响应面法优化斜卧青霉Ju-A10产CMCase的条件

通过响应面分析法对Ju-A10利用不同植物纤维废弃物为碳源生产CMCase条件进行优化,结果显示碳源分别为谷秸、麦秸和造纸废渣,碳源水平分别在9．77％、8．69％、9．97％,300mL三角瓶装液量分别为64．7 mL、54．2 mL、40．8 mL时,CMCase为最高,分别为29．26 IU／mL、29．14 IU／mL、29．81 IU／mL。R2分别达到了0．9117、0．9246、0．8655,说明发酵预测模型可靠性较高,可应用于培养条件的优化。     

响应面法优化斜卧青霉Ju-A10产CMCase的条件*

通过响应面分析法对Ju-A₁₀利用不同植物纤维废弃物为碳源生产CMCase条件进行优化,结果显示碳源分别为谷秸、麦秸和造纸废渣,碳源水平分别在9．77％、8．69％、9．97％,300mL三角瓶装液量分别为64．7 mL、54．2 mL、40．8 mL时,CMCase为最高,分别为29．26 IU／mL、29．14 IU／mL、29．81 IU／mL。R2分别达到了0．9117、0．9246、0．8655,说明发酵预测模型可靠性较高,可应用于培养条件的优化。     

斜卧青霉原生质体制备和再生的研究

研究了斜卧青霉原生质体制备和再生的条件,确定了培养方式、菌龄、渗透压稳定剂、酶的配比、酶解时间等因素对原生质体制备和再生的影响。最佳条件：菌丝体培养12h,用1％溶壁酶＋1％纤维素酶＋1％蜗牛酶的混合酶液酶解,将NaCl作为渗透压稳定剂,酶解时间为1．5h。在此条件下原生质体的形成量达到5．3×10^5个／mL,再生率为19．4％。     

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。     

斜卧青霉去泛素化蛋白酶CREB的缺失提高纤维素酶的生产

斜卧青霉Penicillium decumbens T.是1种重要的产纤维素酶丝状真菌,能有效地降解利用木质纤维素生产第2代生物燃料。为了提高斜卧青霉纤维素酶的产量,构建了去泛素化酶基因creB的敲除盒,并通过同源双交换重组的方法,获得了creB基因缺失突变株ΔcreB。该突变株呈现明显的纤维素酶表达分泌抗葡萄糖代谢阻遏效应,ΔcreB菌株的滤纸酶活、内切纤维素酶活、木聚糖酶活以及外切纤维素酶活分别提高1.8倍、1.71倍、2.06倍以及2.04倍,其胞外蛋白质含量提高了2.68倍。确定了creB基因缺失突变株具有抗碳源代谢物阻遏的生理现象,CREB对斜卧青霉生产纤维素酶的能力具有显著影响,为系统改造丝状真菌高产纤维素酶菌株提供了理论指导。     

蝉拟青霉液体培养条件优化

[目的]建立能够有效提高蝉拟青霉液体培养效率的培养体系,增加其生物量。[方法]以蝉拟青霉生物量作为响应值,通过Plackett-Burman实验设计从8个试验影响因素中筛选出3个主要影响因素,基于3因素3水平的BoxBehnken设计法以及响应面分析法研究影响蝉拟青霉液体培养各因素之间的交互作用。[结果]通过Plackett-Burman设计法确定影响最为显著的因素为接种量、p H和温度,利用最陡爬坡试验确定最佳范围在接种量7%、p H 6.00、温度25℃附近,借助Box-Behnken设计法最终确定最佳液体培养条件为:葡萄糖20 g/L、蛋白胨3 g/L、酵母膏2 g/L、KH2PO41 g/L、MgSO40.5 g/L、接种量6.65%、p H 6.00、温度24.01℃。优化后的培养体系所培养的蝉拟青霉其生物量从优化前的147.106 0 g/L提高到239.475 3 g/L,提高了62.79%。[结论]优化后的培养体系能够有效提高蝉拟青霉生物量,该方法对于优化蝉拟青霉生物量发酵条件是一种有效、可行的方法。     

