可抑制致病菌的益生菌筛选及抑菌物质的初步确定

为了得到可抑制致病菌的益生菌并初步确定其抑菌物质。从土壤、饲料添加剂和肥料添加剂3种样品中初筛能产生抑菌圈的菌株。用牛津杯法,对初筛得到的菌株和实验室保存的菌株进行复筛。对复筛的未知菌株进行形态学、16S r RNA基因序列和生理生化特征鉴定。对复筛菌株的发酵上清液进行酸碱作用验证、高温处理、蛋白酶处理、盐析处理和透析处理,再进行抑菌活力测定。结果显示复筛中有8株对大肠杆菌、金黄色葡萄球菌和藤黄微球菌都有较强抑制作用的菌株。8株菌中有4株已知菌,4株未知菌。已知菌株分别为保加利亚乳杆菌、干酪乳杆菌、鼠李糖乳杆菌和植物乳杆菌。4株未知菌中3株被鉴定为地衣芽孢杆菌,1株被鉴定为乳酸片球菌。地衣芽孢杆菌能产生多种耐高温的抑菌物质,其中包含能透过8-14 k D透析袋的小分子和不能透过8-14 k D透析袋的大分子,初步判定为多肽类物质。5株产酸菌株产生的抑菌物质主要是酸性物质。     

云南传统发酵豆豉中产β-半乳糖苷酶乳酸菌的筛选及其产酶条件的研究

目的从云南豆豉样品中筛选产β-半乳糖苷酶的乳酸菌,并对其产酶条件进行研究。方法从云南省元阳、红河、建水、石屏等地采集豆豉样品,并从中分离得到355株微生物。结果经明胶诱导、脱脂乳平板实验,复筛得到87株蛋白酶产生菌,从中筛选产β-半乳糖苷酶的乳酸菌。通过X-Gal平板实验,共获得34株产β-半乳糖苷酶菌株,通过酶活测定,最终筛选得到1株高产β-半乳糖苷酶菌株GJ-1-3L,经16S rDNA序列分析鉴定为短乳杆菌;GJ-1-3L在以葡萄糖为碳源、多聚蛋白胨为氮源、起始pH 6.5的MRS培养基中,接种量为4%,35℃发酵培养12 h,其β-半乳糖苷酶活性高达6.73 U/mL,Cu2＋、Ba2＋对酶活有抑制作用,而K2HPO4、MgSO4则能促进酶活。结论 GJ-1-3L菌株来源于豆豉,能够产生β-半乳糖苷酶发酵乳糖,同时产生乳酸,其在食品与乳品加工等方面具有很好的应用前景。     

重离子诱变技术选育碱性蛋白酶高产菌株

从采集的土壤样品中分离筛选出一株碱性蛋白酶产生菌G-41,经16S rRNA分子鉴定为芽孢杆菌属菌株。该菌株在发酵培养基中能产生较高产量的胞外碱性蛋白酶(1.7×104U/mL)。以G-41为出发菌株,对其进行重离子辐照诱变处理,获得突变株G-41-68,将该突变株再次经重离子诱变,从大量突变株中筛选出碱性蛋白酶高产菌株15Gy-54,其酶活力达到6.22×104U/mL。与出发菌株相比较,突变株G-41-68和15Gy-54的酶活力分别提高了1.58倍和2.65倍。对突变株15Gy-54的发酵条件进行了优化研究,结果表明,该菌株的碱性蛋白酶活力得到进一步提高,达到7.18×104U/mL,其最适发酵条件为:培养基(g/100mL)为胰蛋白胨1、酵母膏0.5、乳糖5、Na2HPO4·12H2O0.4、KH2PO40.03、Na2CO30.1、MgSO40.0481(4×10-3mol/L)、pH8.0,培养温度41℃,振荡培养时间42-48h。实验结果表明,重离子辐照诱变技术是一种非常有效的微生物诱变育种新技术。     

