Pathway and rate-limiting step of glyphosate degradation by Aspergillus oryzae A-F02

Aspergillus oryzae A-F02, a glyphosate-degrading fungus, was isolated from an aeration tank in a pesticide factory. The pathway and rate-limiting step of glyphosate (GP) degradation were investigated through metabolite analysis. GP, aminomethylphosphonic acid (AMPA), and methylamine were detected in the fermentation liquid of A. oryzae A-F02, whereas sarcosine and glycine were not. The pathway of GP degradation in A. oryzae A-F02 was revealed: GP was first degraded into AMPA, which was then degraded into methylamine. Finally, methylamine was further degraded into other products. Investigating the effects of the exogenous addition of substrates and metabolites showed that the degradation of GP to AMPA is the rate-limiting step of GP degradation by A. oryzae A-F02. In addition, the accumulation of AMPA and methylamine did not cause feedback inhibition in GP degradation. Results showed that degrading GP to AMPA was a crucial step in the degradation of GP, which determines the degradation rate of GP by A. oryzae A-F02.     

黑曲霉固态发酵生产单宁酶的条件优化

研究采用响应面法优化黑曲霉固态发酵生产单宁酶的培养条件。应用Plackett—Burman试验筛选出重要影响因子：五倍子粉含量、（NH4）2SO4浓度以及接种孢子量,最陡爬坡试验逼近最大响应区域。应用Box．Behnken响应面试验对重要影响因子进一步优化。得到最佳培养条件：每250mL三角瓶中装入1．0g五倍子粉、4．4g稻壳和0．5g麸皮、液固比（mL／g）2：1且营养盐溶液组成为（NH4）2s0421g/L、MgSO4·7H2O1g／L、NaCl1g／L,培养基pH自然,接种5．7×10^7个孢子后在30℃温度下培养4d。在此条件下,单宁酶产量从40U／g提高到114U／g,3次重复验证性试验平均值为115U／g,验证了模型的可靠性。     

米曲霉M-4降解己烯雌酚的条件优化

【目的】以米曲霉(Aspergillus oryzae)M-4对己烯雌酚(Diethylstilbestrol,DES)的降解率为响应值,对其降解条件进行优化。【方法】采用Plackett-Burman法对培养基组分和降解条件筛选显著性影响因素,并通过Box-Bohnken设计试验优化降解条件。【结果】最优培养基配方为:蛋白胨1.3%,CaCl_2 0.045%,葡萄糖0.5%,K_2HPO_4 0.15%,KH_2PO_4 0.05%,NaCl 0.05%,Tween 80 0.2%,DES质量浓度44 mg/L;最优培养条件为:初始p H 7.5,种龄72 h,转速140 r/min,培养温度28°C,培养时间72 h。【结论】在最优条件下菌株M-4对DES降解率为83.89%,比优化前(60.98%)提高1.38倍,差异极显著(P0.01)。     

响应面法优化黑曲霉产异淀粉酶的培养条件

在液态发酵条件下,采用单因素实验确定了Aspergillus niger PZ331产异淀粉酶的最适碳源和氮源,分别为蔗糖和硝酸铵。在上述基础上利用Plackett-Burman设计对影响产异淀粉酶的因素进行评价,并筛选出硝酸铵、接种量、培养温度3个主要因素;继而利用响应面设计优化了最佳硝酸铵浓度、接种量和培养温度。最终确定了最优培养条件为：蔗糖10 g/L,硝酸铵10 g/L,磷酸氢二钾3 g/L,硫酸亚铁0.01 g/L,硫酸镁1 g/L,起始p H值4.2;接种量2%（孢子浓度为107cfu/m L）,30℃培养72 h,酶活达137.3μ/m L;比基础培养基的提高了1.71倍左右。     

响应面法优化重组毕赤酵母表达米曲霉碱性蛋白酶诱导条件的研究

为探索重组米曲霉碱性蛋白酶（rAlp）在毕赤酵母中表达的最佳诱导条件,本研究在单因子实验的基础上,运用Box-Behnken设计的响应面试验对诱导条件进行了优化。根据回归分析确定了影响rAlp表达的因子,求得最佳诱导条件为：诱导温度28．53℃,诱导pH6．63,甲醇浓度1．28％。此工艺条件下碱性蛋白酶活力实测值为49．78U／mL,回归模型的预测值与实测值的相对误差〈1％,说明该回归方程与实际情况拟合很好。     

