Agricultural waste from the tequila industry as substrate for the production of commercially important enzymes

Approximately 1 million tons of Agave tequilana plants are processed annually by the Mexican Tequila industry generating vast amounts of agricultural waste. The aim of this study was to investigate the potential use of Agave tequilana waste as substrate for the production of commercially important enzymes. Two strains of Aspergillus niger (CH-A-2010 and CH-A-2016), isolated from agave fields, were found to grow and propagate in submerged cultures using Agave tequilana waste as substrate. Isolates showed simultaneous extracellular inulinase, xylanase, pectinase, and cellulase activities. Aspergillus CH-A-2010 showed the highest production of inulinase activity (1.48 U/ml), whereas Aspergillus niger CH-A-2016 produced the highest xylanase (1.52 U/ml) and endo-pectinase (2.7U/ml) activities. In both cases production of enzyme activities was significantly higher on Agave tequilana waste than that observed on lemon peel and specific polymeric carbohydrates. Enzymatic hydrolysis of raw A. tequilana stems and leaves, by enzymes secreted by the isolates yielded maximum concentrations of reducing sugars of 28.2 g/l, and 9.9 g/l respectively. In conclusion, Agave tequilana waste can be utilized as substrate for the production of important biotechnological enzymes.     

Growth and amylase production by Aspergillus oryzae during solid state fermentation using banana waste as substrate

Aspergillus oryzae, isolated from the starch rich litter soil, produced amylase when banana fruit stalk was used as substrate in a solid state fermentation system. The effects of incubation period, pH, temperature and various carbon sources on the production of amylase were studied. A maximum yield of 380U/mg was recorded on 96th hour of incubation. The amylase is tolerant to wide range of initial culture pH values (3 to 8) and temperature (25 to 35 degrees C), with an optimum pH of 5 and temperature of 35 degrees C. In SSF addition of starch (1%) increased the amylase production, when compared with other carbon sources used.     

Characterization of protease from Alcaligens faecalis and its antibacterial activity on fish pathogens

Alcaligens faecalis AU01 isolated from seafood industry effluent produced an alkaline protease. The optimum culture conditions for growth as well as enzyme production were 37 degrees C and pH 8. The partially purified protease had specific activity of 9.66 with 17.77% recovery with the molecular weight of 33 kDa and it was active between 30-70 degrees C and optimum being at 55 degrees C and pH 9. The enzyme retains more than 85% activity at 70 degrees C and 78% even at pH 10. The enzyme inhibited the growth of fish pathogens such as Flavobacterium sp., Pseudomonas fluorescens, Vibrio harveyi, Proteus sp. and Vibrio parahaemolyticus. From the present study it can be concluded that Alcaligens faecalis AU01 has the potential for aquaculture as probiotic agent and other several applications.     

Effect of temperature on growth and reproduction of the epigeic earthworm,Eudrilus Eugeniae (Kinberg)

Influence of temperature on growth and reproduction of Eudrilus eugeniae has been investigated by laboratory culturing at regulated 25.0 degrees C, 30.0 degrees C, 37.5 degrees C and 40.0 degrees C and in fluctuating (22.7-27.3 degrees C) room temperature of prevailing (winter) season over 16 weeks. All worms died during first and tenth week at 40.0 degrees C and 37.5 degrees C respectively. Weight (biomass) and growth of worms cultured at different temperature varied significantly (P<0.01). The mean growth (mg/g live weight/day) at 25.0 degrees C., 30.0 degrees C, 35.0 degrees C, 37.5 degrees C and in fluctuating temperatures was 1,074.04 +/- 6.07, 1,554.01 degrees 192.37, 148.1 +/- 15.28, 192.83 +/- 25.8 and 1450.4 +/- 162.1 respectively. Growth declined after maturity drastically with coccon production. At 25.0 degrees C though worms are sexually mature, they failed to produce cocoons within 16 weeks whereas, at 35.0 degrees C and 37.5 degrees C they did not sexually mature. Worms attained sexual maturity at a mean weight of about 1000 mg/worm. The mean per cent maturity was higher and earlier in fluctuating temperatures and at 30.0 degrees C than at 25.0 degrees C. Cocoon production was observed only at 30.0 degrees C and in fluctuating temperatures with a mean of 0.9 and 1.5 cocoons/ wom/week and the cumulative cocoon number of 10.8 and 14.7/worm over 16th week respectively. The fluctuating temperature of uncontrolled room environment and 30.0 degrees C were favorable for various life activities of the worms. Eudrilus eugeniae appears to have range of temperature optima more than 25.0 degrees C and less than 28 degrees C. The climatic conditions prevailing in whole of the peninsular India during winter season are favourable for employing this worm in intensive field scale vermiculturing practices.     

