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。     

Variation in polygalacturonase production among Aspergillus flavus isolates.

Pectinase production by Aspergillus flavus was determined by measuring clear zones formed around colonies stained with ruthenium red. Several isolates produced red zones instead of clear zones. Red zones were reproduced with pectinesterase and correlated with absence of specific polygalacturonases. Of 87 isolates tested, 15 produced red zones.     

Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae

Strain improvement is a powerful tool in commercial development of microbial fermentation processes. Strains of Aspergillus sojae which were previously identified as polygalacturonase producers were subjected to the cost-effective mutagenesis and selection method, the so-called random screening. Physical (ultraviolet irradiation at 254 nm) and chemical mutagens (N-methyl-N′-nitro-N-nitrosoguanidine) were used in the development and implementation of a classical mutation and selection strategy for the improved production of pectic acid-degrading enzymes. Three mutation cycles of both mutagenic treatments and also the combination of them were performed to generate mutants descending from A. sojae ATCC 20235 and mutants of A. sojae CBS 100928. Pectinolytic enzyme production of the mutants was compared to their wild types in submerged and solid-state fermentation. Comparing both strains, higher pectinase activity was obtained by A. sojae ATCC 20235 and mutants thereof. The highest polygalacturonase activity (1,087.2?±?151.9 U/g) in solid-state culture was obtained by mutant M3, which was 1.7 times increased in comparison to the wild strain, A. sojae ATCC 20235. Additional, further mutation of mutant M3 for two more cycles of treatment by UV irradiation generated mutant DH56 with the highest polygalacturonase activity (98.8?±?8.7 U/mL) in submerged culture. This corresponded to 2.4-fold enhanced polygalacturonase production in comparison to the wild strain. The results of this study indicated the development of a classical mutation and selection strategy as a promising tool to improve pectinolytic enzyme production by both fungal strains.     

Variation in polygalacturonase production among Aspergillus flavus isolates

Pectinase production by Aspergillus flavus was determined by measuring clear zones formed around colonies stained with ruthenium red. Several isolates produced red zones instead of clear zones. Red zones were reproduced with pectinesterase and correlated with absence of specific polygalacturonases. Of 87 isolates tested, 15 produced red zones.     

响应面法快速优化曲霉Aspergillus sp.F044产脂肪酶培养条件

运用逐因子试验(Seriatim-Factorial Experiment)、Plackett-Burman、Response Surface Methodology(RSM)和单因子试验(Monofactorial Experiment)分析对产脂肪酶曲霉Aspergillus sp.F044产酶培养条件进行了快速优化。首先运用逐因子试验确定Aspergillus sp.F044产脂肪酶最适碳源和氮源,分别为麦芽糖和牛肉膏。在此基础上,通过Plackett-Burrman设计对影响其产酶相关因素进行评估并筛选出具有显著效应的橄榄油、牛肉膏、硫酸镁三个因素。然后通过实验拟合上述三因素与发酵液脂肪酶活力的一阶线性模型,沿着一阶模型给定的路径进行最速上升试验,在上升最高点处由中心组合试验和向应面分析确定其最优培养基组成;最后通过单因子试验确定最适发酵温度和摇床转速。试验优化的最优培养条件为:麦芽糖1.5%(w/v),硫酸铵7‰(w/v),磷酸氢二钾1‰(w/v),牛肉膏1.25%(w/v),硫酸镁2.11‰(w/v),橄榄油1.41%(v/v),自然pH,250r/min和30℃培养72h酶活达32.15U/mL。与初始11.18U相比,酶活提高2.88倍。     

Regulation of the production of polygalacturonase by Aspergillus terreus

The synthesis of polygalacturonase (PG) (EC 3.2.1.15) by a strain of Aspergillus terreus was induced by polygalacturonic acid and repressed by glucose, galactose or fructose even in the presence of the inducer. The production of PG increased when the mycelium was washed free of glucose and incubated in a glucose-free medium containing the inducer, a fact that indicated the reversibility of the repression mechanism. When Actinomycin D and cycloheximide were added to the culture medium, the synthesis of PG ceased. PG synthesis increased 43% with the addition of methionine and 64% both with leucine and with tyrosine. Specific productivity with leucine was 210% higher than that of the control as against 149% with methionine and 70% with tyrosine. The results obtained suggest that PG synthesis is regulated by leucine.     

