Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates

To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the pclA gene encoding a secretion pathway specific (KEX2-like) endo-protease and the disrupted allele of the pg/pi-tp gene encoding a phosphatidylglycerol/phosphatidylinositol transfer protein. The biomass levels produced by the pclA and pg/pi-tp disrupted strains on wheat-based solid media were similar as found for the wild-type strain. However, the pclA disrupted strain showed much more compact colony morphology than the other two strains. Sporulation of the pclA and pg/pi-tp disrupted strains occurred, respectively, 2 days and 1 day later, compared to the wild type during fermentation on ground WK. During surface growth, microscopic analysis revealed that the hyphal growth unit length (L _hgu) of the pclA and pg/pi-tp disrupted strains was, on average, 50 and 74% of that of the wild-type strain. This implies that in both mutant strains, a higher branching frequency occurs than in the wild-type strain. Compared to the wild-type strain, the pclA and pg/pi-tp disrupted strains produced at least 50% more amylase, at least 100% more glucoamylase and at least 90% more protease activity levels after growth on WK. These results support the hypothesis that branching mutants with an increased branching frequency can improve the solid state fermentation process.     

片烟产果胶酶细菌的鉴定及酶活测定

以果胶为碳源, 对津巴布韦片烟烟叶表面产果胶酶细菌进行分离, 采用16S rDNA限制性酶切片段长度多态性分析(ARDRA)和测序方法, 结合形态学、生理生化实验, 对所分离产果胶酶菌株进行鉴定, 同时研究培养时间、温度、起始pH、接种量对菌株产酶的影响。结果表明, 从津巴布韦片烟烟叶表面分离得到的产果胶酶菌株主要为芽孢杆菌属的枯草芽孢杆菌Bacillus subtilis和产碱菌属的粪产碱菌Alcaligenes faecalis。在所分离的菌株中, 枯草芽孢杆菌T10酶活力最高, 以6%的接种量, 在温度为35 °C、起始pH为7.5条件下培养48?56 h, 其果胶酶酶活为571 U/mg, 聚半乳糖醛酸裂解酶酶活为297 U/mg。     

The effect of trehalose on the fermentation performance of aged cells of Saccharomyces cerevisiae

The fermentation process offers a wide variety of stressors for yeast, such as temperature, aging, and ethanol. To evaluate a possible beneficial effect of trehalose on ethanol production, we used mutant strains of Saccharomyces cerevisiae possessing different deficiencies in the metabolism of this disaccharide: in synthesis, tps1; in transport, agt1; and in degradation, ath1 and nth1. According to our results, the tps1 mutant, the only strain tested unable to synthesize trehalose, showed the lowest fermentation yield, indicating that this sugar is important to improve ethanol production. At the end of the first fermentation cycle, only the strains deficient in transport and degradation maintained a significant level of the initial trehalose. The agt1, ath1, and nth1 strains showed the highest survival rates and the highest proportions of non-petites. Accumulation of petites during fermentation has been correlated to low ethanol production. When recycled back for a subsequent fermentation, those mutant strains produced the highest ethanol yields, suggesting that trehalose is required for improving fermentative capacity and longevity of yeasts, as well as their ability to withstand stressful industrial conditions. Finally, according to our results, the mechanism by which trehalose improves ethanol production seems to involve mainly protection against protein oxidation.     

Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase

Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH₄Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.     

Enhanced inulinase production in solid state fermentation by a mutant of the marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> using surface response methodology and inulin hydrolysis

In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl₂. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.     

Biocatalytic potential of protease-resistant phytase of Aspergillus oryzae SBS50 in ameliorating food nutrition

Production of protease-resistant phytase by Aspergillus oryzae SBS50 was optimized in solid state fermentation using wheat bran as substrate. An integrated statistical optimization approach involving the Placket–Burman design followed by response surface methodology was employed. Among all the variables tested, incubation period, triton X-100, moisture ratio, and magnesium sulphate were identified as significant and further optimized using response surface methodology that resulted in 3.35-fold improvement in phytase production from 55.43 to 185.75 U/g dry mouldy bran (DMB). Optimal conditions for maximum phytase production (185.75 U/g DMB) included wheat bran 10 g per 250 ml flask moistened with 35 ml distilled water supplemented with 3.0% triton X-100, 0.04% magnesium sulphate, 1.0% sucrose and 0.5% yeast extract incubated at 30?°C for an incubation time of 48 h. Phytase titers were sustainable (179.55 to 185.75 U/g DMB), when the mould was grown in shake flasks of varied volumes and enamel-coated metallic trays under optimized conditions. Fermentation time was reduced to half from 96 h to 48 h after optimization resulting in a 6.7-fold enhancement in the phytase productivity from 577.39 to 3868.75 U/Kg/h and thus, reducing the cost of enzyme production. Phytase released inorganic phosphate, reducing sugars and soluble proteins from different food samples in a time dependent manner as a result of phytate hydrolysis.     