猪笼草中产蛋白酶内生菌的分离及鉴定

采用研磨法分离纯化猪笼草各组织器官中的内生菌,并采用水解圈法和摇瓶发酵法分别进行初筛与复筛,对筛选出的菌株进行16SrDNA分析鉴定。结果表明:在猪笼草叶片、捕虫囊、根和茎等4种器官中共分离出25株内生菌;进一步从中筛选出2株可产胞外蛋白酶的细菌A3、L5,其中菌株A3的产酶能力较高,水解圈/菌落直径比(D/d)值为6.5,发酵液的蛋白酶活力为17.58U/mL;菌株L5的D/d值为3.0,发酵液的蛋白酶活力为15.77U/mL;16SrDNA鉴定结果表明,菌株A3、L5与芽孢杆菌属成员具有99%的同源性,其中A3是枯草芽孢杆菌(Ba-cillus subtilis),L5可能是其的新变种或新亚种。     

生物合成γ-氨基丁酸曲霉菌株的选育、紫外诱变和发酵条件的研究

从自然发酵的红曲中分离筛选到1株曲霉菌株,其菌丝体可以谷氨酸钠为底物转化富集γ-氨基丁酸(GABA),经初步鉴定该菌株为米曲霉(Aspergillus oryzae)。以该菌株的孢子为对象进行紫外诱变,初筛得到8株产GABA活力不同的菌株,复筛后获1株GABA的产量较高的突变菌株As-8。菌株As-8转化富集GABA的适宜条件为:pH 6.0,以蔗糖为碳源,蛋白胨为唯一氮源或蛋白胨和牛肉膏为复合氮源。产生的GABA含量最高可达4.2g/L,并能稳定遗传。     

菊粉酶产生菌的筛选及60Co诱变选育简

目的：从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法：通过稀释平板涂布法分离微生物;利用^60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果：分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行^60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46．62U/mL,是未诱变菌株酶活的2．72倍。结论：经诱变得到1株高产菊粉酶活力的突变菌株。     

菊粉酶产生菌的筛选及~(60)Co诱变选育

目的:从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法:通过稀释平板涂布法分离微生物;利用60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果:分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46.62 U/mL,是未诱变菌株酶活的2.72倍。结论:经诱变得到1株高产菊粉酶活力的突变菌株。     

产γ -氨基丁酸乳酸菌的筛选及鉴定

从泡菜汁样品中初步筛选得到6株乳酸菌,采用纸层析和分光光度法对含1％谷氨酸的GYP发酵液中GABA含量分析,复筛得到一株产量较高的3#菌株,培养3d其GABA含量可达8.006 g/L.经形态学、生理生化特性分析及16S rDNA鉴定,所筛乳酸菌株为乳酸肠球菌.     

产DHA菌株的诱变育种

以海水中分离得到产DHA的酒香酵母(编号为7-3)作为出发菌株,对其进行单一及复合诱变处理,初筛采用分光光度法确定菌体中油脂的相对含量,选择吸光度相对较高的菌株进行复筛,复筛采用有机溶剂萃取法测定油脂含量、气相色谱法测定DHA含量。单一诱变的菌株H-1的脂肪含量达到43.8%,为对照的4倍,经复合诱变后得到1株编号为H-Z-6-3的菌株其油脂含量达50.4%,诱变后油脂中DHA的含量提高较少。     

两个不同启动子及其组合对碱性蛋白酶AprE异源表达的影响

背景:碱性蛋白酶(alkaline protease)是一种具有广泛用途的工业酶制剂,其发酵活力目前仍不能满足工业生产需要。目的:旨在通过优化启动子及其组合来提高Bacillus subtilis WB600中碱性蛋白酶AprE的产量。方法:以Bacillus subtilis WB600为出发菌株,成功构建了含有4种不同类型启动子(P1、P2、P-1-2、P-2-1)的碱性蛋白酶AprE表达菌株。结果:含不同启动子的4株重组菌均可成功表达碱性蛋白酶,发酵48h,含单一启动子P2的重组菌株表达碱性蛋白酶的活力为4 041U/ml,是P1的1. 23倍。双启动子重组菌B. subtilis WB600/P-2-1-aprE表达的酶活性最高,是双启动子P-1-2的1. 35倍,达到了6 125U/ml。结论:为工业化高产碱性蛋白酶提供了一种有效策略。     