生淀粉糖化酶产生菌Aspergillus niger(6#)的分离筛选及其产酶条件

从大曲中分离到了1株降解生淀粉能力较强的黑曲霉Aspergillusniger(6#).固体发酵酶活可达2461U/g,RDA值为21.47%;液体发酵酶活可达353U/mL,RDA值为20.30%.以6#菌为实验材料,进一步考察了氮源、碳源及pH对生淀粉糖化酶形成的影响.实验结果表明,无机氮源NaNO3较有机氮源蛋白胨更有利于生淀粉糖化酶的形成;pH是影响生淀粉糖化酶形成的重要因素,低pH会阻遏生淀粉糖化酶的形成;玉米粉和麦芽糖较其它碳源更有利于生淀粉糖化酶的形成.图4表2参12     

产中性纤维素酶芽孢杆菌Y106产酶条件优化

筛选得到一株高产中性纤维素酶的芽孢杆菌Ｙ１０６，并对其发酵条件进行了优化，麸皮能够较大幅度地促进产酶，果糖是良好的诱导物，有机氮流促进产酶的效果优于无机氮源，最优的有机氮源是蛋白胨。Ｆｅ＾３＋、Ｆｅ＾２＋、Ｎａ＾＋、Ｃａ＾２＋可以促进纤维素酶的合成，而Ｃｕ＾２＋、Ａｇ＾＋、Ｃｏ＾２＋、Ｈｇ＾２＋则对合成起遏制作用，在酶反应系统中Ｆｅ＾３＋、Ｆｅ＾２＋、Ｃａ＾２＋、Ｍｇ＾２＋能激活纤维素酶活性，Ｃｏ     

一株产耐热纤维素酶菌株的筛选及酶学性质

采用纤维素刚果红平板法从大田蘑菇种植场建堆的稻草样中分离得到了1株能产具有较好温度耐受性和pH稳定性的纤维素酶的放线菌DY3.综合形态、生理生化特征以及16S rDNA序列分析,将其初步鉴定为嗜热裂孢菌(Thermobifida fusca).对该菌所产纤维素酶的性质研究表明:最适催化温度为65℃,在70℃保温60 min后仍有75%以上的活力;最适催化pH为7.5,在pH 5.5～10.0之间该酶的稳定性较好,pH 10.0的条件下仍有80%的活力;最适作用底物是羧甲基纤维素钠.研究可为木质纤维素的生物预处理提供一定参考价值.     

Advances in the study of directed evolution for cellulases

If cellulose can be effectively hydrolyzed into glucose by cellulase, the production costs of hydrogen, ethanol or other chemicals from cellulosic materials will be greatly decreased, and economically viable production of biohydrogen and bioethanol will become feasible. Cellulose is degraded into glucoses by multi-component enzyme systems. Nowadays cellulases are widely used in brewing, food, bioenergy, fodder, textiles, paper, pharmaceuticals, environmental protection and other industries. However, existing cellulases have several problems that limit their wider applications, including the low turnover number for solid cellulosic materials, and low stability in adapting to various application conditions. For example, high temperature, low pH, and so on. Application of directed evolution technology may be one of the most effective ways for improving the characteristics of cellulases. This paper presents a brief review of the cellulose hydrolysis mechanism by cellulase, advances in cellulases (endoglucanase and β-glucosidase) improvement by directed evolution for several characteristics (for instance, thermal stability, pH adaptability and enzyme activity), limitations of directed evolution for cellulases, and the outlook for directed evolution for cellulase.     

Extracellular amylase(s) production by fungi Botryodiplodia theobromae and Rhizopus oryzae grown on cassava starch residue

The fungi Botryodiplodia theobromae and Rhizopus oryzae produce extracellular amylase when grown on a liquid medium containing 2% (WN) soluble starch or cassava starch residue(CSR) (as starch equivalent), a waste generated after extraction of starch from cassava, as the sole carbon source. Using CSR as the sole carbon source, the highest amylase activity of 3.25 and 3.8 units (mg, glucose released x ml(-1) x h(-1)) were obtained in shake flask cultures during the late stationary phase of growth of B. theobromae and R. oryzae, respectively. These values were slightly lower than the values obtained using soluble starch as the carbon source. Maximum enzyme synthesis in CSR incorporated medium occurred at the growth temperature of 30 degrees C and pH 6.0. Presence of inorganic NH4+ salts like ammonium acetate and ammonium nitrate in culture medium yielded more amylase than the other nitrogen sources. Amylase(s) production in the controlled environment of a Table-Top glass Jar Fermenter (2-L capacity) was 4.8 and 5.1 units for B. theobromae and R. oryzae, respectively using CSR as the carbon substrate. It is concluded that CSR, a cheap agricultural waste obtained after starch extraction from cassava could replace soluble starch as carbon substrate for commercial production of fungal amylase(s).     