常压室温等离子体(ARTP)诱变茂源链霉菌菌株

采用常压室温等离子体（ARTP）诱变技术处理茂源链霉菌（Streptoverticillium mobaraense）菌株HS47的孢子,选育微生物谷氨酰胺转胺酶（MTG）高产菌株。菌株的致死率强度和正突变率高低结果表明,在电源功率为120W,处理距离2mm,工作气流量10slpm时,等离子体氦气对茂源链霉菌HS47孢子的最佳处理时间为30s。将诱变后的孢子液稀释涂布后,利用96孔板高通量筛选方法对单菌落进行初筛,选出高产的突变株进行两轮试管复筛,筛选过程中保持对菌株的分离纯化,最终获得一株高产菌株M-8,其MTG酶活由2.8U/ml提高到5.1U/ml,较出发菌株HS47提高了82%。该菌株的摇瓶发酵实验证明,其酶活的提高是单位菌株分泌的MTG有所增加的结果。经过8次传代,证实该菌株具有良好的遗传稳定性。这为谷氨酰胺转胺酶的工业化生产提供了菌种支持和理论支持。     

Role of microalgal ligninolytic enzymes in industrial dye decolorization

Abstract

In this study, the decolorization efficiency of seven microalgae isolates; Nostoc muscorum, Nostoc humifusum, Spirulina platensis, Anabaena oryzae, Wollea saccata, Oscillatoria sp. and Chlorella vulgaris was investigated for dye decolorization. The highest decolorization percentages of Brazilwood, Orange G, and Naphthol Green B dyes (99.5%, 99.5%, and 98.5%, respectively) were achieved by Chlorella vulgaris. However, the maximum efficiency for dye decolorization percentages of CV and malachite green dyes were exhibited by A. oryzae (97.4%) and W. saccata (93.3%). Ligninolytic enzymes activity assay was carried out for laccase and lignin peroxidase enzymes, which revealed a high efficiency of the C. vulgaris, A. oryzae and W. saccata to lignin containing compound degradation. The highest laccase production recorded by C. vulgaris with Brazilwood, Orange G, and Naphthol Green B dyes (665.0, 678.6, and 659.5?U/ml, respectively). Similarly, C. vulgaris gave a high lignin peroxidase enzyme production with the above three dyes respectively (306.00, 298.34, and 311.45?U/ml). In addition, A. oryzae and W. saccata showed the highest production of the laccase enzyme (634.6 and 577.45?U/ml, respectively) with CV and malachite green dyes. The degradation products have been characterized after decolorization and verified using FTIR analysis. The high decolorization percentages achieved by C. vulgaris, A. oryzae and W. saccata make them potential candidates for bioremediation and pre-processing to remove dyes from textile effluents.     

Improvement of β-glucosidase production by co-culture of Aspergillus niger and A. oryzae under solid state fermentation through feeding process

Eleven different Aspergillus strains were evaluated for their ability to produce β-glucosidase using sugar cane bagasse as a sole carbon source under solid state fermentation (SSF). The most potent strains, A. niger NRC 7 (674.6 U/g ds) and A. oryzae NRRL 447 (83 U/g ds), were used in a mixed culture to enhance β-glucosidase production by co-culturing under SSF. In mixed culture, β-glucosidase of the two strains (814 U/g ds) was nearly 1.2- and 9.8-fold than that of monocultures of A. niger NRC 7A and A. oryzae NRRL 447, respectively. Optimization of the culture parameters, initial pH value, moisture content, inoculum size and ratios of the two strains. and incubation time exhibited a significant increase in β-glucosidase production (1,893 U/g ds) than before optimization. Single feeding with citrate-phosphate buffer, succinate buffer, casein. and soybean flour individually after the third day of the fermentation time and controlling the moisture content at 90 % (w/w) induced β-glucosidase production. Maximum enzyme production increased up to 2.1-fold compared to 2,188 U/g ds during normal batch culture. Among nitrogen sources, soybean flour gave the highest β-glucosidase (4,578 U/g ds). while urea reduced β-glucosidase production (1,693 U/g ds). However, the combination of buffers with soybean flour through two fed cycles resulted in a decrease of the enzyme than single fed with buffers or soybean flour alone.     