斜卧青霉转录调控因子BglR的缺失对纤维素酶生产的影响

【目的】斜卧青霉(Penicillium decumbens)作为高效分泌纤维素酶的重要丝状真菌,其纤维素酶的合成与分泌在转录水平上被调控。进一步研究纤维素酶基因表达的转录调控,构建高效高产纤维素酶的工业菌株。【方法】根据斜卧青霉114-2在不同碳源生长条件下基因组表达谱的差异,发现新的转录调控因子BglR(PDE-01706),该蛋白与产黄青霉(Penicillium chrysogenum)Pc20g04780的锌指结构蛋白具有59%同源性。通过基因同源双交换,得到BglR缺失突变株ΔbglR-1,对突变株ΔbglR-1的表型、营养生长、产纤维素酶活、蛋白分泌能力及发酵液pH变化进行研究。【结果】转录调控因子BglR的缺失可导致突变株ΔbglR-1的β-葡萄糖苷酶活力提高40%,并造成其滤纸酶活、内切葡聚糖酶及木聚糖酶活明显降低。【结论】结果表明转录调控因子BglR对于斜卧青霉纤维素酶的调控有重要作用。     

一株高效溶磷产红青霉培养条件优化及其溶磷特性

为了减少农业生产中化学肥料的使用,本研究利用无机磷培养基对富磷的茶树（Camellia sinensis L.）根际微生物进行筛选,获得一株对磷酸三钙具有高效降解能力的真菌菌株JL-1,经鉴定为产红青霉（Penicillium rubens）。通过检测菌株JL-1在溶磷过程中发酵液的pH值、磷含量和有机酸含量变化发现,该菌株在无机磷培养基中,发酵液pH值与葡萄糖酸含量、pH值与磷含量以及葡萄糖酸与磷含量分别呈显著负相关、显著负相关和显著正相关,且在电子显微镜下观察到磷酸三钙颗粒表面存在被侵蚀痕迹,深入分析发现菌株JL-1通过分泌葡糖糖酸实现侵蚀磷酸三钙与溶磷的目的。通过单因素试验、Plackett-Burman设计试验、最陡爬坡试验和中心组合试验等一系列试验对培养条件进行优化发现,菌株JL-1溶解磷酸三钙的最佳碳源和氮源分别是葡萄糖和硫酸铵。葡萄糖含量、磷酸三钙含量和温度是影响菌株JL-1溶磷能力的主要因素,在葡萄糖29.8 g/L,磷酸三钙7.1 g/L,温度31.9℃的条件下,菌株JL-1在无机磷培养基中的溶磷能力达到最佳,溶磷量达到1 194.15 mg/L,是初始值的近3倍。在...     

斜卧青霉L-06内切葡聚糖酶Ⅰ基因的克隆与表达

【目的】克隆斜卧青霉L-06的内切葡聚糖酶Ⅰ基因(egI),并实现其在大肠杆菌内的高效表达。【方法】利用RT-PCR技术克隆了斜卧青霉L-06的内切葡聚糖酶Ⅰ基因(egI),并将egI基因克隆到原核表达载体中,构建了重组质粒pET32a-egI。【结果】转化至大肠埃希菌Rosetta(DE3),经IPTG诱导重组蛋白表达,SDS-PAGE检测结果表明:重组表达产物的相对分子质量约为80 kD,与预期相符。重组表达的菌悬液,经破碎离心,取其上清液,进行纤维素酶活性染色,获得了活性条带。DNS法测得内切酶活力为2.56 IU/mL。【结论】构建了斜卧青霉L-06内切葡聚糖酶Ⅰ的原核表达系统。     

