维生素C发酵中伴生菌对氧化葡糖杆菌的影响

通过分析维生素C二步发酵过程中活菌数、产酸量、pH、糖酸转化活力等 ,研究了蜡状芽孢杆菌 (俗称大菌 )对氧化葡糖杆菌 (俗称小菌 )生长和产酸的影响。结果表明 ,在大菌存在情况下 ,小菌的活菌数约为单菌培养条件下的 5倍 ,产酸量为单菌培养条件下的 2～ 3倍 ,糖酸转化活力为单菌培养条件下的 2～ 3倍 ,提示在混合菌发酵条件下大菌仅仅是通过刺激小菌的生长而促进小菌产酸。用小菌休止细胞进行的糖酸转化实验结果也表明 ,无论大菌的发酵上清液还是破碎的菌体 ,都未发现对小菌产酸产生直接影响。     

贵州白柄鸡Zong菌菌丝体培养条件研究

本文报道筛选了优良的固体培养基配方,对不同ｐＨ值、温度、碳源、氮源条件下白柄鸡Ｚｏｎｇ菌菌丝体的生长进行测定。结果表明：白柄鸡Ｚｏｎｇ菌以碳源葡萄糖、氮源花生 、ＰＨ４．６、温度３０℃淡最适生长条件。目前,白柄鸡Ｚｏｎｇ菌在实验室条件下生长良好,这对人工栽培和利用白柄鸡Ｚｏｎｇ菌具有实际经济意义。     

鸡胴体中沙门菌的定量检测及风险评估

目的通过鸡胴体中沙门菌的定量检测,了解长春市市售鸡胴体中沙门菌的污染状况,为沙门菌的定量检测评估提供依据。方法前增菌:鸡胴体缓冲蛋白胨水淋洗后培养;增菌:TT肉汤和RV肉汤;分离:接种XLT4培养基;鉴定:API20E和沙门多价血清。结果沙门菌全年都有检出,阳性率15%~100%不等。7月份阳性率最高为100%;其次8月份为90%;6月份为80%。冷藏储存条件下鸡胴体样品中沙门菌的检出率及其含菌量均高于冷冻条件下。农贸市场鸡胴体样品中沙门菌的检出率及其含菌量均高于大型超市。要加强市售鸡胴体中各环节中沙门菌污染的监管。结论长春市市售鸡胴体中沙门菌的污染率为64.6%(155/240)。冷藏储存条件下鸡胴体样品中沙门菌的检出率及其含菌量均高于冷冻条件下。农贸市场鸡胴体样品中沙门菌的检出率及其含菌量均高于大型超市。要加强市售鸡胴体中各环节中沙门菌污染的监管。     

阿萨希毛孢子菌通过分泌铁载体在低铁限制性条件下生长

目的探讨阿萨希毛孢子菌在低铁限制性条件下生长的机制,为后续联合常用抗真菌药物治疗播散性毛孢子菌病提供实验室依据。方法 200μmol/L的去铁胺加入酵母浸出粉胨葡萄糖培养基(Yeast Extract Peptone Dextrose Medium,YPD)培养基中制备低铁限制性培养基,检测阿萨希毛孢子菌临床分离株在该培养基中的生长曲线,并检测低铁条件下,阿萨希毛孢子菌产生铁载体的情况。结果阿萨希毛孢子菌的生长曲线不受低铁限制性条件的影响,且其在低铁环境下可以产生铁载体。结论阿萨希毛孢子菌通过产生铁载体在低铁限制性条件下正常生长。     