产低温脂肪酶菌株CZW001发酵培养基优化研究

【目的】研究产低温脂肪酶菌株CZW001发酵培养基。【方法】在单因素试验的基础上, 采用Plackett-Burman (P-B)设计, Box-Behnken (B-B)设计和响应面试验设计(RSM), 在20 °C、pH 8.0、160?r/min发酵2 d条件下, 对发酵培养基进行优化。【结果】该菌株最适产酶培养基为(g/L): 葡萄糖7.68, 橄榄油21.93, 硫酸铵2.0, 磷酸二氢钾1.0, 硫酸镁0.27, 氯化钙0.3, 氯化钠20.0, 吐温-80 1.0。其最高酶活为62.8 U/mL, 比优化前提高了3.14倍。【结论】通过对产低温脂肪酶菌株CZW001发酵培养基优化研究, 明显提高低温脂肪酶活力。     

Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.     

一株产褐藻胶裂解酶的需钠弧菌筛选及酶解产物分析

[背景]褐藻胶裂解酶种类丰富、降解机制多样,是高效环保降解褐藻胶、制备褐藻寡糖的工具酶,成为褐藻植物高值化开发利用的研究热点.[目的]从海泥中筛选获得褐藻胶裂解酶高效产酶菌株,确定菌株发酵产酶最优条件,鉴定和分析酶降解产物,进而解析该酶的降解特性.[方法]以褐藻胶为唯一碳源,从海带养殖场附近海泥中筛选菌株,通过形态学观...     

Production and characterization of a milk-clotting enzyme from Aspergillus oryzae MTCC 5341

Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. Aspergillus oryzae MTCC 5341 was identified to produce the highest milk-clotting activity during screening of 16 fungal strains. Solid state fermentation using wheat bran along with 4% defatted soy flour and 2% skim milk powder as substrate was optimal for growth of A. oryzae and production of the enzyme. Nearly 40,000 U/g bran of milk-clotting activity was present at the end of 120 h. The enzyme could be recovered by percolating the bran with 0.1 M sodium chloride for 60 min at 4°C. The decolorized enzyme preparation had high ratio of milk clotting to proteolytic activity. Affinity precipitation with alginate and subsequent elution with 0.5 M sodium chloride containing 0.2 M CaCl₂ resulted in an enzyme preparation with specific activity of 3,500 U/mg and 72% yield. Optimum pH and temperature for activity of the enzyme were characterized as 6.3 and 55°C, respectively. Milk-clotting enzyme showed differential degree of hydrolysis on casein components. High ratio of milk clotting to proteolytic activity coupled with low thermal stability strengthens the potential usefulness of milk-clotting enzyme of A. oryzae MTCC 5341 as a substitute for calf rennet in cheese manufacturing.     

Effect of glyphosate toxicity on growth, pigment and alkaline phosphatase activity in cyanobacterium Anabaena doliolum: a role of inorganic phosphate in glyphosate tolerance

Response of glyphosate toxicity on photoautotrophic cyanobacterium A. doliolum and its mutant strain was investigated. Chlorophyll a content of both the wild type and mutant strain in the presence of glyphosate (N-phosphonomethyl glycine) initially showed an increasing trend when supplemented with Pi and a declining tendency under the Pi-starved condition. The results suggested that both the wild type and mutant strains were more sensitive to glyphosate in the absence of phosphate. Alkaline phosphatase activity of wild type strain in the presence of Pi, enhanced in response to addition of glyphosate (40 microg/ml), but the activity remained unaltered by addition of glyphosate in the Pi-starved cells, whereas the alkaline phosphatase activity in the mutant strain under both Pi-starved as well as unstarved conditions was stimulated (approximately 5.4 and 3.1-fold, respectively) by addition of glyphosate. The results on alkaline phosphatase activity indicated a glyphosate-induced depletion in the phosphate content of the cells, particularly in the mutant strain, as evident from the stimulated activity of alkaline phosphatase. It is suggested that enzyme activity in the Pi-starved wild type cells may not be influenced any further by glyphosate, as cellular phosphate reserve might not be available for further depletion.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Fermentation optimization and industrialization of recombinant Saccharopolyspora erythraea strains for improved erythromycin a production