山苍籽油抑制黑曲霉生长的细胞学机制

采用电镜、多维显微、快速显微图像分析，并与计算机技术相结合，研究天然山苍籽香精油抗黑曲霉的机理，获得了单纯使用光学显微镜或电镜难以获得的结果，表明该香精油能直接通过损伤黑曲霉质膜，改变其膜的物理参数和选择通透性而进入细胞，诱发细胞的一系列因素，从而导致孢子失去萌发力，并抑制菌丝体生长．图2表1参12     

Population dynamics of the tropical cladoceran Ceriodaphnia rigaudi Richard, 1894 (Crustacea: Anomopoda). Effect of food type and temperature

The knowledge of population effects of food on tropical, filter-feeding cladocerans is scarce because a reduced number of species has been extensively studied. Ceriodaphnia rigaudi Richard 1894, a small-sized cladoceran distributed mainly in tropical and subtropical regions of the world, was studied. The aim of this study was to contribute to the knowledge of the reproductive biology of a poor-known Cladoceran; for this we assessed the effect of feeding and temperature on the reproduction and life cycle of this species. Three microalga species (Pseudokirchneriella subcapitata, Ankistrodesmus falcatus, and Chlorella vulgaris) were supplied as food each at a concentration of 12 mg l(-1) (dry weight, equivalent to 1.3 x 10(6), 0.4 x 10(6) and 1.35 x 10(6) cell m1(-1), respectively, and equivalent to 7.8 microg C ml(-1), at two temperatures (20 and 25 degrees C). We evaluated, among other responses, longevity, total progeny, survival, life expectancy at birth and fecundity. Organisms fed with the microalgae A. falcatus and P subcapitata presented both higher longevity (30.7 +/- 5.91, 26.6 +/- 3.59 days, respectively) and total progeny (45 +/- 13.80, 40.7 +/- 0.66 neonates female (-1) values than those organisms fed C. vulgaris (13.5 +/- 4.63 days and 17.6 +/- 6.19 neonates female (-1), respectively). On the other hand, temperature affected significantly the population parameters of C. rigaudi, recording maximal longevity values (56.1 +/- 9.41 days) at 20 degrees C in organisms fed A. falcatus; however, age at first reproduction and total progeny were negatively affected by this temperature: sexual maturation of the females was delayed until the age of 16 days and the number of neonates produced was smaller (9.8 +/- 3.45 with C. vulgaris; 24.7 +/- 6.01 with P subcapitata, and 35.5 +/- 8.59 neonates female(-1) with A. falcatus). The best reproductive responses for C. rigaudi in this study were obtained with A. falcatus at degrees 25 degrees C.     

Seasonal variations in abundance of nitrifying bacteria in fish pond ecosystem

Seasonal changes in abundance of nitrifiers (ammonia-oxidizing and nitrite-oxidizing bacteria) in surface and bottom water of freshwater ponds were examined with respect to temperature, DO, pH as well as concentration of ammonia and nitrite. The most probable number (MPN) of ammonia-oxidizers in different ponds varied from 1297 +/- 3.6 to 1673.23 +/- 0.36 ml(-1) in bottom and 720.5 +/- 8.1 to 955.3 +/- 10.8 ml(-1) in surface water during the rainy season while the MPN ranged from 1074 +/- 1.07 to 1372.17 +/- 4.6 ml(-1) in bottom and 515 +/- 10.1 to 678 +/- 11.8 ml(-1) in surface water in winter. However, the MPN were greatly reduced in summer and ranged from 435.05 +/- 15.7 to 547.54 +/- 2.12 ml(-1) in bottom and 218.7 +/- 7.3 to 368.4 +/- 9.32 ml(-1) in surface water. Similar seasonal trends were also observed in MPN of nitrite-oxidizers. Among all the physico-chemical parameters, abundance of nitrifiers was more positively correlated with ammonia and nitrite concentration in all the seasons. The abundance of nitrifiers in surface and bottom water was highest in rainy season followed by winter and modest in summer. The potential nitrification activities and oxidation rates were shown to be linear and activity of ammonia-oxidizing and nitrite-oxidizing bacteria was highest during rainy season.     

