Extraction of rifamycin B from native and aqueous solutions]

Optimal conditions for extraction of rifamycin B from aqueous solutions and fermentation broth filtrates at pH values within 2.0-7.0 were determined. When the antibiotic was extracted from the aqueous solutions, the highest yield was obtained at pH 2.0. When the antibiotic was extracted from the fermentation broth filtrates, it was found that chloroform was the most selective solvent with respect to rifamycin B, the chloroform selectivity being increased at pH 3.5-4.0. It was shown that rifamicin B passed from the buffer solutions with a concentration of 3-20 mg/ml to chloroform in amounts of 6-7 mg/ml and to ethylacetate and butanol in amounts of 20 mg/ml. Such conditions of chloroform and butanol (9 : 1) increased the rifamycin B contents in the extract up to 40 mg/ml.     

毕赤酵母工程菌高密度发酵中氨态氮含量的测定

首次尝试将钠氏法应用于毕赤酵母高密度发酵中,并与靛酚蓝检测法进行了比较,证实钠氏法在0~10 mg/L的范围内,相关性好(r2=0.996 5),精确度(RSD=2.14%~5.32%)和精密度(Prec ision=97%~105%)高,更适合于毕赤酵母发酵中氨态氮含量的检测。并对其检测条件进行优化,确定检测波长为400 nm,最佳的显色反应为10 min。该方法用于毕赤酵母高密度发酵表达重组人血清白蛋白胸腺肽的结果表明,在发酵液氨基氮含量低于1.5 mg/kg,重组目的蛋白的表达出现了明显的降解,改善并控制发酵液中氨态氮的含量后,可以明显控制目的蛋白的降解,提高产品的产率。结果证实该方法用于发酵液中氨态氮的检测时,准确可靠,切实可行。     

Development of balanced medium composition for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3

Aim:  To develop optimum fermentation environment for enhanced rifamycin B production by isolated Amycolatopsis sp. RSP-3.
Methods and Results:  The impact of different fermentation parameters on rifamycin B production by isolated Amycolatopsis sp. RSP-3 was investigated using Taguchi methodology. Controlling fermentation factors were selected based on one variable at a time methodology. The isolated strain revealed more than 25% higher production compared to literature reports. Five different nutritional components (soyabean meal, glucose, potassium nitrate, calcium carbonate and barbital) and inoculum concentration showed impact on rifamycin B production at individual and interactive level. At optimized environment, 65% contribution was observed from selected fermentation parameters.
Conclusions:  Soyabean meal and calcium carbonate were the most significant factors among the selected factors followed by barbital and potassium nitrate. Glucose, however, showed the least significance on rifamycin B production with this strain. A maximum of 5·12 g l⁻¹ rifamycin B production was achieved with optimized medium containing (g l⁻¹) soyabean meal, 27; glucose, 100; potassium nitrate, 4; calcium carbonate, 3 and barbital, 1·2.
Significance and Impact of the Study:  The present study signifies identification of balanced medium component concentrations for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3. This strain requires organic and inorganic nitrogen sources for effective product yield. Yet at individual level, organic nitrogen source has c. nine-fold higher influence compared to inorganic one.     

The role of bitoxibacillin components in the manifestation of its biological activity

New methods were developed and applied to quantitative determination of beta-exotoxin and antibiotic activity of delta-endotoxin with respect to Micrococcus spp. in bitoxibacillin (BTB) and the fermentation broths prepared under industrial conditions. The biosynthesis of beta-exotoxin in the period of its maximum accumulation during the fermentation was estimated. It was shown that the primary biological effect of BTB on insects consisted in the actions of beta-exotoxin and delta-endotoxin. Biological activity of each of the entomocidal components of the entomocidal components of BTB did not practically correlated with the number of viable spores. There was a correlation between the antibiotic activity of crystalline B. thuringiensis subsp. thuringiensis solutions and the insecticidal activity of the entomopathogenic preparations. Determination of beta-exotoxin and antibiotic activity of delta-endotoxin might be used as a complex procedure for testing the quality of BTB. The method for estimating antibiotic activity of the crystal solutions allowed one to assay the biological activity of other preparations based on Bacillus thuringiensis non-synthesizing beta-exotoxin.     