Proteases from Bacillus: a new insight into the mechanism of action for rhizobacterial suppression of nematode populations

AIMS: The aim of this study was to investigate the role of proteases in Bacillus spp. of rhizobacteria in suppressing nematode populations and to understand their mechanism of action. METHODS AND RESULTS: Rhizobacteria with nematicidal activity were isolated from soil samples of five root knot nematode-infested farms. Among these strains, nematotoxicities of Bacillus strains were intensively analysed. Further assays of nematicidal toxins from Bacillus sp. strain RH219 indicated an extracellular cuticle-degrading protease Apr219 was an important pathogenic factor. The Apr219 shared high similarity with previously reported cuticle-degrading proteases from Brevibacillus laterosporus strain G4 and Bacillus sp. B16 (Bacillus nematocida). The cuticle-degrading protease genes were also amplified from four other nematicidal Bacillus strains isolated from the rhizosphere. In addition to Apr219, a neutral protease Npr219 from Bacillus sp. RH219 was also investigated for activity against nematodes. CONCLUSIONS: The wide distribution of cuticle-degrading proteases in Bacillus strains with nematicidal activity suggested that these enzymes likely play an important role in bacteria-nematode-plant-environment interactions and that they may serve as important nematicidal factors in balancing nematode populations in the soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased understanding of the mechanism of action of Bacillus spp. against nematodes could potentially enhance the value of these species as effective nematicidal agents and develop new biological control strategies.     

Phenoloxidase Activity in Hemolymph of Plasmodium-Melanizing and Nonmelanizing Strains of Anopheles gambiae

ABSTRACT Phenoloxidase (PO) activity was compared in two strains of the mosquito, Anopheles gambiae , one of which melanotically encapsulates and kills malaria parasites ( Plasmodium spp.) and a second which permits normal development of these parasites. The enzyme assay, based on the formation of dopachrome from L-dopa, was linear with respect to enzyme concentration and time and was as inhibitable by phenylthiourea. There were no significant differences in the K m or V max of hemolymph samples taken from 3 days old naive mosquitoes of each strain. An age course demonstrated that PO activity was slightly higher in the refractory strain on Days 1 and 2 after emergence but decreased to similar stable levels in both strains by Day 3. PO activity differed in the two strains following an uninfected blood meal. While PO activity significantly increased by 48 h post blood feeding in Plasmodium -susceptible strain, activity remained level or slightly decreased in the Plasmodium -refractory strain. Finally, protease inhibitors and proteases were tested for effects on PO activity. Leupeptin, TLCK, and TPCK all inhibited PO activity in both strains. PO in both strains was activated by bovine trypsin but not by bovine chymotrypsin.     

巴里坤湖和玛纳斯湖嗜盐菌的分离及功能酶的筛选

目的:了解新疆巴里坤湖与马纳斯湖中嗜盐菌及功能酶的多样性。方法:从两湖中采集水样进行菌种分离,采用PCR方法扩增出其16S rRNA基因(16S rDNA),并测定了基因的序列。对分离菌株进行了蛋白酶、淀粉酶、酯酶、脂肪酶、以及纤维素酶的筛选。结果:从两湖水样共分离得到51株嗜盐菌。基于16SrDNA序列的同源性比较和系统发育学分析,发现从两湖分离获得的中度嗜盐菌分别属于Planococcaceae、Bacillacea、Staphylococcus、Halomonadaceae、Salicolaceae以及Pseudomonadacaeae 6个属。分离得到的极端嗜盐古菌属于Halobacteriaceae属。功能酶筛选结果表明产蛋白酶的嗜盐菌共有15株,产酯酶的共有23株,产淀粉酶的共有8株,未获得产脂肪酶和纤维素酶的嗜盐菌。结论:新疆巴里坤湖和马纳斯湖中有丰富的嗜盐微生物资源及酶资源,有重要的研究意义和应用前景。     

