枯草芽孢杆菌中性β—甘露聚糖酶的产生及性质

由土壤中分离出一株产中性β甘露聚糖酶的枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）,编号ＢＭ９６０２。该菌在液体培养条件下,产生中性β甘露聚糖酶。多糖能作为碳源,而单糖不能作为碳源;有机氮源优于无机氮源。产酶最适培养基组成：魔芋粉４％,牛肉蛋白胨和酵母膏各１％。产酶最适培养条件：培养基起始ｐＨ８５,３５℃,振荡培养３６ｈ。以槐豆胶为底物,培养滤液中性β甘露聚糖酶活力为９６ＩＵ／ｍＬ。酶在ｐＨ５０～１００和５０℃下稳定;作用最适条件为ｐＨ６０和５０℃;水解魔芋粉和槐豆胶均产生寡聚糖。     

Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by the endophytic fungus Monotospora sp. were evaluated. The optimal temperature and initial pH for laccase production by Monotospora sp. in submerged culture were found to be 30 degrees C and 8.5, respectively. Maltose (2 g l(-1)) and ammonium tartrate (10 g l(-1)) were the most suitable carbon and nitrogen source for laccase production. Under optimal culture medium, the maximum laccase activity was determined to be 13.55 U ml(-1), which was approximately four times higher than that in basal medium. This is the first report on laccase production by an endophytic fungus.     

Utilization of ram horn peptone in the production of glucose oxidase by a local isolate Aspergillus niger OC-3

Glucose oxidase (GO) is an enzyme that is used in many fields. In this study, ram horn peptone (RHP) was utilized as the nitrogen source and compared with other nitrogen sources in the production of GO by Aspergillus niger. To obtain higher GO activity, 14 A. niger strains were isolated from soil samples around Erzurum, Turkey. Among these strains, the isolate that was named A. niger OC-3 achieved the highest GO production. The production of GO was carried out in 100 mL scaled batch culture. The fermentation conditions such as initial pH, temperature, agitation speed, and time were investigated in order to improve GO production. The results showed that the cultivation conditions would significantly affect the formation of GO, and the utilization of the RHP achieved the highest enzyme production (48.6 U/mL) if compared to other nitrogen sources. On the other hand, the maximum biomass was obtained by using the fish peptone (7.2 g/L), while RHP yielded 6.4 g/L. These results suggest that RHP from waste ram horns could effectively be used in the production of GO by A. niger OC-3.     

Production of glucose oxidase using Aspergillus niger and corn steep liquor

Glucose oxidase production was optimized using an isolated strain of Aspergillus niger and an economical nutrient source, corn steep liquor (CSL). The culture produced 580 +/- 30 units/ml of the enzyme using 70 g/l sucrose as the carbon source. Using CSL as the sole nutrient source enzyme synthesis was increased to 640 +/- 36 units/ml. None of the nitrogen sources (nitrates of calcium, sodium, ammonium, potassium and yeast extract, malt extract, and peptone) was beneficial to the enzyme synthesis. Aeration and agitation enhanced enzyme synthesis to 850 +/- 45 units/ml. Glucose oxidase has numerous applications in food industry and clinical fields.     

Effect of nitrogen source and nitrogen concentration on the production of pyruvate by Torulopsis glabrata

The effect of nitrogen sources including yeast extract, peptone, soybean hydrolyzate and some inorganic nitrogen sources, as well as the nitrogen concentration on the fermentative production of pyruvate by Torulopsis glabrata WSH-IP12 was investigated. The addition of yeast extract greatly inhibited pyruvate accumulation, while peptone was shown to be the most favorable nitrogen source. In flask culture, 15 g l(-1) peptone was needed to consume 80 g l(-1) glucose with 23.4 g l(-1)of pyruvate accumulated. Pyruvate production was markedly dependent on the ratio of carbon to nitrogen (C:N), its production was improved by increasing the concentration of glucose and peptone proportionally and reduced by exclusively increasing the glucose concentration. In a glucose fed-batch culture, cell growth and pyruvate production slowed after 28 h. However, cell growth and pyruvate production recovered after further nitrogen, in the form of peptone and ammonium sulfate, was added to the culture. A final concentration of pyruvate of 54.5 g l(-1) was achieved at 64 h (yield to glucose consumed of 0.471 g g(-l)). By using aqueous ammonia instead of potassium hydroxide for pH control, 57.3 g l(-1) pyruvate with a yield of 0.498 g g(-1) was produced by 55 h. This result further indicates that nitrogen level plays an important role in the production of pyruvate.     

