复合菌株发酵猪血粉的条件研究*

利用筛选出的2株高产蛋白酶的米曲霉(Aspergillus oryzae)菌株AS100、AS102作猪血粉发酵的主发酵菌株,配以1株酵母菌(Saocharomyces cereisiae)菌株Y113和1株细菌(Bacilus sp．)菌株Asp007为辅助菌株,研究了这四株菌的产酶性能,同时确定了它们在发酵血粉过程中的一系列参数。通过猪血粉发酵,获得了一种高蛋白发酵饲料,其香味浓郁,蛋白质含量高达69％,且富含游离氨基酸,VitD3、烟酸等维生素及Fe等无机元素,可作为禽畜高蛋白源或饲料添加剂。     

一株耐盐酵母的分离、鉴定及其生物学特性

以洋河酒厂的酒曲为材料,首先利用盐浓度梯度富集培养和氯化三苯四唑(TTC)染色法初筛获得18株酵母菌株,再通过杜氏小管产气及摇瓶发酵实验,从18株初筛菌株中分离得到1株在1. 4 mol/L NaCl浓度下生长性能较好的菌株,命名为W58。经形态观察和26S rDNA基因序列比对分析,该酵母菌株被鉴定为1株库德里阿兹威氏毕赤酵母(Pichia kudriavzevii)。对菌株的生长性能及耐受性进行研究发现:该菌最适生长温度为30℃,最适pH为6. 0,在高盐条件下(1. 4 mol/L NaCl),其胞内海藻糖的积累显著增加,比对照菌株高28. 6%。此外,菌株W58在高盐条件下,其ATPase活性比对照菌显著提高(约3倍)。综上所述,菌株W58在高盐条件下能通过胞内积累海藻糖以及维持一定的ATPase活性抵抗外界环境压力。     

酵母菌株对芒果酒理化特性及抗氧化特性的影响

采用5株广为使用的酵母菌株(EC1118, D254, Red fruit, Aroma white, SY)分别作为芒果酒的发酵菌株。通过对比它们的发酵力,起发时间以及对芒果酒的酒精度、干浸出物、挥发酸、总糖、可滴定酸含量、pH值以及抗坏血酸、总酚、抗氧化活性(DPPH, FRAP)、感官得分的影响,选取得到芒果酒发酵的最适酵母菌株。结果表明,5株酵母所发酵的芒果酒酒精度相差不大,且干浸出物与总糖含量之间没有显著性差异(p0.05)。Aroma white具有较强的发酵力,所发酵的芒果酒挥发酸含量较低,抗氧化能力较强,感官得分最高。因此确定在这5株菌株中,它最适宜作为芒果酒的发酵菌株。此外,运用主成分分析(principal component analysis,PCA)对5株发酵菌株生产的芒果酒进行了区分,结果表明此法可将5种芒果酒进行明显区分。     

口腔黏膜真菌鉴定方法的比较

比较常见用于黏膜真菌菌种鉴别的多种方法,探寻最佳的鉴别方法。采集230例普通人群口腔黏膜样本,分别用玉米吐温-80培养观察厚膜孢子法、糖发酵生化反应法、CHROMagar假丝酵母菌显色培养基法、ITS基因的PCR-RFLP(聚合酶链反应-限制性片段长度多态性)法、ITS测序菌种鉴定法,鉴别真菌各菌株。结果显示:有56例菌株至少通过1种方法检出真菌;玉米吐温-80分离培养假丝酵母菌37株;50例菌株ITS基因测序共鉴定出8个菌种,白假丝酵母菌(C.albicans)29株,近平滑假丝酵母菌(C.parapsilosis)10株,热带假丝酵母菌(C.tropicalis)5株,Candida metapsilosis 1株,Lodderomyces elongisporus 1株,克柔假丝酵母菌(Candida krusei)1株,乙醇假丝酵母菌(C.ethanolica)1株,季也蒙毕赤酵母菌(Pichia guilliermondii)2株;CHROMagar假丝酵母菌显色培养基法鉴定出3种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌;PCR-RFLP法检出5种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌、季也蒙毕赤酵母菌、克柔假丝酵母菌,与基因的测序鉴定一致率为91%;糖发酵生化反应法阳性标本占被检出真菌例数的46.4%(26/56)。结果表明:ITS基因的测序法可以准确鉴定真菌各个菌种;PCR-RFLP法能鉴定常见的菌种,但操作繁琐;CHROMagar假丝酵母菌显色培养基法能快速准确鉴别3种常见假丝酵母菌菌种;玉米吐温-80可以准确培养鉴别白假丝酵母菌;糖发酵生化反应法,缺乏足够的敏感度和特异性,难以准确鉴别各个菌种。     

