混菌状态对新菌系产生VC前体KGA的影响

通过测定氧化葡萄糖酸杆菌产酸变化,研究了新菌系混菌状态对产酸的影响。结果显示：新混菌体系中,种液KGA为4.1-5.5mg/ml,混合菌菌群氧化葡萄糖酸杆菌/掷孢酵母为26-35：1,混菌生物量达0.46-0.60(OD值）,有利于二菌在发酵中相互协调,促进产酸,调节接种生物量可增加产酸速度,缩短产酸周期,但不影响最终产酸量。     

抗生素和干扰素

950112 由产黄,I『一生产青霉素的生物化学和分子遗传学[会,英2/Martin,J.F.I Ann.N.Y.Acad.s01.-1991,646.-132~201[译自DBA,1992,儿(13),92-07338_'] 将产黄青霉AS-P-78酰基辅酶A;6一氨基青霉烷酸一酰基转移酶纯化至均质并鉴定之。用含粗糙脉孢菌(Neurospora crassa)PY r4基因和产贝菏霉pyrG基因的质粒载体作选择标记,可获得产黄青霉的高频转化。克隆、表达并测序了产黄青霉AS-P-78的异青霉素-N-合酶基因,还克隆及测序了该菌的酰基辅酶A t 6一氨基青霉烷酸一酰基转移酶和ACV-合成酶基因。产黄青霉DNA的噬菌体EMBL3基因文库…     

肠道细菌产酸克雷伯氏菌及其挥发性物质对斑翅果蝇成虫的引诱效果

【目的】明确肠道细菌产酸克雷伯氏菌Klebsiella oxytoca对斑翅果蝇Drosophila suzukii的引诱效果,并鉴定和验证该菌挥发性物质对斑翅果蝇成虫的引诱效果。【方法】在无寄主植物和有寄主植物（巨蜂葡萄）的条件下,测定并分析产酸克雷伯氏菌和NB培养基(对照)的上清液对斑翅果蝇成虫的诱集比例以及被引诱成虫的雌雄性比;利用气相色谱质谱联用法(HS-SPME-GC-MS)分别鉴定产酸克雷伯氏菌和NB培养基(对照)上清液中的挥发性物质,并检测浓度较高的4种产酸克雷伯氏菌挥发性物质对斑翅果蝇成虫的引诱效果。【结果】产酸克雷伯氏菌上清液对斑翅果蝇成虫具有引诱作用。其中,无寄主植物条件下,产酸克雷伯氏菌上清液对雄虫引诱作用强于对雌虫,但是在有寄主植物条件下产酸克雷伯氏菌上清液诱集的雌雄成虫数量差异不显著。在产酸克雷伯氏菌上清液中检测到21种挥发性物质,其中浓度较高的为3-甲基-1-丁醇、2-苯乙醇、乙酸异戊酯和吲哚,斑翅果蝇成虫对3-甲基-1-丁醇、2-苯乙醇和吲哚具有趋向性,而对乙酸异戊酯则有趋避性,且3-甲基-1-丁醇可吸引更多的雄虫。【结论】肠道微生物产酸克雷伯氏菌上清液可用于引诱斑翅果蝇成虫,3-甲基-1-丁醇是产酸克雷伯氏菌代谢物中引诱雄虫的重要物质。本研究为斑翅果蝇的防治提供了参考依据。     

一组鸡源乳酸菌产乳酸及其耐受特性研究*

研究了12株（K9、D17、C1、C12、D11、D14、C2、D9、K6、C21、D1和D7）分离自肉鸡肠道的乳酸菌的产乳酸能力及其中3株产酸能力强的菌株的耐受特性。12株乳酸菌产乳酸结果表明：12h内,K6产乳酸速度最快,其次为K9和C1,24h时,D17乳酸浓度最高,48h时C1终乳酸浓度最高。K9、D17和C1的耐受试验结果表明：C1菌株耐酸能力最强,pH2时,C1菌株培养3h后还能检测到活菌,D17和K9菌株培养1h后就已经检测不到活菌。在胆盐浓度0．08％-0．40％范围内,C1、D17和K9均有一定的耐受能力,随着胆盐浓度的升高,C1、D17和K9的存活数呈现缓慢的下降趋势。3株菌中D17耐热能力最强,经80％处理后仍有10^4.9／mL存活数,而K9和C1已检测不到活菌;C1对热最敏感,65℃处理后存活数由10^8／mL降为10^3／mL。     