Preparation and assay of C-glucosyltransferase from roots of Pueraria lobata

C-glucosyltransferase (EC 2.4.1.X) is one of the key enzymes for the biosynthesis of puerarin. This paper describes the methodology in purification and assay of the enzyme for the first time in Puerarin lobata (Wild.) Ohwi. C-glucosyltransferase from roots of P. lobata was extracted and partially purified by (NH4)2SO4 saturation. The effects of pH, temperature, and substrate concentration on the activity of the enzyme were investigated. The properties of the puerarin produced by C-glucosyltransferase were studied by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The peak activity of C-glucosyltransferase was detected in fraction of by 80% saturation of (NH4)2SO4 and the optimal conditions for enzymatic reaction were 35.5 micromol l(-1) of isoliquiritigenin and 560 micromol l(-1) of UDP-G at pH 8.1, 28 degrees C for 1 h. Mn2+ at 1 mmol l(-1) and Al3+ at 1 mmol l(-1) increased the enzyme activity, while Mg2+ inhibited its activity. The enzyme activity in Nicotiana tabacum and P. lobata were detected under the above assay conditions. Higher activity was found in roots than in leaves and stems of P. lobata, while no enzyme activity was detected in leaves of N. tabacum. It was the first time that activity of C-glucosyltransferase, which transforms isoliquiritigenin to puerarin, was detected in P. lobata.     

Enzymes from marine phycophages that degrade cell walls of seaweeds

Agarase, cellulase and alginate lyase activities from crude extracts of Aplysia dactylomela Rang, Haliotis coccinea canariensis Nordsieck, Littorina striata King et Broderip and Diadema antillarum Phillipi were measured in vitro to compare digestive efficiencies against several components of complex seaweed cell walls. Commercial abalone acetone powder (AAP, an extract from Haliotis sp.; Sigma, Ref. A-7514) and purified (Sigma, Ref. A-6306) and non-purified agarases from Pseudomonas atlantica were used with the same objective. Optimum conditions for agarase and cellulase activities were 40°C and pH 6.0. For alginate lyase, optimum temperature and pH were species-dependent. Highest reducing sugar release was shown by crude extracts from A. dactylomela. These crude extracts displayed high agarase activity compared with bacterial agarases at 40°C, and were significantly higher at 25°C. AAP and crude extracts from L. striata and D. antillarum exhibited high specific activities on all the substrates. Cold extract from Gracilaria spp. was the best substrate with which to measure agarase activity.     

Influence of culture media and environmental factors on mycelial growth and sporulation of Lasiodiplodia theobromae (Pat.) Griffon and Maubl

Lasiodiplodia theobromae, a common tea (Camellia sinensis) pathogen, usually does not sporulate or sporulates poorly in common media, which makes spore production difficult. In this study the effects of culture media, carbon source, nitrogen source, temperature, pH and light on mycelial growth and sporulation were evaluated. Among several carbon sources tested, glucose and sucrose were found superior for growth. Potassium nitrate supplemented media showed maximum growth amongst the tested inorganic nitrogen sources while peptone produced maximum growth among the tested organic nitrogen sources. Tea root extract supplemented potato dextrose agar medium was found to be the most suitable for mycelial growth and sporulation of L. theobromae. The fungus grow at temperatures ranging from 40 to 36 degrees C, with optimum growth at 28 degrees C and no growth was noted at 40 degrees C. There was no significant effect of different light period on growth of L. theobromae, but light enhanced sporulation. The fungus grow at pH 3.0-8.0 and optimum growth was observed at pH 6.0. Tea root extract supplemented potato dextrose agar medium with pH 6.0 was the most suitable for production of conidia of L. theobromae at 28 degrees C. Hence this media may be recommended for inoculum production for further studies.     