聚半乳糖醛酸酶高产菌的产酶条件研究

通过单因素及正交试验对米曲霉（Aspergillus orza）C491产生聚半乳糖醛酸酶的发酵条件进行了优化。该菌株的摇床发酵滤液以桔子果胶为底物时酶活力可达344．8U／mL。产酶最适培养基组成为：麦芽汁（糖度6％）中添加6％桔皮粉,2％硫酸铵（w/v）。最适培养条件：起始pH4（灭菌前）,30℃,r/min,培养112h。Tween80可以促进C－491产酶。     

米曲霉发酵纯化无患子皂苷的工艺研究

采用微生物发酵法对无患子皂苷水提取液进行纯化.比较了采用自然发酵、接种酵母菌发酵和接种米曲霉发酵纯化无患子皂苷的效果.结果表明,提取液不灭菌,接种米曲霉发酵纯化效果较为明显,优化后的发酵条件为:温度30℃、接种龄12 h、接种量为3％、摇床转速150 r/min,发酵7d后,皂苷含量稍有下降,但皂苷纯度可从48.71％提高到82.47％.米曲霉发酵法明显优于水提醇沉法、絮凝法和正丁醇萃取法.     

高产洛伐他汀棒曲霉菌株的筛选、鉴定及发酵条件优化

从不同生境(食品、土壤、空气、有机质等)收集到的自然发酵样品中分离得到150株曲霉属菌株.用高效液相色谱(HPLC)法检测发酵液中洛伐他汀(Lovastatin)含量,筛选获得1株稳定高产Lovastatin的曲霉菌株(编号:Ac-32).根据菌落形态特征并结合18 S rDNA测序,鉴定其为棒曲霉(Aspergillus clavatus).通过摇瓶发酵单因素实验优化了碳氮源种类、碳氮源含量、碳氮比(C/N)、发酵温度、初始pH、转速、种龄和接种量,确定了棒曲霉菌株Ac-32摇瓶发酵产Lovastatin的适宜条件为:乳糖为碳源、蛋白胨为氮源、碳源含量为100 g/L、氮源含量为12 g/L、碳氮比(C/N)为15:1.8、温度28℃、转速180 r/min、初始pH 5.2、种龄4 d、接种量6%.采用Minitab 17软件的P-B实验设计法,筛选对Lovastatin产量有显著影响的因素为:温度、pH、碳源含量和氮源含量.根据P-B实验结果,运用响应面法分析,确定棒曲霉菌株Ac-32产Lovastatin的最优条件为:碳源含量100 g/L,氮源含量11.8 g/L,温度28℃,pH 5.2.在此条件下,Lovastatin最高产量为236.221μg/mL.     

粘性嗜热链球菌ST-1产胞外多糖发酵条件优化

应用响应面法对一株粘性嗜热链球菌ST-1的产胞外多糖的发酵条件进行了优化.根据单因素的实验结果,选取接种量,发酵温度,发酵时间作为考察因素,以胞外多糖的产量作为响应值,利用Box-Behnken实验设计方法,建立了胞外多糖产量与三个考察因素之间的回归方程并得到最佳发酵条件为:接种量5%,发酵时间14 h,发酵温度42℃...     

响应面法优化荷叶离褶伞菌丝体产漆酶条件

为了对荷叶离褶伞产漆酶条件进行优化,在单因素实验基础上,通过最陡爬坡实验(PB)对培养基8因素进行筛选,获得影响产漆酶的3个显著性因素:葡萄糖,pH和KH₂PO₄;通过中心组合(CCD)设计及响应面分析确定了最优发酵条件:葡萄糖20.09g/L,酪蛋白1.5g/L,酵母提取物1.5g/L,MgSO₄ 3g/L,CuSO₄ 3.75mg/L,KH₂PO₄ 3.97g/L,pH 4.98,VB₁ 0.1g/L,愈创木酚12mg/L,该条件下,漆酶酶活为829.83U/mL,较未优化对照提高46.6%.     