Solid-state production of polygalacturonase by Aspergillus sojae ATCC 20235

The effect of solid substrates, inoculum and incubation time were studied using response surface methodology (RSM) for the production of polygalacturonase enzyme and spores in solid-state fermentation using Aspergillus sojae ATCC 20235. Two-stage optimization procedure was applied using D-optimal and face-centered central composite design (CCD). Crushed maize was chosen as the solid substrate, for maximum polygalacturonase enzyme activity based on D-optimal design. Inoculum and incubation time were determined to have significant effect on enzyme activity and total spore (p<0.01) based on the results of CCD. A second order polynomial regression model was fitted and was found adequate for individual responses. All two models provided an adequate R(2) of 0.9963 (polygalacturonase) and 0.9806 (spores) (p<0.001). The individual optimum values of inoculum and incubation time for maximum production of the two responses were 2 x 10(7) total spores and 5-6 days. The predicted enzyme activity (30.55 U/g solid) and spore count (2.23 x 10(7)spore/ml) were very close to the actual values obtained experimentally (29.093 U/g solid and 2.31 x 10(7)spore/ml, respectively). The overall optimum region considering the two responses together, overlayed with the individual optima. Solid-state fermentation provided 48% more polygalacturonase activity compared to submerged fermentation under individually optimized conditions.     

Regulation of the production of polygalacturonase by Aspergillus niger

Synthesis of ethylene in static cultures as well as the effect of endogenous and exogenous ethylene on the synthesis of polygalacturonase byAspergillus niger were determined. This strain produced maximum ethylene amounts when cultured at 30 °C for 3 d. The effect of adding ethylene precursors (citrate-cycle intermediates) on ethylene production was investigated. Best intracellular and extracellular polygalacturonase production was obtained with 2-oxoglutaric, pyruvic and fumaric acids, and with glutamic acid too. Addition of ethylene to the culture medium also increased the synthesis of polygalacturonase, although to a lower degree than when glutamic acid was added.     

Growth characteristics and eicosapentaenoic acid production by Nannochloropsis sp. in mixotrophic conditions

Nannochloropsis sp. was grown under mixotrophic conditions with 30 mM glucose as carbon source, reaching 507 mg dry wt l(-1) after 8 d. This was 1.4-fold of that obtained under photoautotrophic conditions. Under mixotrophic and photoautotrophic cultivations, the net photosynthetic rate of Nannochloropsis sp. did not change but the respiratory rate increased in the mixotrophic cultivation. The yield of eicosapentaenoic acid was 22 mg l(-1) in the mixotrophic cultivation and 20 mg l(-1) under the photoautotrophic cultivation.     

Acidic condition-inducible polygalacturonase of Aspergillus kawachii

Aspergillus kawachii IFO 4308, which can grow in an extremely acidic condition (pH 2), produced some extracellular polygalacturonases (PGase). However, pH 2 and pH 5 culture filtrates showed different pH PGase activity profiles. Anion exchange chromatographies revealed that the PGase compositions of the two culture filtrates were different, and dominant enzymes (PGase-A1 and -A2 in the pH 2 culture and PGase-B in the pH 5 culture) were purified and characterized. The optimal pH was pH 4 for A1, pH 3 for A2, and pH 5 for B. PGase-A1 and -A2 were more stable at low pH than PGase-B. Molecular masses of PGase-A1, -A2, and -B were 43, 83, and 71 kDa, respectively. The N-terminal amino acid sequence of PGase-B was similar to those of other fungal PGases, but distinctly different from those of PGase-A1 and -A2. These results suggest that PGase-A1 and -A2 may be acidic condition-inducible enzymes and that a pH-regulated expression system is involved in the PGase production of A. kawachii.     