一株红景天内生细菌的筛选及初步研究

【目的】从红景天根部筛选并鉴定一株产酪醇的细菌,初步研究其产酪醇特性,为寻找红景天替代资源提供新途径。【方法】用NA培养基从大花红景天根部中分离内生细菌,通过薄层层析(TLC)、高效液相色谱(HPLC)、气相色谱-质谱联用(GC-MS)筛选出产量最大的菌株,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析其分类学地位。单因素实验确定初始pH、培养温度、发酵时间及接种量对菌株产酪醇活力的影响。【结果】从大花红景天根部分离出14株内生细菌,其中8株能产酪醇,筛选出酪醇产量最大的菌株B3,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析初步鉴定为水生拉恩氏菌(Rahnella aquatilis)。研究其发酵条件,其最适pH为6.0,最适温度为32 °C,最佳发酵时间为42 h,最佳接种量为15%。在最适发酵条件下,用改良NA培养基发酵,B3菌株酪醇的产量为15.68 mg/L。【结论】B3菌株是一株具有产酪醇能力的细菌,在最适发酵条件下酪醇产量达到15.68 mg/L,具有潜在的开发价值。     

高产糖化酶菌株的筛选与诱变育种

从土壤及霉变淀粉质材料等样品中分离获得到产糖化酶活性较高的菌株L-3,经鉴定为一种紫红曲霉;通过紫外线和硫酸二乙酯分别进行诱变,得到4株性能良好的糖化酶变异株,其中菌株7性能最好,命名为M.F7。对该菌株进行发酵性能测试,结果表明该菌株糖化酶活力比原菌株提高了20.8%;经过继代培养,证明其性状能稳定遗传。通过正交试验,确定菌株7最佳培养温度为30℃,最适pH值为5.5～6.0,最适培养时间为5 d。     

基于微生物同化作用的D-丙氨酸生产工艺研究

以L-丙氨酸为唯一碳氮源,从采集的若干土壤中初筛出能够降解L-丙氨酸的菌株;再以D-丙氨酸为唯一碳氮源,复筛出降解L-丙氨酸而不降解D-丙氨酸的菌株。依据菌种对DL-丙氨酸的不对称降解活性,筛选出具有最高的L-丙氨酸降解活性的菌株,并对菌株同化L-丙氨酸的反应条件进行了研究。结果表明:编号为ALA-D82的菌株具有最高的降解L-丙氨酸的能力,经鉴定为酵母菌属。在30℃,控制pH6.0,通气比1:1(V/V)和转速900 r.min-1的条件下,L-丙氨酸降解的速度最大。在最适条件下,1500 g DL-丙氨酸分两部分添加入7 L的反应液中。反应72 h后溶液中的L-丙氨酸被完全降解,提取得到D-丙氨酸晶体,产率和光学纯度分别达到92.13%和99%。     

生淀粉高浓度酒精发酵的研究

本研究利用国内常用的糖化酶制剂糖化生玉米面中的淀粉,同时接种酵母菌,在30℃下,探讨了玉米淀粉的高浓度酒精发酵工艺。选择到了一株产高浓度酒精酵母菌,H0菌株。发酵温度为30℃、pH4一s、加糖化酶量为每克原料300单位、酵母接种量3％(v／v)和原料加量为33．0％(w／v)时,这株酵母菌在70小时内可产生17．5％(v／v)的乙醇。如果原料加量为36．0％(w／v)时,该菌株在96小时内可以产生18．O％(v／v)的乙醇。在前一种加料情况下,成熟发酵醪中的pH为5、残还原糖为O．19％、残总糖为3．5“。在后一种加料情况下,成熟发酵醪中的pH为5、残还原糖为0．81％、残总糖为5．1％。     