Use of a polarographic method for determining trichothecin]

The polarographic behaviour of trichothecin was studied. It was shown that the antibiotic could be detected in solutions at concentrations of 7.10(-7) moles with the help of the polarographic method. Conditions for the polarographic determination of trichothecin in fermentation broth were developed. The error was not more than 3 per cent. The reliability of the results was shown by statistical treatment of data performed in accordance with the requirement of the USSR State Pharmacopeia, X ed., prescribing that the precision of the assay is such that the fiducial limits at p = 95 per cent deviate from the average value by not more than 5 per cent. Comparison of the results of trichothecin determination in the fermentation broth with the polarographic and biological methods showed no significant difference. Therefore, the polarographic method may be recommended for trichothecin determination in the fermentation broth.     

β-glucosidases from a new Aspergillus species can substitute commercial β-glucosidases for saccharification of lignocellulosic biomass

β-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for β-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus ) showed 10 times greater β-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial β-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis-Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3?U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4?°C for Novozym 188. The significant thermostability of strain AP β-glucosidases was further confirmed when compared with Cellic CTec. The β-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for β-glucosidase activity.     

Spectrophotometric determination of rifampicin S in the presence of rifampicin O]

The limits of the use of the methods of direct photometric and differential spectrophotometric determination of rifamycin S containing rifamycin O as an admixture were discussed. The method of direct photometry did not provide determination of rifamycin S in the presence of rifamycin O since instability of the latter under the analysis conditions. A possibility of using the differential-spectrophotometric method for determination of rifamycin S in the presence of not more than 5 per cent of rifamycin O was shown.     

Differential spectrophotometric method of analysing levorin in the culture broth and in the mycelium]

It was shown on model experiments that the microbiological method was not applicable for determination of levorin content in industrial intermediate products containing in addition levoristatin, since the presence of the latter made higher the results of the microbiological assay. Because of this till to the present date the quantitative content of levorin in the industrial intermediate products was determined photometrically using alcohol (pure solvent) as the reference solution. Still, this method also made higher the results of the assay, especially when the content of levorin was determined in the fermentation broth. In the solid phase levorin is contained in the mycelium which occupies only 1 to 2 per cent of the fermentation broth, while the liquid phase or the fermentation broth filtrate occupies 98 to 99 per cent. It was found that the fermentation broth filtrate contained protein admixtures which coagulated on addition of alcohol to the fermentation broth and formed fine colloid solutions. As a result the absorption values became higher. In the present study not the pure solvent but an extract of the fermentation broth filtrate containing neither levorin, nor levoristatin was used as the reference solution. Such a differential method provided elimination of all errors due to the presence of various metabolites in the fermentation broth filtrate which varied both qualitatively and quantitatively.     

等离子体作用结合氧限制模型选育利福霉素SV高产菌株

利福霉素SV毒性低、疗效高、抗菌谱广,主要由地中海拟无枝酸菌发酵生产,其发酵过程属于耗氧发酵,供氧直接影响产物形成.为减少发酵过程氧限制影响,进一步提高利福霉素发酵产量,通过构建定向氧限制模型,将常温常压等离子体诱变和无水亚硫酸钠氧限制筛选模型相结合,建立了利福霉素生产菌株24孔板快速培养的高通量筛选方法,高效选育出能...     

Improvement of rifamycin B production using mutant strains of Amycolatopsis mediterranei

Two mutant strains of Amycolatopsis mediterranei VA17 and VA18 were isolated using physical (UV) and chemical (NTG) mutagens gave high rifamycin B than the parent type when grown in the same fermentation medium with a pH of 7.2, temperature 32v°C for a period of 12 days. The cultural conditions of both mutant strains are similar to the parent strain except temperature which was higher by 4v°C. By this mutation and selection study, rifamycin B production was improved from 1400 mg/l to 2450 mg/l.     