Morphology engineering of Aspergillus oryzae for l-malate production

Aspergillus oryzae is a competitive natural producer for organic acids, but its production capacity is closely correlated with a specific morphological type. Here, morphology engineering was used for tailoring A. oryzae morphology to enhance l -malate production. Specifically, correlation between A. oryzae morphology and l -malate fermentation was first conducted, and the optimal range of the total volume of pellets in a unit volume of fermentation broth (V value) for l -malate production was 120–130 mm³/ml. To achieve this range, A. oryzae morphology was improved by controlling the variation of operational parameters, such as agitation speed and aeration rate, and engineered by optimizing the expression of cell division cycle proteins such as tyrosine-protein phosphatase (CDC14), anaphase promoting complex/cyclosome activator protein (CDC20), and cell division control protein 45 (CDC45). By controlling the strength of CDC14 at a medium level, V value fell into the optimal range of V value and the final engineered strain A. oryzae CDC14(3) produced up to 142.5 g/L l -malate in a 30-L fermenter. This strategy described here lays a good foundation for industrial production of l -malate in the future, and opens a window to develop filamentous fungi as cell factories for production of other chemicals.     

响应面法对微拟球藻产微藻蛋白的发酵条件优化

以稳定期微藻蛋白浓度为评价指标,利用响应面设计对微拟球藻(Nannochloropsis gaditana)的分批发酵条件进行优化。在单因素试验的基础上,选取温度、p H、搅拌速度及通气量为影响因子,采用四因素三水平的Box-Benhnken中心组合法设计试验。结果表明:微拟球藻的最佳发酵条件为温度30℃、p H 6.9、搅拌速度340 r/min以及通气量0.65 vvm,在此优化条件下得到微藻蛋白浓度为6.18 g/L,与模型预测值基本相符,较优化前提高了9.18%。     

Adenine Auxotrophic Mutants of Aspergillus oryzae: Development of a Novel Transformation System with Triple Auxotrophic Hosts

adeA and adeB genes homologous to Saccharomyces cerevisiae ADE1 and ADE2, respectively, were cloned from Aspergillus oryzae. AdeA and AdeB share 62.8% and 52.5% identities with S. cerevisiae Ade1 and Ade2, respectively. In order to obtain triple auxotrophic mutants from A. oryzae, 12 red-colored mutant colonies were isolated by UV mutagenesis of a double auxotrophic host, NS4 (niaD ^?, sC ^?), as a parent strain. All the mutants exhibited adenine auxotrophy and showed fluorescence in the vacuoles due to accumulation of a purine biosynthetic pathway precursor. Adenine auxotrophy of all the mutants was restored by introduction of either A. oryzae adeA or adeB genes. Sequence analysis demonstrated that substitutions or deletions of a single base pair occurred, inducing substitutions or frame shifts of amino acid sequences in both ade genes complementing the mutants. This study provides a novel host-vector system with triple auxotrophy in A. oryzae.     

Cultivation characteristics of immobilized Aspergillus oryzae for kojic acid production

Aspergillus oryzae in situ grown from spores entrapped in calcium alginate gel beads was used for the production of kojic acid. The immobilized cells in flask cultures produced kojic acid in a linear proportion while maintaining the stable metabolic activity for a prolonged production period. Kojic acid was accumulated up to a high concentration of 83 g/L, at which the kojic acid began to crystallize, and, thus, the culture had to be replaced with fresh media for the next batch culture. The overall productivities of two consecutive cultivations were higher than that of free mycelial fermentation. However, the production rate of kojic acid by the immobilized cells was suddenly decreased with the appearance of central cavernae inside the immobilized gel beads after 12 days of the third batch cultivation.     