玉米浆对Vc二步发酵产2-酮基-L-古龙酸影响的研究

通过在培养基中添加不同量的玉米浆,研究其对氧化葡萄糖酸杆菌（俗称小菌）生产Vc前体2-酮基-L-古龙酸的影响,并研究玉米浆成分中的12种主要氨基酸对小菌产酸的影响。结果表明：每100 mL发酵培养基中添加2.5 g左右过滤除菌玉米浆时,2-酮基-L-古龙酸产量高达26.84 mg/mL,小菌活菌数为不添加玉米浆时小菌单菌发酵下的9.74倍。过量玉米浆抑制小菌产酸。12种氨基酸单独与氧化葡萄糖酸杆菌发酵培养及全部混合后与氧化葡萄糖酸杆菌发酵培养对产酸及菌体生长无影响。     

同时产多种基因型ESBLs肺炎克雷伯菌的分析

目的分析汕头大学医学院第一附属医院临床分离的肺炎克雷伯菌同时产多种基因型ESBLs的情况。方法采用PCR扩增对产ESBLs的肺炎克雷伯菌初步分型,然后PCR扩增阳性产物克隆测序分析,脉冲场凝胶电泳（PFGE）分型;用浓度梯度法检测10种抗菌药物对同时产多种基因型ESBLs的肺炎克雷伯菌的最低抑菌浓度（MIC）。结果83株产ESBLs的肺炎克雷伯菌中,发现9株同时产TEM、SHV和CTX-M型,测序结果为CTX-M-3、TEM-1及多种SHV型（SHV12及SHV28）。将这9株细菌作PFGE分析,结果共被分成7个型。药敏结果表明,9株菌除对亚胺培南敏感外,对氨曲南、头孢曲松、头孢噻肟、头孢他啶、丁胺卡那和四环素均高度耐药,对哌拉西林／三唑巴坦、头孢吡肟、环丙沙星极少菌株敏感。结论发现CTX—M-3超广谱-及多种SHV型超广谱-和TEM-1广谱β-内酰胺酶基因同时存在该院临床分离的肺炎克雷伯菌中,耐药十分严重,应引起重视。     

巨大芽孢杆菌B．m2980产孢对氧化葡萄糖酸杆菌产酸的影响

为确定维生素C二步发酵中巨大芽孢杆菌（伴生菌）芽孢形成对氧化葡萄酸杆菌（产酸菌）产酸的影响,本研究通过对巨大芽孢杆菌生长特性分析,选取培养12h（未形成芽孢）和36h（芽孢大量形成）巨大芽孢杆菌B．m2980,检测其胞外液、胞内液以及混合液对产酸菌生成2-酮基-L-古龙酸的影响。结果表明,在未开始形成芽孢时,伴生菌胞外液、胞内液及混合液对产酸菌的生长和产酸有较低的促进作用,其中胞内液的促进能力大于胞外液;在芽孢生成后,胞外液以及混合液对产酸菌生长和产酸的促进能力显著提高。     

L-苏氨酸发酵条件的研究及发酵产物的鉴定

采用逐步诱变处理与单菌落分离相结合,选育出一株产L-苏氨酸量较多的突变株C. cre-natum m-85 (AHV,Met-)。试验证明,生物素与蛋氨酸为其亲株d20～23生长必需因子,同时蛋氨酸又是积累L-苏氨酸的促进因子,硫胺素是生长和积累苏氨酸的促进因子。当生物素、蛋氨酸、硫胺素相配合,菌株产酸能力得以充分显示出来。通气量是L一苏氨酸发酵外部控制的主要条件。L-苏氨酸积累需气量较大。在合适的培养条件下,该菌可在发酵液中积累L一苏氨酸达13.4g／1。 发酵产物的结晶经旋光测定,红外光谱分析,纸上层析及生物鉴定证明是L-苏氨酸。     