产β-甘露聚糖酶Aspergillus sp. LQ21的分离、鉴定及发酵条件的研究

β-甘露聚糖酶是一类能够水解甘露聚糖、葡甘露聚糖、半乳甘露聚糖的半纤维素酶类,广泛存在于动植物和微生物中,此酶在食品、医药、饲料、造纸、石油等方面已得到广泛应用;近年来,其作为食品和饲料添加剂方面倍受关注。对采自土壤和树皮的样品通过富集培养、平板初筛和摇瓶复筛,得到1株具产β-甘露聚糖酶能力的曲霉属菌株LQ21,结合形态特征、培养特征及18S rRNA基因序列分析,将该菌株鉴定为Aspergillussp.真菌。考察了培养时间、起始pH、培养温度、碳源和氮源对该菌株产酶的影响。初步确定了其最适产酶培养基组成:魔芋粉0.5%,蛋白胨1%,NaNO30.2%,K2HPO40.1%,KCl 0.05%,MgSO4.7H2O 0.05%,FeSO4.7H2O 0.001%;最适培养条件:初始pH4.5,温度35℃,转速200 r/min,培养60 h发酵液上清中酶活达到最高。     

Production of red pigment by submerged culture of Paecilomyces sinclairii

AIMS: From a survey of submerged culture of edible mushrooms, a high pigment-producing fungus Paecilomyces sinclairii was selected and its optimal culture conditions investigated. METHODS AND RESULTS: The optimal culture conditions for pigment production were as follows: inoculum age, 3 d; temperature, 25 degrees C; initial pH, 6.0; carbon source, 1.5% (w/v) soluble starch; nitrogen source, 1.5% (w/v) meat peptone. Although addition of 10 mmol l(-1) CaCl2 to the culture medium slightly increased pigment production, most of the bio-elements examined had no notable or detrimental effect on pigment production. CONCLUSIONS: Under the optimal conditions obtained in the flask culture tested, a ninefold increase in pigment production (4.4 g l(-1)) was achieved using a 5(-l) batch fermenter. Paecilomyces sinclairii secreted water-soluble red pigment into the culture medium. The pigment colour was strongly dependent on the pH of the solution: red at pH 3-4, violet at pH 5-9 and pink at pH 10-12. SIGNIFICANCE AND IMPACT OF THE STUDY: The high concentration of pigment (4.4 g l(-1)) produced by P. sinclairii demonstrates the possibility of commercial production of pigment by this strain, considering its relatively high production yield and light stability.     

碳源和氮源对彩绒革盖菌液体发酵合成漆酶的影响

研究了碳源、氮源对彩绒革盖菌液体发酵合成漆酶的影响。结果表明,在所选的几种碳、氮源中,麸皮为试验菌株合成漆酶的较好碳源;酵母浸膏、酒石酸铵、蛋白胨均是比较理想的氮源。不同的碳氮比对彩绒革盖菌漆酶的产量有着显著的影响,高碳高氮是其生产漆酶的最佳条件。在适宜的培养条件下该菌株能合成高活力的漆酶,其酶活力可达298 U/mL以上,产酶周期为6~7 d。     

Production of laccase by a newly isolated strain of Trametes modesta

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by Trametes modesta were evaluated using the one-factor-at-a-time method. The final optimisation was done using a central composite design resulting in a four-fold increase of the laccase activity to 178 nkat ml(-1). Response-surface analysis showed that 7.34 g l(-1) wheat bran, 0.87 g l(-1) glucose, 2.9 g l(-1) yeast extract, 0.25 g l(-1) ammonium chloride, an initial pH of 6.95 and an incubation temperature of 30.26 degrees C were the optimal conditions for laccase production. Laccase produced by T. modesta was fully active at pH 4 and at 50 degrees C. The laccase was very stable at pH 4.5 and at 40 degrees C but half-lives decreased to 120 and 125 min at higher temperature (60 degrees C) and lower pH (pH 3).     