Optimization of production and reaction conditions of polygalacturonase from Byssochlamys fulva

In the present study, the optimization of production and reaction conditions of polygalacturonase produced by a fungus Byssochlamys fulva MTCC 505 was achieved. The production of polygalacturonase with a considerable activity of 1.28 IU/ml was found when the culture was shaken at 30°C for 5 days in 100 ml of medium containing (w/v) 10 g/l pectin, 2 g/l NaNO?, 1 g/l KH?PO?, 0.5 g/l KCl, 0.5 g/l MgSO?. 7H?O, 0.001 g/l FeSO?. 7H?O, 0.001 g/l CaCl?. The best carbon and nitrogen source for this enzyme were pectin (1%) and Ca(NO?)? (0.1%), respectively. The enzyme gave maximum activity at incubation time of 72 h, temperature of 30°C and pH 4.5. During the optimization of reaction conditions, the enzyme showed maximum activity in sodium citrate buffer (50 mM) of pH 5.5 at 50°C reaction temperature for 15 minutes of incubation. The enzyme showed greater affinity for polygalacturonic acid as substrate (0.5%). Km and Vmax values were 0.15 mg/ml and 4.58 μmol/ml/min. The effect of various phenolics, thiols, protein inhibitors and metal ions on the enzyme activity was investigated. The enzyme was quite stable at 4°C and 30°C. At 40°C the half life of the enzyme was 6 h and at 60°C it was 2 h.     

Enhanced polygalacturonase production by Aspergillus niger NRRL-364 grown on supplemented citrus pectin

Enhanced levels of extracellular polygalacturonase activity were obtained when Aspergillus niger NRRL-364 was grown on pectic substances as sole carbon sources in a submerged culture. Among the factors affecting enzyme production those of carbon source concentration, nitrogen source, initial pH and time of cultivation were found to be the most important ones. Under optimum growth and activity conditions yields as high as 14.5 U (measured as reducing groups) ml^-1 of growth medium were obtained, comparing favourably with those reported for fungi grown under similar conditions and used in food processes.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

裂殖壶菌Schizochytrium sp. 20888产DHA培养条件优化

【目的】为了优化裂殖壶菌产DHA的培养条件,提高油脂中DHA含量。【方法】采用单因素试验和正交设计试验方案,针对分批培养时间、培养基碳、氮源的种类和浓度以及培养温度开展试验,采用重量法测定生物量、采用索氏提取法测定油脂总量,采用气相色谱法测定油脂DHA含量,考察培养条件对细胞油脂DHA含量的影响。【结果】最适培养时间为4 d,培养温度23°C,最优碳氮源组成为(g/L):葡萄糖65、甘油80、蛋白胨6、酵母粉4和谷氨酸钠8。【结论】裂殖壶菌Schizochytrium sp.20888在葡萄糖和甘油组成的复合碳源和由蛋白胨、酵母粉和谷氨酸钠组成的复合氮源的培养基中可以得到最优的DHA产量,细胞DHA含量能达到33.68%。     

Culture conditions of Aspergillus oryzae for production of enzyme preparation

Submerged culture was better than solid culture in the production of proteinase and peptidases from Aspergillus oryzae 460. On the contrary, solid culture was better than submerged culture in the production of α-amylase, carboxymethyl cellulase, and pectinlyase from the same fungus.The soy souce mash (moromi) made with the enzyme preparation from submerged culture was highly viscous and the soy sauce produced was characteristic in low contents of alcohol and reducing sugar, low pH value, and less aroma. Soy sauce made with the enzyme preparation from solid culture was superior on these points to that from submerged culture.Wheat bran was best as the raw material for the enzyme preparation in easy koji making, large amount produced, and low cost.In enzyme production from a solid culture, addition of urea (0.8% to wheat bran) nearly doubled the leucine aminopeptidase for Leu-Gly-Gly. The incubation period was reduced to 30 to 40 h from 50 to 60 h using germinated spores and moisture-controlled culture with forced aeration.     