Chromium (VI) biosorption by immobilized Aspergillus niger in continuous flow system with special reference to FTIR analysis

Aspergillus niger was treated with acid and immobilized in calcium alginate matrix. The dynamic removal of Cr (VI) ion was studied using continuously fed column packed with immobilized biosorbent beads. Column experiments were carried out to study the effect of various bed heights (20, 30, 40 cm) under different flow rates (5, 7.5, 10 ml min(-1)) on efficiency of biosorption. The maximum time (1020 minutes; 17 hr) before breakthrough point was observed in case of 40 cm bed height with flow rate of 5ml min(-1). FTIR analysis of acid treated immobilized A. niger was used fora qualitative and preliminary analysis of chemical functional groups present on its cell wall which provided the information on nature of cell wall and Cr (VI) interaction during the process of biosorption. The IR spectra of biosorbent recorded before and after chromium biosorption had shown some changes in the band patterns, which were finally analyzed and was found that chemical interaction such as ion-exchange between carboxyl (-COOH), hydroxyl (-OH) and amine (-NH2) group of biosorbent and Chromium ion were mainlyinvolved in biosorption of Cr (VI) onto A. niger cell wall surface. The biosorbed metal was eluted from biosorbent by using 0.1 M H2SO4 as eluant. Immobilized biosorbent could be reused for five consecutive biosorption and desorption cycles without apparent loss of efficiency after its reconditioning. Considering all above factors together this paper discusses the efficient chromium biosorption process carried out by immobilized A. niger biosorbent.     

短小芽孢杆菌A—30耐碱性木聚糖酶基因的分子生物学研究

采用PCR方法对短小芽孢杆菌A－30菌株的耐碱性木聚糖酶基因进行克隆。在木聚糖选择平板上用刚果红染色法筛选出阳性克隆，提取阳性克隆的重组质粒进行酶切鉴定和测序。该基因在大肠杆菌中表达，过夜培养物胞外、胞内和周质空间的木聚糖酶酶活分别为0．159IUmL^-1、0.322IUmL^-1和0.007IUmL^-1。此木聚糖酶表现出较宽的pH作用范围，最适作用pH7左右，在pH9时仍有60％以上的酶活性。图4参14     

Antimicrobial activity of methanol extract of Origanum majorana L. (Sweet marjoram)

In-vitro microbicidal activity of the methanol extract of Origanum majorana L. was tested against seven fungi (Fusarium solani, Candida albicans, Aspergillus niger, A. parasiticus, Rhizopus oryzae, Rhizoctonia otyzae-sativae and Altemaria brassicicola) and six bacteria (Bacillus subtilis, B. megaterium, Escherichia coil, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus). The methanol extract of O. majorana can be used as an effective herbal protectant against different pathogenic bacteria and fungi. High toxicity against the growth of Aspergillus niger was diagnosed.     

Effect of phosphogypsum amendment on soil physico-chemical properties, microbial load and enzyme activities

Phosphogypsum (PG) is produced as a solid waste from phosphatic fertilizer plants. The waste slurry is disposed off in settling ponds or in heaps. This solid waste is now increasingly being used as a calcium supplement in agriculture. This study reports the effectof PG amendmenton soil physico chemical properties, bacterial and fungal count and activities of soil enzymes such as invertase, cellulase and amylase over an incubation period of 28 days. The highest mean percent carbon loss (55.98%) was recorded in 15% PG amended soil followed by (55.28%) in 10% PG amended soil and the minimum (1.68%) in control soil. The highest number of bacterial colonies (47.4 CFU g(-1) soil), fungal count (17.8 CFU g(-1) soil), highest amylase activity (38.4 microg g(-1) soil hr(-1)) and cellulase activity (38.37 microg g(-1) soil hr(-1)) were recorded in 10% amended soil. Statistically significant difference (p<0.05) has been recorded in the activities of amylase and cellulase over the period of incubation irrespective of amendments. Considering the bacterial and fungal growth and the activities of the three soil enzymes in the control and amended sets, it appears that 10% PG amendment is optimal for microbial growth and soil enzyme activities.     

黑曲霉耐热木聚糖酶XYNB在毕赤酵母中的高效表达

高比活木聚糖酶的高效表达是进一步提高木聚糖酶发酵效价、降低生产成本的有效途径.将黑曲霉木聚糖酶基因XynB(不含信号肽)克隆到分泌型表达载体pPIC9K上,线性化后电击转化巴斯德毕赤酵母GS115,G418和PCR鉴定的阳性转化子经0.5%甲醇、在28℃诱导表达.SDS-PAGE分析表明,该蛋白相对分子质量为20×103左右.优化的诱导表达条件为,每隔12 h添加0.5%的甲醇,发酵5 d后,比活达4 757 U/mg;其最适温度为55℃,最适pH为5.0,80℃处理30min后仍有74%的残余酶活.