人干扰素α—2b生产工艺的研究

人干扰素α-2b的简易、高效生产工艺研究,包括高效率、小型化的发酵技术,不用单克隆抗体亲和层析的纯化工序,以及大肠杆菌表达的人干扰素α-2b包涵体蛋白的天然构型复性等问题,从10L基因工程大肠杆菌发酵液所得1000g菌体中得人干扰素α-2b约500mg,效价为1×10~8u/mg,设备投资少,生产成本低,产品表现了高纯度、高效价。适宜大量生产,为广大人民群众提供廉价高质量药品创造了条件。     

Determination of the biological activity of heliomycin by a method of diffusion in agar]

A biological method for determination of geliomycin activity using agar diffusion is described. A nutrient medium containing Hottinger broth up to 35 mg per cent of amine nitrogen, 1.5 percent of agar-agar, tap water, pH 7.8 to 8.0 was used. Bacillus subtilis ATCC 6633 served as the test-culture. Geliomycin was dissolved in 0.1 N sodium hydroxide solution.     

Product inhibition and its removal on josamycin fermentation by Streptomyces narbonensis var. josamyceticus

Josamycin formation was shown to be closely associated with the degree of mycelial growth. Media using glycerol or glucose were slight in josamycin accumulation. However, the mycelial growth and product formation were interrupted in the course of the fermentation. The self-resistance level of the producing organism against josamycin was shown to be not higher than 1 mg per ml. In media using fatty oil, the organism grew smoothly and gave a concentration of josamycin at least 5 times higher than the self-resistance levelMost of the josamycin was distributed in the oily phase of the broth. The josamycin concentration of the water phase of the broth was less than the self-resistance level, except in the later period of the fermentation. Unsaturated fatty acids remaining in the broth were found to be the best extractors of josamycin. It was concluded that the enhanced josamycin accumulation of this fermentation resulted from the removal of the product inhibition.     

人小肠三叶因子在毕赤酵母中克隆表达及对肠粘膜修复作用的研究

为了研究人肠三叶因子(hITF)对肠粘膜的保护作用,利用RT-PCR从肠粘膜中扩增出hITF基因片段,与诱导分泌型毕赤酵母载体pPIC9连接构建了重组质粒pPIC9hITF,重组质粒转化至宿主菌GS115,经过PCR鉴定和转化子发酵筛选,得到一个重组毕赤酵母高产菌株GS115/pPIC9hITF。在5L发酵罐中用基本盐培养基培养重组菌株,添加甲醇诱导表达hITF,离心收集的上清液通过离心交换层析纯化得到hITF。质谱鉴定结果表明纯化的hITF与天然提取产品在N端序列上完全相同。细胞实验和动物实验结果表明重组hITF能够促进细胞迁移,并可以保护肠粘膜免受有害因子的侵袭,保持了较好的生物学活性。     

土壤拮抗放线菌的分离筛选及其抑菌物质特性的研究

目的从土壤中筛选拮抗能力强且抑菌特性稳定的放线菌菌株。方法采用双层琼脂法筛选出4株拮抗放线菌菌株,然后采用杯碟法测这4株菌株发酵液提取物的抗菌谱、最小抑菌浓度、热稳定性和酸碱稳定性。结果 4株菌株发酵液提取物都能抑制金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌和白色念珠菌的生长。以金黄色葡萄球菌为指示菌测发酵液提取物的最小抑制浓度,6#和9#拮抗作用较强,发酵液提取物稀释0.125mg/ml仍有抑菌作用。6#菌株在100℃处理30min后仍有40%的抑菌活性。6#菌株发酵液提取物在碱性环境条件下比在酸性环境条件下稳定。结论 4株菌株中6#菌株发酵液提取物具有拮抗能力强、最小抑菌浓度低和在碱性条件下活性较稳定的特点。     

Production of Bacillus thuringiensis Berliner var. kurstaki Grown in Alternative Media

