastewater using microfungi. Three fungal strains, Trichoderma viride WEBL0702, Aspergillus niger WEBL0901 and Aspergillus oryzae WEBL0401, were selected in terms of microbial capability for FBP production and COD reduction. T. viride appeared to be the best strain for FBP production due to high productivity and less nitrogen requirement. More than 5 g/L of fungal biomass was produced in shake fermentation using T. viride without nitrogen addition, and by A. oryzae and A. niger with addition of 0.5-1.0 g/L (NH4)2SO4. The FBP production process corresponded to 84-90% COD reduction of winery wastewater. Fungal biomass contained approximately 36% protein produced by two Aspergillus strains, while biomass produced by T. viride consisted of 19.8% protein. Kinetic study indicated that maximum fungal cell growth could be achieved in 24h for T. viride and 48 h for A. oryzae and A. niger. Current results indicated that it could be feasible to develop a biotechnological treatment process integrated with FBP production from the winery waste streams.     

Diversity of propanil-degrading bacteria isolated from rice rhizosphere and their potential for plant growth promotion

The herbicide propanil has long been used in rice production in southern Brazil. Bacteria isolated from contaminated soils in Massaranduba, Santa Catarina, Brazil, were found to be able to grow in the presence of propanil, using this compound as a carbon source. Thirty strains were identified as Pseudomonas (86.7%), Serratia (10.0%), and Acinetobacter (3.3%), based on phylogenetic analysis of 16S rDNA. Little genetic diversity was found within species, more than 95% homology, suggesting that there is selective pressure to metabolize propanil in the microbial community. Two strains of Pseudomonas (AF7 and AF1) were selected in bioreactor containing chemotactic growth medium, with the highest degradation activity of propanil exhibited by strain AF7, followed by AF1 (60 and 40%, respectively). These strains when encapsulated in alginate exhibited a high survival rate and were able to colonize the rice root surfaces. Inoculation with Pseudomonas strains AF7 and AF1 significantly improved the plant height of rice. Most of the Pseudomonas strains produced indoleacetic acid, soluble mineral phosphate, and fixed nitrogen. These bacterial strains could potentially be used for the bioremediation of propanil-contaminated soils and the promotion of plant growth.     

Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS

Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidutans histone H2B protein tagged by EGFP (H2B::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B::EGFP, fluorescence was detected in nuclear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66% of conidia had two, 24% had one, and 10% had three or more in the A. oryzae transformant. The conidia expressing H2B::EGFP were put through FACS (fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were successfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied number of nuclei existed in each conidium and some strains had a small number of uninucleate conidia.