Production of phytase (myo-inositolhexakisphosphate phosphohydrolase) by Aspergillus niger van Teighem in laboratory-scale fermenter

The growth and production pattern of phytase by a filamentous fungus, Aspergillus niger van Teighem, were studied in submerged culture at varying agitation rates and controlled and uncontrolled pH conditions. Allowing the initial culture to grow under neutral condition with subsequent decline in pH resulted in increased phytase productivity. A maximum of 141 nkat/mL phytase was obtained when the broth pH was maintained at pH 2.5 as compared to 17 nkat/mL units at controlled pH 5.5. The culture morphology and rheological properties of the fermentation broth significantly varied with the agitation rate. The volumetric oxygen transfer coefficient was determined at different phases of fungal growth during batch fermentation using static gassing out and dynamic gassing out methods. The oxygen transfer coefficient (k(L)a) of the fermenter was found to be 125 h(-)(1) at 500 rpm as compared to 38 h(-)(1) at 200 rpm. The oxygen transfer rates at different phases of growth were significantly affected by cell mass concentration and fungal morphology. During the course of fermentation there was a gradual decline of k(L)a from 97 h(-)(1) on day 2 to 63 h(-)(1) on day 6 of fermentation, after which no significant change was observed. The degree of agitation considerably influenced the culture morphology where shear thinning of filamentous fungus was observed with the increase in agitation.     

L-Glutamate oxidase from Streptomyces cremeus 510 MGU: effect of nitrogen sources on enzyme secretion]

Effect of nitrogen sources (organic complexes and mineral salts) on L-glutamate oxidase synthesis by Streptomyces cremeus 510 MGU was studied. Optimal enzyme production was not provided by any single nitrogen source. The most effective combination of nitrogen sources (soy flour, peptone, ammonium sulfate) was elaborated by the mathematical planning method. The results of experiment allowed to enhance biosynthesis of extracellular L-glutamate oxidase to 2.4-2.6 U/mL. It was shown that L-glutamate oxidase of Streptomyces cremeus 510 MGU is highly specific to substance and is stable during storage of filtrated culture with pH 6.8-9.0.     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

β-Mannanase production and kinetic modeling from carob extract by using recombinant Aspergillus sojae

The main objectives of this study were to optimize β-mannanase fermentation conditions by using Response Surface Methodology (RSM) and to model kinetically using the kinetic models. Based on the results, the optimum fermentation conditions were found to be initial sugar concentration of 10°Bx, whey concentration of 0.75% [w/v], and inoculum size of 8% (v/v). Under optimized conditions, β-mannanase activity (P), sugar consumed (ΔS), maximum β-mannanase production rate (Q_P), and sugar utilization yield (SUY) were 687.89 U/mL, 47.38 g/L, 118.54 U mL^–1 day^–1, and 69.73%, respectively. Kinetic models were employed to describe the optimum β-mannanase fermentation process. The kinetic analysis of β-mannanase fermentation showed that β-mannanase fermentation is growth associated because the α value (U/mgX) is approximately 330-fold higher than the β value (U/mgX·hr). Nevertheless, maintenance value (Z) was lower than γ value, thus showing that Aspergillus niger mainly utilizes the sugars for β-mannanase production and fungal growth. Consequently, carob extract and whey powder could be used to be cost-effective carbon and organic nitrogen sources, respectively. It was clearly indicated that the suggested kinetic models can successfully describe the fungal growth, β-mannanase production, and substrate consumption.     