纤维素降解菌L-06的筛选、鉴定及其产酶条件的分析

从用于堆肥的水稻秸秆中初筛出一株高效纤维素降解菌L-06, 根据18S rRNA基因序列和菌株形态分析, 初步鉴定该菌为斜卧青霉(Penicillium decumbens)。研究了液体发酵培养基中氮源、碳源、表面活性剂、培养温度、起始pH以及接种量对该菌株各纤维素酶活力的影响。在最适条件下, 该菌的b-葡萄糖苷酶(BGL)、外切纤维素酶(CBH)于培养第3天酶活力分别达到1662 u/mL和2770 u/mL; 内切纤维素酶(EG)、滤纸糖化力(FPase)于培养第4天酶活力分别达到18064 u/mL和4035 u/mL。在产酶优化实验中, 该菌的EG和CBH在pH10的培养条件下分别保持了70%和43%的酶活性; 在50oC培养条件下EG和CBH分别保持了68%和59%的酶活性。表现出了耐热, 耐碱的特性。     

Genome shuffling improves production of cellulase by Penicillium decumbens JU‐A10

Aims: Improvement of cellulase production of Penicillium decumbens by genome shuffling of an industrial catabolite‐repression‐resistant strain JU‐A10 with its mutants. Methods and Results: After two rounds of genome shuffling, three fusants, GS2‐15, GS2‐21 and GS2‐22, were obtained, showing 100%, 109% and 94% increase in FPase activity than JU‐A10 respectively. The cellulase production of the fusants on various substrates, such as corn stover, wheat straw, bagasse and the corncob residue, was studied. The maximum productivities of GS2‐15, GS2‐21 and GS2‐22 were 92·15, 102·63 and 92·35 FPU l^?1 h^?1 on the corncob residue at 44 h respectively, which were 117%, 142% and 118% higher than that of JU‐A10 (42·44 FPU l^?1 h^?1, at 90 h). The improvements of the fusants were possibly because of their enhanced growth rates, earlier cellulase synthesis and higher secretion of extracellular proteins. Conclusions: The fusants obtained after genome shuffling could produce abundant cellulase much earlier, and they could be potential candidates for bioconversion process. Significance and Impact of the Study: This is the first report on the improvement of cellulase production in fungi by genome shuffling, and this is a good technique to improve other important phenotypes in fungi.     

Purification and characterization of a novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production

An acidic Cel6A, cellobiohydrolase (CBH) II, was purified from Penicillium decumbens and designated as PdCel6A. The deduced internal amino acid sequence of the novel CBH has a high degree of sequence identity with the CBH II from Aspergillus fumigatus. Surprisingly, PdCel6A exhibits characteristics comparable to that of CBH I, as well as CBH II. Similar to CBH I, the novel CBH has a specific activity of 1.9 IU/mg against p-nitrophenyl-β-d-cellobioside. The enzyme retains about 80% of its maximum activity after 4h of incubation at pH 2.0. Using delignified corncob residue as the substrate, ethanol concentration increased by 20% during simultaneous saccharification and fermentation when supplemented with low doses of PdCel6A (0.2mg/g substrate). To our knowledge, this is the first report involving a CBH I-like CBH II. The present paper provides new insight into the role of CBH II in cellulose degradation.     

Process optimization for biodiesel production from mahua (Madhuca indica) oil using response surface methodology

A central composite rotatable design was used to study the effect of methanol quantity, acid concentration and reaction time on the reduction of free fatty acids content of mahua oil during its pretreatment for making biodiesel. All the three variables significantly affected the acid value of the product, methanol being the most effective followed by reaction time and acid catalyst concentration. Using response surface methodology, a quadratic polynomial equation was obtained for acid value by multiple regression analysis. Verification experiments confirmed the validity of the predicted model. The optimum combinations for reducing the acid level of mahua oil to less than 1% after pretreatment was 0.32 v/v methanol-to-oil ratio, 1.24% v/v H2SO4 catalyst and 1.26 h reaction time at 60 degrees C. After the pretreatment of mahua oil, transesterification reaction was carried out with 0.25 v/v methanol-to-oil ratio (6:1 molar ratio) and 0.7% w/v KOH as an alkaline catalyst to produce biodiesel. The fuel properties of mahua biodiesel so obtained complied the requirements of both the American and European standards for biodiesel.     