Serine proteases and metalloproteases associated with pathogenesis but not host specificity in the Entomophthoralean fungus Zoophthora radicans

The protease activity of a Zoophthora radicans strain that was highly infective toward Pieris brassicae (cabbage butterfly) larvae was compared with that of isogenic strains that were adapted to Plutella xylostella (diamondback moth) larvae through serial passage. All strains produced three distinct serine proteases ranging in size from 25 to 37 kDa; however, the original strain from P. brassicae also produced large amounts of an approximately 46 kDa metalloprotease. Subsequently, a cDNA encoding a 43 kDa (mature enzyme) zinc-dependent metalloprotease, ZrMEP1, was isolated from the original fungal strain and most likely corresponds to the 46 kDa protease observed with in-gel assays. ZrMEP1 possessed characteristics of both the fungalysin and thermolysin metalloprotease families found in some pulmonary and dermal pathogens. This is the first report of this type of metalloprotease from an entomo pathogenic fungus. A cDNA encoding a trypsin-like serine protease, ZrSP1, was also identified and was most similar to a serine protease from the plant pathogen Verticillium dahliae. In artificial media, ZrMEP1 and ZrSP1 were found to be differentially responsive to gelatin and catabolite repression in the fungal strains adapted to P. brassicae and P. xylostella, but their expression patterns within infected larvae were the same. It appears that while these proteases likely play a role in the infection process, they may not be major host specificity determinants.     

Detection of protease specifically splitting actin in revertants of Shigella flexneri L-forms

Proteolytic activity of cell extracts from revertants of Shigella flexneri L-forms as well as biochemical properties of these strains and their sensitivity to antibiotics were studied. The protease found earlier in cells of strain E. coli A2 was shown to be synthesized by one of 8 revertants under study. This protease split actin and did not split some other proteins, its activity was inhibited by inhibitors of metalloproteases. Strain 5a2c which produced the protease was similar to the strain E. coli A2 and differed from other revertants in some biochemical properties, resistance to ampicillin and sensitivity to furazolidone. Thus the protease activity can be a marker of structural and functional transformation of Sh. flexneri under the influence of furazolidone.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Screening and isolation of an organic solvent-tolerant bacterium for high-yield production of organic solvent-stable protease

Forty-three strains were screened from crude oil-contaminated samples by toluene and cyclohexane enrichment in medium. Ten of these strains demonstrated high protease activity on skim-milk agar. Among them, the PT121 isolate, identified as Pseudomonas aeruginosa, was selected based on its extracellular protease stability in the presence of hydrophilic organic solvents. The crude protease also retained most of its activity up to at least 14 days in the presence of various organic solvents at 50% concentration, and the protease activity in production medium was 10,876U/ml after 72h incubation. This protease showed high activity as a catalyst for aspartame precursor Cbz-Asp-Phe-NH2 synthesis in the presence of 50% dimethylsulfoxide (DMSO).     

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.     

津巴布韦烟叶中淀粉酶和蛋白酶产生菌的分离及鉴定

目的:从津巴布韦烟叶中分离产蛋白酶菌和产淀粉酶能力最高的菌株,并对其进行鉴定。方法:采用淀粉富集培养基和酪蛋白富集培养基分别分离津巴布韦烟叶中的产淀粉酶和产蛋白酶菌株,通过生理生化实验和16SrRNA序列分析鉴定分离的菌株。结果:产蛋白酶菌株菌体不透明、表面有褶皱,蛋白酶酶活为52.10±0.13 U/mL;产淀粉酶菌株菌体表面呈黏状,淀粉酶酶活为3.69±0.07 U/mL;产蛋白酶与产淀粉酶的2株菌均与枯草芽孢杆菌的16S rRNA序列有100%的相似性,结合生理生化指标初步鉴定为枯草芽孢杆菌。结论:获得的2株菌在降解烟叶的蛋白质和淀粉过程中可能起重要作用。