木聚糖酶高产菌株的诱变*

出发菌株Aspergillus niger M1经过紫外线诱变得到一株木聚糖酶活力提高30％的突变株A．niger J506。木聚糖酶谱带检测发现,突变株成熟发酵液中有3种类型的木聚糖酶,而出发菌株中只有两种。经过正交试验得出突变株产酶的最佳发酵条件为：主碳源浓度4％、麸皮与玉米芯的比例为5：5、葡萄糖浓度0．1％、草酸铵浓度2．0％,培养基初始pH为5．0,250mL三角瓶的装液量为100mL。     

Optimization of submerged culture conditions for the mycelial growth and exo-biopolymer production by Cordyceps militaris

AIMS: The objective of the present study was to determine the optimal culture conditions for exo-biopolymer production by Cordyceps militaris in shake flask culture. METHODS AND RESULTS: The optimal temperature and initial pH for both mycelial growth and exo-biopolymer production by Cordyceps militaris in shake flask culture were found to be 20 degrees C and 6.0, respectively. Sucrose (40 g x l(-1)) and corn steep powder (10 g x l(-1)) were the most suitable carbon and nitrogen source for both mycelial growth and exo-biopolymer production. CONCLUSION: Under optimal culture conditions, the maximum exo-biopolymer concentration in a 5-l jar fermenter indicated 10.3 g x l(-1), which was approximately three times higher than that in shake flask culture. SIGNIFICANCE AND IMPACT OF THE STUDY: This process can have a significant impact on the industrial scale when sucrose and corn steep powder were used as carbon and nitrogen source.     

嗜碱芽孢杆菌NTT33产碱性β-甘露聚糖酶发酵条件的研究及高产菌株选育

研究了嗜碱芽孢杆菌（ａｌｋａｌｏｐｈｉｌｉｃＢａｃｌｕｓｓｐ．）ＮＴＴ３３发酵产生胞外碱性β－甘露聚糖酶的条件,其最佳碳源为１％槐豆角,最佳氮源为１％蛋白胨＋０．２％酵母膏,发酵培养３６ｈ产酶量最高（达６１．３υ／ｍＬ）。甘油,葡萄糖,甘露糖等对产酶有强的阻遏作用。该菌株经紫外诱变处理后,采用透明圈法初步筛选出在含葡萄糖和不含葡萄糖的槐豆胶培养基上同时产生透明圈的菌株,进一步测定其产酶进程曲线,最后筛选到一株（ＮＴＴ３３－ｒ６）部分消除葡萄糖代谢阻遏高产β－甘露聚糖酶的菌株,其酶活力比出发菌株提高５０％,,达到９６．３Ｕ／ｍｌ;。     

Lasiodiplodan, an exocellular (1→6)-β-d-glucan from Lasiodiplodia theobromae MMPI: production on glucose, fermentation kinetics, rheology and anti-proliferative activity

Lasiodiplodan, an exopolysaccharide of the (1→6)-β-D: -glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose) and nitrogen sources (KNO(3), (NH(4))(2)SO(4), urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan's anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05 ± 0.05 g L(-1)), maltose (2.08 ± 0.04 g L(-1)) and yeast extract (2.46 ± 0.06 g L(-1)) produced the highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74 ± 0.006 vs. 0.22 ± 0.008 g g(-1)). A comparison of the fermentative parameters of L. theobromae MMPI in shake flasks and a stirred-tank bioreactor at 120 h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01 ± 0.07 g L(-1)) with a specific yield of 0.25 ± 0.57 g g(-1) and a volumetric productivity of 0.06 ± 0.001 g L(-1) h(-1). A factorial 2(2) statistical design developed to evaluate the effect of glucose concentration (20-60 g L(-1)) and impeller speed (100-200 rpm) on lasiodiplodan production in the bioreactor showed the highest production (6.32 g L(-1)) at 72 h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60°C in the presence of CaCl(2). Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time- and dose-dependent with an IC(50) of 100 μg lasiodiplodan mL(-1).     