响应面法优化黑曲霉HDF05产β-葡萄糖苷酶过程参数

为获得黑曲霉Aspergillus niger HDF05菌株较高的β-葡萄糖苷酶酶活,对其发酵条件进行了优化。采用Plackett-Burman实验设计考察关键发酵操作参数对产酶的影响。继而采用最陡爬坡路径逼近最大响应区域,并结合中心组合实验和响应面对4个显著性因素进行分析。Plackett-Burman实验结果表明,发酵温度、装液量、麦麸和 (NH4)2SO4浓度对β-葡萄糖苷酶合成影响显著。通过响应面分析得到一元二阶方程,对方程求解得到优化的发酵过程参数：发酵温度为28 ℃,装液量为71.4 mL/250 mL,麸皮浓度为36 g/L,(NH4)2SO4浓度为5.5 g/L。采用该优化的过程参数,菌株的最大产β-葡萄糖苷酶活力可达60.06 U/mL,较优化前提高了23.9％。将黑曲霉HDF05产生的β-葡萄糖苷酶用于酸解玉米芯纤维残渣的酶解实验中,可明显降低纤维二糖的积累,48 h内可使玉米芯纤维素残渣酶解得率达到80.4％。     

Parameter optimization for production of ligninolytic enzymes using agro-industrial wastes by response surface method

Lignin and manganese peroxidase (LiP, MnP) and laccase production by Phanerocheate chrysosporium was optimized by response surface methodology for brewery waste and apple pomace. The effect of moisture, copper sulphate, and veratryl alcohol (VA) concentrations on enzyme production was studied. Moisture and VA had significant positive effect on MnP and LiP production and the viability of P. chrysosporium (p < 0.05) and copper sulphate produced a negative effect. However, moisture and copper sulphate had a significant positive (p < 0.05) effect on laccase production, but VA had an insignificant positive effect (p < 0.05). Higher values of MnP, LiP and viability of P. chrysosporium on apple pomace (1287.5 U MnP/gds (units/gram dry substrate), 305 U LiP/gds, and 10.38 Log 10 viability) and brewery waste (792 U MnP/gds and 9.83 Log 10 viability) were obtained with 80% moisture, 3 mmol/kg VA, and 0.5 mmol/kg copper. LiP production in brewery waste (7.87 U/gds) was maximal at 70% moisture, 2 mmol/kg VA, and 1 mmol/kg copper. Higher production of laccase in apple pomace (789 U/gds) and brewery waste (841 U/gds) were obtained with 80% moisture, 3 mmol/kg VA, and 1.5 mmol/kg copper. Thus, moisture along with VA and copper sulphate was pertinent for the production of ligninolytic enzymes and increased cell viability.     

Fermentation medium for collagenase production by Penicillium aurantiogriseum URM4622

Medium composition and culture conditions for maximal collagenase production by Penicillium aurantiogriseum URM4622 were optimized using a response surface approach. A full two-level design on three factors (initial medium pH, soybean flour concentration, and temperature) was employed to identify the most significant fermentation parameters for collagenase production, and a subsequent central composite design (CCD) was used to find the optimal levels of the two most significant factors (initial medium pH and soybean flour concentration). The design results indicated that the initial medium pH and the temperature had significant negative main effects, whereas the substrate concentration had a positive effect on the collagenase production. The maximum collagenolytic activity predicted by the fitted response surface was expected to occur at pH 7.21, 1.645% soybean flour concentration and 24°C. Three replicate experiments were run at these conditions and yielded an activity response of 283.36 ± 1.33 U, which not only is the highest obtained in this study but also represents a 5-fold increase over the lowest response observed in the initial design. Since all experiments were carried out with an inexpensive substrate, the final results point out to a cost-effective medium for collagenase production with potential industrial-scale applications.     

Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology

Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.