染料脱色菌与芳胺降解菌的筛选及降解染料研究

从印染厂废水处理系统的曝气池中分离到17株对多种染料有较高脱色能力的细菌,其脱色率都在80%以上,但偶氮染料脱色后产生的中间产物多数为无色的芳香胺类化合物,大多数细菌不能将其进一步降解。为此,通过富集培养和梯度驯化又筛选到一株以对硝基苯胺为唯一碳、氮源的菌株J18,该菌株虽对染料脱色能力很弱,却能够降解芳香胺类化合物。将芳香胺降解菌J18与染料脱色菌H两菌株混合培养,在最适条件下,结果使染料的脱色率和芳胺降解率均到达90%和85%以上,从而达到了彻底降解染料的目的。     

一株黄瓜根际促生菌的筛选、鉴定及其发酵特性

Salkowski比色法评价菌株发酵产吲哚乙酸(IAA)的能力;采用平皿、盆栽方法检测菌株的促生能力;对典型菌株生理生化测定及16S rRNA基因序列系统发育分析,初步确定菌株的分类地位;进一步采用正交设计探索不同碳源、氮源对菌株产IAA的影响。结果表明从黄瓜植株根部分离得到菌株18株,其中8株为产IAA菌株。菌株SGM7产IAA能力最强,产量达23.59 mg/L;1%SGM7菌悬液对盆栽黄瓜幼苗有明显促生效果(P0.05);初步鉴定SGM7为短小芽孢杆菌(Bacillus pumilus);其最适产IAA发酵培养基配方为:蛋白胨1%、玉米粉2%、麸皮0.25%、硫酸铵0.05%、硝酸钾0.05%;其发酵液IAA产量高达35.87 mg/L。     

H+-ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate

A mutant of Corynebacterim glutamicum ('Brevibacterium flayum') ATCC14067 with a reduced H+-ATPase activity, F172-8, was obtained as a spontaneous neomycin-resistant mutant. The ATPase activity of strain F172-8 was reduced to about 25% of that of the parental strain. Strain F172-8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using ajar fermentor. It was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than in the parent. The respiration rate per cell of the mutant also increased to twice as much as that of the parent. However, the growth rate of the mutant was lower than that of the parent. Under those conditions, the parent produced more than 40 g/l glutamic acid, while the mutant hardly produced any glutamic acid. Instead the mutant produced 24.6 g/l lactic acid as the main metabolite of glucose. Remarkably, the accumulation of pyruvate and pyruvate-family amino acids, i.e., alanine and valine, was detected in the mutant. On the other hand, the parent accumulated alpha-ketoglutaric acid and a glutamate-family amino acid, proline, as major by-products. It was concluded that the decrease in the H+-ATPase activity caused the above-mentioned metabolic changes in strain F172-8, because a revertant of strain F172-8, R2-1, with a H+-ATPase activity of 70% of that of strain ATCC14067, showed a fermentation profile similar to that of the parent. Sequence analyses of the atp operon genes of these strains identified one point mutation in the gamma subunit in strain F172-8.     

Alanine production in an H+-ATPase- and lactate dehydrogenase-defective mutant of Escherichia coli expressing alanine dehydrogenase