Agro - industrial residues and by - products available in southeastern Brazil were used as ingredients for low - cost culture media for liquid fermentation of Bacillus thuringiensis var. kurstaki. Highest spore yield was obtained with a medium containing cheese whey , soya bean milk and molasses (WSM) . Crystals and spores were produced in all media and potency of the final product was highest for nutrient broth + yeast extract medium (NBY) . There was no correlation between the number of spores in the fermented media and the potency of the preparations . Considering all three factors , the potencies , costs and yields of the final products , lowest relative cost was obtained with BMM medium ( Bombyx mori pupae + molasses) . NBY and WSM had intermediate relative cost approximately nine times higher than BMM . The cost analysis suggests that BMM medium should be preferred for local production of B. thuringiensis var . kurstaki in comparison to other media tested . The results also demonstrate the importance of considering yields , cost and potency of the B. thuringiensis preparations in selecting the production medium .     

蜡状芽孢杆菌发酵南极磷虾生成活性物质的研究

南极磷虾具有分布量广、营养价值高、贮藏量大等特点,是一种重要的海洋蛋白新资源,有待开发利用。从深海泥样中分离筛选出一株能够以南极磷虾为唯一碳氮源生长的蜡状芽孢杆菌,利用该菌发酵南极磷虾。对发酵条件进行了初步优化,研究了发酵液的抗氧化能力以及发酵液中风味物质的种类和含量的变化情况。结果显示,30℃恒温培养48 h,DPPH清除率为44.05%,总酚含量为0.059 2 mg/m L,亚铁离子螯合能力为92.23%,还原力为200.13μmol/L,总蛋白含量为2.952 g/100 g,有机酸组成丰富,游离氨基酸含量(227.540 2 mg/m L)比发酵前(106.5796 mg/m L)增加一倍。本研究使南极磷虾这一丰富的资源得到最大限度的利用,同时也使南极磷虾的生物活性物质能够在生产中发挥潜在的作用。     

Estimation of pullulan by hydrolysis with pullulanase

A novel method for the estimation of pullulan was developed in which pullulan was hydrolysed by pullulanase. The hydrolysed product was mainly maltotriose and was determined colorimetrically using 3,5-dimethylsalicylic acid. This gave good linearity with respect to the concentration of pullulan in the fermentation broth. The content of pullulan determined in this way was less than that determined by a coupled enzyme assay and was comparable to that determined by an HPLC method. The new method was specific for estimation of pullulan, demonstrated high accuracy, and could assay pullulan from up to 3.2 mg/ml.     

中国被毛孢发酵液的活性成分分析及清除自由基和抗白色假丝酵母活性的研究

测定处于不同生长期的中国被毛孢Hirsutella sinensis菌株RCEF0273胞外多糖含量、发酵液中5种核苷代谢量和氨基酸含量,并对其菌株次生代谢产物清除自由基活性和抑菌效果进行了测定分析,发现:中国被毛孢胞外多糖含量最高可达到1.9480g/L,发酵液中尿苷、鸟苷、肌苷、胸腺嘧啶核苷和腺苷5种核苷含量最高分别为10.4132mg/g、12.1929mg/g、2.2698mg/g、1.1000mg/g和1.8181mg/g,其菌株发酵液冻干粉中氨基酸总量为3.2560mg/g,活性测定结果显示中国被毛孢发酵液的正丁醇相有很强的清除自由基活性和抑菌活性。在10mg/mL的测试浓度下,37℃孵育15min后其自由基清除率可达93.25%,在2mg/mL的测试浓度下,其抗白色假丝酵母Candida albicans的抑菌圈直径达到21.42mm。     

Isolation of a cyclitol antibiotic: 2,3,4,5-tetrahydroxycyclohexanemethanol

The isolation and structure determination of a new microbial product, (+)- (123/45)-2,3,4,5-tetrahydroxy-1-cyclohexanemethanol is described. This product was detected in the fermentation broth of a newly isolated actinomycete by its antibacterial activity. A novel isolation method was developed and crystalline product was obtained in good yield. The structure was determined by spectroscopic examination of the product and its acetyl and trimethylsilyl derivatives. The racemic form of this compound had already been synthezised by G. E. McCasland et. al., as analog of galactose.