土霉素菌渣源酵母菌的筛选及其培养条件的优化

【背景】随着制药行业的蓬勃发展,中国每年产生大量的抗生素菌渣,已造成了不可忽视的环境污染和资源浪费等问题。由于抗生素菌渣中含有丰富的蛋白质等有机物质,利用其蛋白质等进行二次生产或将成为解决抗生素菌渣处理问题的一种有效方法。【目的】从土霉素菌渣中筛选出天然酵母菌菌株,并利用土霉素菌渣作为酵母菌生长的主要培养基成分,通过其培养条件的优化,实现土霉素菌渣的资源化利用。【方法】以土霉素菌渣为样品,运用稀释涂布法进行酵母菌的筛选,通过其形态学观察和18S rRNA基因序列分析等方法对菌株进行鉴定。通过单因素试验与Box-Behnken Design试验结合,对培养条件进行优化,确定筛选菌株在以土霉素菌渣为主要成分培养基中的最佳生长条件。【结果】筛选的土霉素菌渣源酵母菌为一株希腊接合囊酵母菌(Zygoascus hellenicus)Y1,其最佳培养条件为:土霉素菌渣添加量为5%,葡萄糖添加量为0.5%,接菌量为2%,在pH 5.0、32℃、160r/min条件下培养24h。【结论】筛选到一株希腊接合囊酵母菌Y1,该菌能够很好地利用土霉素菌渣进行生长,实现了土霉素菌渣的资源化利用,大大减少了菌渣的...     

甲型副伤寒沙门菌培养条件的优化

优化甲型副伤寒沙门菌的培养条件,提高菌体产量。方法通过单因素及正交试验,对影响甲型副伤寒沙门菌生长的培养温度、NaCl浓度和pH等条件进行优化。结果甲型副伤寒沙门菌在NaCl浓度0.75%、温度35℃、pH6.5时菌体产量最高。结论通过对甲型副伤寒沙门菌培养条件进行优化,获得较高的菌体产量,为后期诊断试剂盒的开发奠定了基础。     

聚磷菌的诱变选育及其生长特性

方法:采用氮离子注入技术对巢湖底泥中筛选出的一株细菌进行辐照诱变处理,选育出2株高效聚磷菌,并对这两株菌的生长特性进行了研究,根据生理生化及生长试验,并参照《伯杰氏细菌手册》进行菌种分类鉴定,这两株菌被鉴定为假单胞菌属细菌(Pseudomonas sp),并研究了菌量、温度、pH、氧、碳源对两株假单胞菌(Pseudomonas sp)的生长特性及聚磷代谢的影响。结果:试验表明,诱变后的聚磷率明显高于试验出发菌,为出发菌的1.43~3.89倍。两株菌的最适生长温度为30℃,最适pH和菌量分别为6、8和OD=0.6、0.4。最适菌量在厌氧条件下出现放磷现象,好氧条件下过量摄磷现象,经过先厌氧条件的培养其去磷率明显高于直接好氧培养条件下的去磷率,以乙酸钠为碳源时其聚磷率高于乳糖、甲醇、乙醇为碳源时的摄磷量。     

肺炎克雷伯氏菌荚膜多糖表达量评价指标的建立及发酵条件优化

建立了特异性强的肺炎克雷伯氏菌荚膜多糖全菌ELISA检测方法,检测结果与多糖表达量相关性好;以全菌ELISA值结合菌数为评价指标,对影响荚膜多糖表达的培养基组成及发酵条件进行了优化,优化后的摇瓶培养条件下发酵液活性和生物量分别比优化前提高72.7和33倍,并经7L罐放大实验,绘制发酵动力学曲线,为肺炎克雷伯氏菌荚膜多糖进一步开发打下基础。     

贵州白柄鸡菌菌丝体培养条件研究

本文报道筛选了优良的固体培养基配方,对不同ｐＨ值、温度、碳源、氮源条件下白柄鸡菌（ＴｅｒｍｉｔｏｍｙｃｅｓａｌｂｉｃｅｐｓＨｅ．）菌丝体的生长进行测定。结果表明：白柄鸡菌以碳源葡萄糖、氮源花生粉、ｐＨ４６、温度３０℃为最适生长条件。目前,白柄鸡菌在实验室条件下生长良好,这对人工栽培和利用白柄鸡菌具有实际经济意义     