Previously, we reported that pyruvate production was markedly improved in TBLA-1, an H⁺-ATPase-defective Escherichia coli mutant derived from W1485lip2, a pyruvate-producing E. coli K-12 strain. TBLA-1 produced more than 30 g/l pyruvate from 50 g/l glucose by jar fermentation, while W1485lip2 produced only 25 g/l pyruvate (Yokota et al. in Biosci Biotechnol Biochem 58:2164–2167, 1994b). In this study, we tested the ability of TBLA-1 to produce alanine by fermentation. The alanine dehydrogenase (ADH) gene from Bacillus stearothermophilus was introduced into TBLA-1, and direct fermentation of alanine from glucose was carried out. However, a considerable amount of lactate was also produced. To reduce lactate accumulation, we knocked out the lactate dehydrogenase gene (ldhA) in TBLA-1. This alanine dehydrogenase-expressing and lactate dehydrogenase-defective mutant of TBLA-1 produced 20 g/l alanine from 50 g/l glucose after 24 h of fermentation. The molar conversion ratio of glucose to alanine was 41%, which is the highest level of alanine production reported to date. This is the first report to show that an H⁺-ATPase-defective mutant of E. coli can be used for amino acid production. Our results further indicate that H⁺-ATPase-defective mutants may be used for fermentative production of various compounds, including alanine.     

癫痫患者血浆中游离氨基酸水平分析

对７１例癫痫患者血浆中１８种游离氢基酸与３６例正常人血浆中游离氨基酸的水平对照。分析表明,抑制性氨基酸（γ－氨基丁酸、甘氨酸）水平明显升高的癫痫患者,其它氨基酸水平绝大多数低于正常人;兴奋性氨基酸（谷氨酸、天门冬氨酸、丙氨酸、谷氨酰胺）水平明显升高的癫痫患者,其它氨基酸水平低于正常人。     

放线菌Streptomyces sp．FJ3的核糖体工程改良与活性产物的分离

[目的]利用核糖体工程抗性筛选技术,获得有抗菌活性突变株,并对突变株新产生活性物质进行研究.[方法]以三峡库区筛选出的无抗菌活性放线菌野生株为出发菌,通过单菌落挑选与平板划线培养,分离筛选具有链霉素和利福平抗性突变株;通过摇瓶发酵和对发酵液进行纸片法活性测定,获得抗金葡菌活性突变株;采用高效液相色谱法(HPLC)分析其发酵液组分,通过LC-MS对变化峰进行分析;进行16S rDNA及形态学鉴定.[结果]链霉素和利福平对放线菌菌株FJ3的MIC分别为0.5μg/mL和110μg/mL;在FJ3突变菌株中,共获得24株链霉素突变菌株和20株利福平突变菌株,抗菌活性筛选显示6株具有抗菌活性,其中2株链霉素突变菌株对金葡菌有强抑菌活性,采用Doskochilova溶剂系统纸层析结果表明,该活性物质为一种核酸类抗生素,HPLC和LC-MS显示该活性物质可能为硫藤黄菌素.[结论]利用核糖体工程技术可以改变放线菌的次级代谢,获得具有生物活性的突变株,拓展药源放线菌活性菌株新资源.     

Stimulation of alanine amino transferase (AlaAT) gene expression and alanine accumulation in embryo axis of the model legume Medicago truncatula contribute to anoxia stress tolerance

The efficiency of ethanolic fermentation in anoxia tolerance under sugar-limiting conditions, as in the field is still matter of debate. Due to higher rates of glycolysis and ethanol fermentation, faster depletion of sugar stores leads to decreased survival. In the present work the hypothesis that alanine amino transferase ( AlaAT ) fermentation be involved in anoxia tolerance was explored in Medicago truncatula during germination and seedling establishment. Expression of AlaAT and two low oxygen-responsive genes, alcohol dehydrogenase ( ADH ) and lactate dehydrogenase ( LDH ) were determined by real time quantitative RT-PCR and AlaAT activity was determined by ¹⁵N-Glutamate labelling coupled to amino acids analysis by gas chromatography–mass spectrometry and HPLC. Under anoxia not only ADH and LDH levels of expression increased but also AlaAT expression increased substantially. In parallel in vivo AlaAT activity increased and resulted in an increase in alanine synthesis that accumulated as the major amino acid instead of asparigine. These findings support the hypothesis that AlaAT expression and alanine accumulation contribute efficiently to anoxia tolerance. By competing with ethanolic fermentation for pyruvate, under sugar-limiting conditions alanine synthesis saves C3 skeletons avoiding a shortage in carbon availability and limits accumulation of acetaldehyde, a toxic compound. On another hand, increase in alanine was accompanied by an increase in γ-amino butyric acid, both amino acids may intervene in cytosolic pH regulation. Finally the role of alanine in anoxia tolerance was strengthened by the fact that when alanine synthesis was impaired germination and seedling development failed under anoxia.     