寄生曲霉CICC40365利用木糖产L-苹果酸的发酵条件优化

【目的】为提高L-苹果酸产量及木糖利用率,以寄生曲霉(Aspergillus parasiticus CICC40365)为菌种,木糖为碳源,对其发酵工艺及木糖代谢途径进行初步研究。【方法】采用单因素试验和响应曲面法(Box-Behnken设计)对培养基和发酵条件进行优化。【结果】获得最佳培养基配方为:木糖100.0 g/L、硫酸铵2.0 g/L、酵母浸粉3.0 g/L、硫酸镁0.20 g/L、硫酸锰0.15 g/L、硫酸亚铁0.08 g/L、碳酸钙80.00 g/L,L-苹果酸的产量为53.58 g/L,较优化前提高40.5%。发酵条件较好组合为:接种量为8%(体积比)、摇瓶装液量60 mL/250 mL、发酵温度32°C、摇床转速170 r/min、发酵周期8 d,L-苹果酸的产量为55.47 g/L。Mg2+、Mn2+对木糖代谢中相关酶的影响研究结果表明,木酮糖激酶在该菌株代谢木糖过程中起着重要作用。【结论】寄生曲霉CICC40365能够较好地利用木糖发酵产L-苹果酸,其产量及木糖的利用效率均得到提高。     

高产纤维素酶的拟康宁木霉菌株8985固态发酵条件优化

木霉是重要的产纤维素酶真菌,在其可利用性评价筛选过程中,获得了一株在实验室条件下高产纤维素酶的拟康宁木霉菌株8985.采用响应面法对8985产纤维素酶的固态发酵条件进行了研究,以滤纸酶活为响应值,通过Plackett-Burman设计对11个因素进行了筛选,包括温度、湿度、发酵时间、K2HPO4、(NH4)2SO4、T...     

黑曲霉产菊粉酶菌株的选育及培养基优化

为获得高产菊粉酶的黑曲霉菌株,以Aspergillus niger YH-1为出发菌株,经过亚硝基胍(NTG)诱变,以高温高菊芋粉相结合的方式进行梯度驯化,选育出一株产菊粉酶菌株YH-3,并运用响应面实验方法对该菌株的培养基进行优化。确定了最佳培养基组成:菊芋粉25.2 g/L、豆饼粉40 g/L、蔗糖酯4.9 g/L、NaCl 5.5 g/L。发现内切菊粉酶活力(I)由60.9 U/mL提高到165.0 U/mL,比出发菌株提高了1.7倍。研究证明蔗糖酯对于黑曲霉YH-3发酵产菊粉酶是一种有效的促进剂。     

Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae

We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD-sC-). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD-sC-adeA-) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD-sC-DeltaargB adeA- or niaD-sC-DeltaargB adeB-) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD-sC-DeltaargB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.     

基于响应面法优化匍枝根霉TP-02液态发酵纤维素酶条件

利用Plackett-Burman设计法(Plackett-Burman,PB),对影响根霉TP-02液态发酵产纤维素酶的8个因子进行了筛选,结果表明,影响该菌发酵产纤维素酶的主要因子为麸皮与稻草的比例、槐糖、Tween 80。利用最陡爬坡试验逼近最大响应区域,在此基础上,采用响应面法(ResponseSurface Methodology,RSM)对这3个因子的影响进行研究,得出纤维素酶产量的数学模型,通过对二次多项回归方程求解,得到3个因子的最优用量:麸皮稻草比例为:3.7:1,槐糖量为:0.62%,Tween 80为0.68 g/L,在优化后的条件下培养96 h,纤维素酶滤纸酶活可达到8.13 IU/mL比优化前提高了38.97%。     

响应面法优化产酰胺酶发酵培养基

采用响应面分析方法,对阿萨希丝孢酵母（Trichosporon asahii）ZZB-1产酰胺酶的发酵培养基进行了优化。运用单N子试验筛选出麦芽糖和酵母浸膏为最适碳源、氮源,金属离子Ca^2＋、Mn^2＋可提高发酵酰胺酶产量;通过最陡爬坡实验逼近以上4个因子的最大响应区域后,采用Box—Behnken响应面分析法,确定产酰胺酶最佳发酵培养基为麦芽糖18．84g／L、酵母浸膏9．55g／L、NaC15g／L、KH2PO41g／L、MgSO4·7H2O0．2g／L、FeS040．001g／L、CaC0370．84μmol／L、MnS0465．39肚mo[／L（1％丙烯酸诱导）,NH4·H2O调节pH至7．0。培养基优化后酰胺酶产量由初始2554U／L提高到4156U／L,为原始发酵培养基配方酶活产量的1．63倍。