嗜酸菌及其应用

自然界大多数环境的pH值为5～9,它适合多数微生物生长。嗜酸菌是一种能在低pH条件下生长和繁殖的极端环境微生物［‘－’］,通常在pHZ～5生长很好,pHS．5以上生长不好。有些嗜酸菌在中性pH条件下根本不生长,如氧化硫硫杆菌（Thiobacillusthiootidans）,酸热硫化叶菌（deghlobusacidocaldarius）,酸热芽抱杆菌O沏ciousacidoca儿brius）等,最佳生长pH是2．0～3．0,这些都是专性嗜酸菌。一些真菌也能在pHS．0或更低条件下生长,实际上是耐酸菌。l嗜酸菌生态分布及其对环境适应机制嗜酸菌生长在酸性环境,这主要与硫或硫化物的存在…     

运用定点突变提高重组木聚糖酶在毕赤氏酵母中的表达

运用PCR介导的定点突变对米曲霉(Aspergillus　oryzae)来源的木聚糖酶在毕赤酵母中的重组表达进行了研究,获得一表达量远远高于亲本的突变株I156A,对其进行了提纯并研究其酶学特性,除热稳定性外其余与亲本基本一致。突变株I156A所产木聚糖酶XynFl的分子量为35kD,在pH 4～9范围内稳定,最适pH为70,最适温度为45℃,在50℃以下稳定性略高于亲本。     

木聚糖酶XYNB分子中折叠股B1和B2间的疏水作用对酶热稳定性的影响

对来源于Streptomycesolivaceoviridis的高比活木聚糖酶XYNB进行同源建模,并结合嗜热木聚糖酶氮末端芳香族氨基酸疏水作用的结构分析,设计了XYNB的T11Y定点突变,观察XYNB分子中折叠股B1和B2的疏水作用对酶的热稳定性的影响。将突变酶XYNB′在毕赤酵母中表达,表达的XYNB′经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明,XYNB′的耐热性比XYNB有明显的提高,但最适温度与原酶一样为60℃。另外,XYNB′的最适pH、Km值及比活性均有一定的改变。实验证实了木聚糖酶XYNB的氮端芳香族氨基酸之间的疏水相互作用与其热稳定性相关,为进一步的结构与功能研究提供了优良的基因材料。     

斜卧青霉纤维素酶和木聚糖酶高产菌株的选育

以纤维素酶高产菌株斜卧青霉A50为出发菌株,通过紫外诱变原生质体获得1株木聚糖酶活力提高80%而纤维素酶活力没有改变的6号菌。蛋白质电泳和酶谱检测结果显示,纤维素酶谱基本无差别,而木聚糖酶谱显示6号菌比A50多了一条带。6号菌优化后的产酶培养基组成为:麸皮7%、葡萄糖0.1%,该条件下,纤维素酶活为19.7IU/mL,木聚糖酶活力为215.4IU/mL。     

Cloning, disruption, and expression of two endo-beta 1, 4-xylanase genes, XYL2 and XYL3, from Cochliobolus carbonum.

In culture, the filamentous fungus Cochliobolus carbonum, a pathogen of maize, makes three cationic xylanases, XYL1, which encodes the major endoxylanase (Xyl1), was earlier cloned and shown by gene disruption to encode the first and second peaks of xylanase activity (P. C. Apel, D. G. Panaccione, F. R. Holden, and J. D. Walton, Mol. Plant-Microbe Interact. 6:467-473, 1993). Two additional xylanase genes, XYL2 and XYL3, have now been cloned from C. carbonum. XYL2 and XYL3 are predicted to encode 22-kDa family G xylanases similar to Xyl1. Xyl2 and Xyl3 are 60% and 42% identical, respectively, to Xyl1, and Xyl2 and Xyl3 are 39% identical. XYL1 and XYL2 but not XYL3 mRNAs are present in C. carbonum grown in culture, and XYL1 and XYL3 but not XYL2 mRNAs are present in infected plants. Transformation-mediated gene disruption was used to construct strains mutated in XYL1, XYL2, and XYL3. Xyl1 accounts for most of the total xylanase activity in culture, and disruption of XYL2 or XYL3 does not result in the further loss of any xylanase activity. In particular, the third peak of cationic xylanase activity is still present in a xyl1 xyl2 xyl3 triple mutant, and therefore this xylanase must be encoded by yet a fourth xylanase gene. A minor protein of 22 kDa that can be detected immunologically in the xyl1 mutant disappears in the xyl2 mutant and is therefore proposed to be the product of XYL2. The single xylanase mutants were crossed with each other to obtain multiple xylanase disruptions within the same strain. Strains disrupted in combinations of two and in all three xylanases were obtained. The triple mutant grows at the same rate as the wild type on xylan and on maize cell walls. The triple mutant is still fully pathogenic on maize with regard to lesion size, morphology, and rate of lesion development.     