Induction of nutritional mutants ofMicrococcus glutamicus and their amino acid accumulation

Micrococcus glutamicus ATCC 13032, a glutamic acid-producing organism, was treated with 0.2M ethylmethane sulfonate, the auxotrophs isolated showing varied patterns of extracellular amino acids. Eighty auxotrophic strains were obtained, out of which 31 excreted 1.0-4.0 mg threonine per ml and all the auxotrophs required biotin for growth and production of the amino acid. Eleven auxotrophs produced 1.5 to 3.0 mg alanine per ml and these auxotrophs required amino acids for their growth. Other auxotrophs lost their excretion capacity in subsequent fermentation trials. Further mutation of the biotin-requiring auxotroph Micrococcus glutamicus EM with gamma rays resulted in the isolation of 89 auxotrophic strains, out of which 28 excreted threonine (up to 5.0 mg per ml) higher than the parent auxotroph. Exposure to X-rays yielded 97 auxotrophs, out of these 35 producing 1.0-3.0 mg methionine per ml and requiring biotin for growth and production of the amino acid. Other auxotrophs produced alanine (0.5 to 2.0 mg per ml) and threonine (2.0 to 3.3 mg per ml). Irradiation with gamma rays favoured the development of threonine producing auxotrophs while X-rays favoured methionine-producing auxotrophs.     

Mechanism of Tet repressor induction by tetracyclines: length compensates for sequence in the alpha8-alpha9 loop

Natural Tet repressor (TetR) variants are alpha-helical proteins bearing a large loop between helices 8 and 9, which is variable in sequence and length. We have deleted this loop consisting of 14 amino acid residues in TetR(D) and rebuilt it stepwise with up to 42 alanine residues. All except the mutant with the longest alanine loop show wild-type repression, but none is inducible with tetracycline. This demonstrates the importance of the alpha8-alpha9 loop and its amino acid sequence for induction. The induction efficiencies increase with loop length, when the more tightly binding inducer anhydrotetracycline is used. The largest increase of inducibility was observed for TetR mutants with loop lengths between eight and 17 alanine residues. Since loop residues Asp/Glu157 and Arg158 are conserved in the natural TetR sequence variants, we constructed a mutant in which all other residues of the loop were replaced by alanine. This mutant exhibits increased anhydrotetracycline induction compared to the corresponding alanine variant. Thus, these residues are important for induction. Binding constants for the anhydrotetracycline-TetR interaction are below the detection level of 10(5) M(-1) for the mutant with a loop of two alanine residues and increase sharply until a loop size of ten residues is reached. TetR variants with longer loops have similar anhydrotetracycline-binding constants, ranging between 2.6 x 10(9) M(-1) and 8.0 x 10(9) M(-1), about 500-fold lower than wild-type TetR. The increase of the affinity occurs at shorter loop lengths than that of inducibility. We conclude that the induction defect of the polyalanine variants arises from two increments: (i) the loop must have a minimal length-to allow efficient inducer binding; (ii) the loop must structurally participate in the conformational change associated with induction.     

胶质芽孢杆菌HM8841紫外线诱变育种研究

以胶质芽孢杆菌HM8841作为出发菌株,通过紫外线诱变和突变菌株性能测定,选育出3株适合生产发酵的优良菌种。与出发菌株相比,突变株具有缩短发酵周期,提高发酵水平,增强芽孢抗逆性能等特点。     

Amino acid accumulation by an analogue sensitive mutant ofCorynebacterium glutamicum

Summary A fluoroacetate/fluoropyruvate-sensitive mutant was derived from the parent strainCorynebacterium glutamicum ATCC 21513. Accumulation of various amino acids in the fermentation broth using the two strains was compared. The FA/FP-sensitive mutant accumulated about 26.5 g/L L-lysine and 2.2 g/L aspartic acid which was about 3-fold and 10-fold respectively, more than the amount produced by the parent strain.