Isolation and characterization of an active mannanase-producing anaerobic bacterium, Clostridium tertiumKT-5A, from lotus soil

Of 10 strains of mannanase-producing anaerobicbacteria isolated from soils and methanogenic sludges, Clostridium tertium KT-5A,which was isolated from lotus soil, produced high amounts of extracellular β-1,4-mannanase. The isolate was an aerotolerant anaerobe without quinon systems; the cell growthcultivated with no addition of reducing agents was also stable. High yields of mannanasewere obtained by inducing enzyme production with galactomannan guar gum and beef extract/peptone as carbon and nitrogen sources, respectively. Fermentation endproducts on galactomannan fermentation were formate, acetate, lactate, butyrate, carbondioxide and hydrogen. The extracellular mannanase displayed high activity ongalactomannans of locust bean gum galactose/mannose (G/M) ratio 1:4 and spinogum (G/M 1:3), but weak activity on guar gum galactomannan (G/M 1:2) and konjac glucomannan. As far as is known, this is the first report on the isolation of an activemannanase-producing anaerobic bacterium from natural environments.     

Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and beta-xylosidase while maintaining low protease production

A growth medium was developed for maximal production in batch culture of extracellular xylanase and beta-xylosidase by Aspergillus awamori CMI 142717 and a mutant (AANTG 43) derived from the wild-type strain. The optimum pH for the production of xylanase and beta-xylosidase was 4.0. The best temperature of xylanase production was 30 degrees C; 35 degrees C was optimal for beta-xylosidase. Protease production was never completely suppressed under any of the conditions tested. However, protease titre was 3.5-fold less than the control in medium in which proteose peptone and yeast extract were omitted: the level of xylanase was not affected (8.6 U mL(-1)) but beta-xylosidase titre was increased 4.7-fold to 1.5 U mL(-1). When corn steep liquor was used as the sole nitrogen source, xylanse and beta-xylosidase titres were further increased by 1.5- and 1.9-fold, respectively. Of the carbon sources investigated, ball-milled oat straw or oat spelt xylan produced the highest titres of xylanse and beta-xylosidase. None of the soluble carbon sources investigated produced the high titres of xylanase or beta-xylosidase induced by either oat straw for xylanse and beta-xylosidase was 2% and the optimum spore inoculum was between 10(6) and 10(7) spores/mL(-1) final concentration. The level of xylanse activity obtained in the culture filtrates of the mutant was a remarkable 820 U mL(-1) when the reducing sugar released was measured by the dinitrosalicylic acid method. This enzyme titre would appear to be the highest reported so far. The xylanases system contained the correct balance of enzymes to effect extensive hydrolysis of oat spelt xylan. The protease titre was very low.     

Bimutation breeding of Aspergillus niger strain for enhancing β-mannanase production by solid-state fermentation

A parent strain Aspergillus niger LW-1 was mutated by the compound mutagenesis of vacuum microwave (VMW) and ethyl methane sulfonate (EMS). A mutant strain, designated as A. niger E-30, with high- and stable-yield β-mannanase was obtained through a series of screening. The β-mannanase activity of the mutant strain E-30, cultivated on the basic fermentation medium at 32 °C for 96 h, reached 36,675 U/g dried koji, being 1.98-fold higher than that (18,501 U/g dried koji) of the parent strain LW-1. The purified E-30 β-mannanase, a glycoprotein with a carbohydrate content of 19.6%, had an apparent molecular weight of about 42.0 kDa by SDS–PAGE. Its optimal pH and temperature were 3.5 and 65 °C, respectively. It was highly stable at a pH range of 3.5–7.0 and at a temperature of 60 °C and below. The kinetic parameters K_m and V_max, toward locust bean gum and at pH 4.8 and 50 °C, were 3.68 mg/mL and 1067.5 U/mg, respectively. The β-mannanase activity was not significantly affected by an array of metal ions and EDTA, but strongly inhibited by Ag⁺ and Hg²⁺. In addition, the hydrolytic conditions of konjak glucomannan using the purified E-30 β-mannanase were optimized as follows: konjak gum solution 240 g/L (dissolved in deionized water), hydrolytic temperature 50 °C, β-mannanase dosage 120 U/g konjak gum, and hydrolytic time 8 h.