木聚糖酶XYNB的N46D突变、表达及酶学性质变化

对来源于Streptomyces olivaceoviridis的高比活木聚糖酶XYNB进行同源建模和同源序列比较,发现第11族木聚糖酶的催化结构域在β折叠股A3和B3之间存的一个保守的氨基酸位点,该位点与木聚糖酶的pH特性有关.据此设计了XYNB的N46D定点突变.将突变酶XYNBN46D在毕赤酵母中表达,表达的XYNBN46D经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明, XYNBN46D的最适pH值由5.2下降到4.2,pH稳定性也向酸性pH偏移,同时,热稳定性和最适温度也有一定的提高, 但酶的比活性显著下降.结果证实,木聚糖酶XYNB的第46位Asn与其最适pH值相关.对导致酶学性质改变的可能因素进行了分析,结果为进一步的结构与功能研究提供了资料.     

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus for simultaneous biosynthesis of xylanase and laccase under solid-state fermentation

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching.     

Inducible character of β‐xylanase in a hyperproducing mutant of Thermomyces lanuginosus

Ten strains of Thermomyces lanuginosus from various culture collections were evaluated for extracellular endo‐β‐1,4‐xylanase production. The best xylanase producer (5771±173 nkat/mL) T. lanuginosus SK, was subjected to UV and N‐methyl‐N‐nitro‐N‐nitrosoguanidine mutagenesis. A mutant strain T. lanuginosus MC134, that showed on oatspelts xylan a 1.5 fold higher xylanase production than the parent strain SK, was subjected to a study of the regulation of xylanase synthesis during growth on various carbohydrates and during induction in glucose‐grown cells. In the growth experiments the highest production of xylanase was observed in the presence of xylans, however, an appreciable amount of the enzyme, about 10%, was also produced during growth on xylose. Xylobiose was found to be the most efficient xylanase inducer in the glucose‐grown cells. Its induction efficiency was followed by xylose, beechwood and birchwood xylan. Xylanase induction by polysaccharides started several hours later but proceeded for a longer time than that induced by the low molecular mass inducers, indicating that the polysaccharides serve as more sustainable source of inducers and that they have to be first hydrolyzed by the low level of constitutively synthesized xylanase. The repression of the induction of xylanase by glucose confirmed that the xylanase synthesis in the mutant strain is similar to the parent strain and exhibits an induction‐repression regulation mechanism.     

Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10128 micromol glucose x min(-1) x (mg protein)(-1) at pH 6.0. It was more thermostable at 60 degrees C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.     

G1 versus G2 cell cycle arrest after adriamycin-induced damage in mouse Swiss3T3 cells

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-beta-D-xylobioside substrate was increased by 10(3)-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the alpha-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.     

木聚糖酶高产菌株的诱变*

出发菌株Aspergillus niger M1经过紫外线诱变得到一株木聚糖酶活力提高30％的突变株A．niger J506。木聚糖酶谱带检测发现,突变株成熟发酵液中有3种类型的木聚糖酶,而出发菌株中只有两种。经过正交试验得出突变株产酶的最佳发酵条件为：主碳源浓度4％、麸皮与玉米芯的比例为5：5、葡萄糖浓度0．1％、草酸铵浓度2．0％,培养基初始pH为5．0,250mL三角瓶的装液量为100mL。