Continuous production of glucose oxidase with Aspergillus sp. under ultrasound waves

Glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4, GOD) was continuously released from Aspergillus sp. under mild ultrasound waves (20 kHz, 15 W). However, GOD was not released from the cells under normal conditions because of their thick wall. GOD production under ultrasound waves was optimum at pH 7.5 and 30°C and decreased with increasing ultrasonic frequency. Ultrasonic cavitation accelerated GOD release from the cells. Microscopic observation and determination of ATP and nucleic acids in the broth revealed that the mycelia were not broken during a 5 h reaction under ultrasound waves (15 W). About 10% of GOD produced in cells was released during the reaction.     

Growth and pigment production byArthrobacter pyridinolis n. sp.

A new bacterium capable of growing on 2-hydroxypyridine as sole source of carbon and nitrogen was isolated from soil. During its growth on solid medium, approximately 50% of this substrate was converted to a brilliant blue crystalline pigment which was deposited extracellularly in the colony mass. The pigment was identical to that produced byArthrobacter crystallopoietes during its growth on 2-hydroxypyridine. The new isolate exhibited the typical cycle of morphogenesis characteristic of the genusArthrobacter. The organism is different from all other reported species ofArthrobacter. It is proposed that the organism be namedArthrobacter pyridinolis n. sp.List of Abbreviations MSP mineral salts phosphate basal culture medium containing 2-hydroxypyridine, yeast extract and trace salts - 2-HP 2-hydroxypyridine - PFU plaque forming units - G+C guanine+cytosine - T _m midpoint of thermal denaturation     

Influence of environmental conditions on the production of pectinesterase and polygalacturonase byAspergillus niger

Summary A pectinesterase- and polygalacturonase-producer strain ofAspergillus niger showed a diauxic growth when cultivated in a medium containing pectin as the carbon source. Diauxie was suppressed by adding yeast extract to the medium. The specific production of polygalacturonase was decreased by increasing the initial concentration of pectin, while that of pectinesterase was enhanced with up to 15 g pectin/l. Yeast extract increased the production of biomass but decreased that of the enzymes. The temperature of incubation (25 to 40°C) did not affect the production of biomass or that of polygalacturonase, but production of pectinesterase was markedly affected, 30°C being the optimal temperature.

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Influence de la composition du milieu et les conditions des cultures sur la production de pectinestérase et polygalacturonase par Aspergillus niger

Résumé Aspergillus niger que produit pectinestérase et polygalacturonase, quand est developpé dans un milieu avec pectine comme source de carbone a presenté le phenomene de diauxie. Cela ne succedait pas quand le milieu avait aussi extrait de levure dans sa composition. L'effect de la concentration initiale de pectine sur la production specifique des enzymes des cellules (unité d'enzyme g^–1 de cellules poid sec) a été différent selon la phase du developpement de la culture, sugérant une rélation étroite parmi la synthèse des enzymes et l'état physiologique des cellules. L'augmentation de la concentration de pectine a troublé beaucoup la production spécifique de polygalacturonase. Au contraire, jusqu'à une concentration de 15 g/l a augmenté la synthèse de pectinestérase. La grandeur et structure des pellets ont changé avec la variation de la concentration de pectine, ces modifications peuvent avoir eu influence sur les valeurs des coefficients de transfert de masse et en conséquence sur la cinétique de production des enzymes. L'augmentation de la concentration iniciale d'extrait de levure dans le milieu que contenait pectine comme source de carbone a augmenté la production de biomasse, mais a baisse la synthèse des enzymes surtout celle de pectinestérase. Températures parmi 25°C et 40°C n'ont pas modifié la production de biomasse et non plus polygalacturonase. Au contraire la production de pectinesterase a été bien différente selon la température utilisé ayant un optimum de 30°C.

     

Aspergillus asperescens n. sp.

Summary A newAspergillus species,Aspergillus asperescens, is described. Three strains of this fungus have been isolated from soil and dung of bats in caves in Great-Britain and Poland. It is characterized by a yellow green colour, radiate conidial heads, two series of sterigmata, a brown conidiophore, hülle cells and subglobose conidia, which are smooth in young and rough in old cultures.