Improvement of Erythromycin A (Er-A) production and purity by metabolic engineering of the industrial erythromycin-producing strains Saccharopolyspora erythraea strians ZL1004 and ZL1007, in which the amounts of tailoring enzymes EryK (a P450 hydroxylase) and EryG (an S-adenosylmethionine-dependent O-methyltransferase) for biotransformation of Erythromycin D to Er-A were modulated, was performed in a 50 L fermentor. Addition of 15 g/L of corn steep liquor to the medium increased Er-A production; maximum Er-A production was 8,196 U/mL at 191 h, which was 81.8% higher than that of control (4,507 U/mL at 184 h). Er-B impurities were completely eliminated, whereas Er-C impurities were only 153 U/mL at 191 h. Analysis of intra- and extracellular metabolites and key enzyme activities in central carbon metabolism revealed that the pool of TCA cycle intermediates was enhanced by the addition of corn steep liquor and induced an increase in erythromycin biosynthesis. There were no significant differences between strains ZL1004 and ZL1007 regarding Er-A production and impurity accumulation. Compared to wild type strain, Er-A production was improved by 23.9% while Er-C was reduced by 83.9% and Er-B was completely eliminated. Furthermore, fermentation of recombinant strain ZL1004 was successfully scaled up from laboratory scale (50 L fermentor) to industrial scale (25 and 132 m³), with similar levels of Er-A production and purity obtained.     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

微生物转谷氨酰胺酶的生产菌种诱变和发酵生产分析

对本研究室从土壤分离得到的使霉菌(Streptomyces sp.)WZFF．W-12菌株的斜面孢子预培养处于初萌发状态后,以亚硝基胍(NTG)进行诱变育种试验,并根据诱变处理后菌落的某些形态变化状况与产酶能力相结合的特征,初步判断产酶性能,挑选高酶活菌株,再经过初筛和复筛,获得一性能良好的产酶突变菌株WZFF.W-12.var MN-35,转谷氨酰酶活达0．53U／mL,比原始菌株提高了1．2倍。然后在摇瓶条件下,对其发酵过程中的主要培养基组成及各种培养条件对菌体生长和产酶的影响作用进行了研究,结果表明该菌株发酵生产转谷氨酰酶的适宜破源为可溶性淀粉 葡萄糖,氮源是多价胨外加少量的酵母膏,优化工艺条件为种龄时间24h、接种量10％、初始以值6．5、温度30℃和搅拌速度200r／min,产酶能力显著提高,用小型生化反应器可以稳定生产2.0U／mL以上的酶产品。     

Pectinase production by a Brazilian thermophilic fungus <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> N31 in solid-state and submerged fermentation

Thermophilic organisms produce thermostable enzymes, which have a number of applications, justifying the interest in the isolation of new thermophilic strains and study of their enzymes. Thirty-four thermophilic and thermotolerant fungal strains were isolated from soil, organic compost, and an industrial waste pile based on their ability to grow at 45°C and in a liquid medium containing pectin as the only carbon source. Among these fungi, 50% were identified at the genus level as Thermomyces, Aspergillus, Monascus, Chaetomium, Neosartoria, Scopulariopsis, and Thermomucor. All isolated strains produced pectinase during solid-state fermentation (SSF). The highest polygalacturonase (PG) activity was obtained in the culture medium of thermophilic strain N31 identified as Thermomucor indicae-seudaticae. Under SSF conditions on media containing a mixture of wheat bran and orange bagasse (1 : 1) at 70% of initial moisture, this fungus produced the maximum of 120 U/ml of exo-PG, while in submerged fermentation (SmF) it produced 13.6 U/ml. The crude PG from SmF was more thermostable than that from SSF and exhibited higher stability in acidic pH.     

黑曲霉生产菊粉酶工艺条件的研究

从徐州市沛县河口镇秦庄村牛蒡种植基地取得的土壤样本中,筛选出产菊粉酶的菌株,对从土壤中分离到的40株产菊粉酶的各类微生物进行酶活的测定。通过透明圈法初筛及摇瓶复筛,获得产菊粉酶能力较高的霉菌菌株3株,为C122803、D081506和D081513,这3株菌株的酶活分别达到：C122803：1.411 U/ml;D081506 ：1.895U/ml;D081513 ：1.792U/ml。其中D081506的酶活最高,为1.895U/ml;对D081506号黑曲霉产菊粉酶的发酵条件进行了研究,确定了优化的发酵条件为：牛蒡汁2%,酵母膏1.6%,(NH4)2SO41.6%,NaCl 0.5%,K2HPO4 0.5%,pH 5.0,在27℃、140 r/min条件下,摇瓶培养24h, D081506酶活力为2.958U/ml,酶活力提高56.09%。