产L-苏氨酸重组大肠杆菌的构建和发酵性能

【背景】大肠杆菌由于生长性能优良、遗传背景清晰,常被用作苏氨酸生产菌。【目的】敲除大肠杆菌Escherichia coli THR苏氨酸合成途径的非必需基因,并异源表达苏氨酸合成必需的关键酶,构建一株苏氨酸高产菌株。【方法】利用FLP/FRT重组酶系统,敲除E. coli THR中lysC、pfkB和sstT,同时进行谷氨酸棒杆菌中lysC~(fbr)、thrE和丙酮丁醇梭菌中gapC的重组质粒构建并转化到宿主菌中。【结果】以E. coli THR为出发菌株,敲除其苏氨酸合成途径中表达天冬氨酸激酶Ⅲ (AKⅢ)的基因lysC、磷酸果糖激酶Ⅱ基因pfkB及苏氨酸吸收蛋白表达基因sstT,使菌株积累苏氨酸的产量达到75.64±0.35g/L,比出发菌株增加9.9%。随后异源表达谷氨酸棒杆菌中解除了反馈抑制的天冬氨酸激酶(lysC~(fbr))、苏氨酸分泌转运蛋白(thrE)及丙酮丁醇梭菌中由gapC编码的NADP+依赖型甘油醛-3-磷酸脱氢酶,获得重组菌株E. coli THR6菌株。该菌株积累苏氨酸的产量提高到105.3±0.5 g/L,糖酸转化率提高了43.20%,单位产酸能力提高到5.76 g/g DCW,最大生物量为18.26 g DCW/L。【结论】单独敲除某个基因或改造某个途径不能使苏氨酸大量合成和积累,对多个代谢途径共同改造是构建苏氨酸工程菌的最有效方法。     

日本氨基酸生产研究又获进展

在1987年日本农艺化学会年会上,协和发酵工业公司防府工厂技术研究所报告说,仅通过突变育种技术就能大幅度提高L-苏氨酸产生菌(大肠杆菌)的产酸量。原株一旦积累苏氨酸,回复变异就易发生,发酵生产24次中正常生产只能维持5次,产酸浓度为20g/L左右。现在只通过突变育种获得了发酵68小时可稳产58g/L的菌种,达到工业化     

Stabilization of threonine production in an Escherichia coli overproducing strain using tightly regulated T7 promoter system

The industrial production of compounds by E. coli strains is often accompanied with variability in yield levels. To investigate the mechanism of such instability the over-production of threonine was used as a model. The instability in this strain appears to be caused by a metabolic burden resulting in occurrence of low producing revertants. A successful application of the tightly regulated T7 expression system is presented as a possible solution providing a substantial stabilization of the threonine production.     

Improved production of potato microtubers using a temporary immersion system

A temporary immersion system for potato microtuber production was designed using 4-l vessels. This culture technique showed several advantages compared to solid cultures: i.e., three fold increase in shoot length, more internodes per plant and improved vigor. In the tuber induction stage, microtubers can be induced at all plant nodes, indicating that the tuberization is not restricted to specific regions. For both cultivars tested, Desiree and Atlantic, an average of 3.1 and 2.8 tubers per single node cutting was achieved after 9 weeks in culture. The size and weight of the tubers were higher than on solid media. Scale up was performed with cv. Atlantic in 10-l polycarbonate flasks and 12 units were mounted containing 150 single nodal cuttings each. An average of 2.6 tubers per inoculated cutting was obtained, with 1.3 g fresh weight per microtuber. Temporary immersion is a valuable option for potato microtuber production, as well as for shoot production during the planting season. This revised version was published online in June 2006 with corrections to the Cover Date.     

CARBOHYDRATE-PEPTIDE LINKAGES IN GLYCOPROTEINS AND MUCOPOLYSACCHARIDES FROM BRAIN

Abstract— Treatment of glycopeptides, prepared from glycoproteins of rat and rabbit brain, with NaOH-NaBH₄ leads to the destruction of a portion of the serine, threonine and galactosamine present, and the appearance in acid hydrolysates of alanine, α-aminobutyric acid and galactosaminitol. These results indicate that N-acetylgalactosamine at the reducing end of oligosaccharide chains in brain glycoproteins is linked O-glycosidically to the hydroxyl groups of serine and threonine residues. 2-acetamido-1-(L-β-aspartamido)-l,2-dideoxy-β-D-glucose was also detected after partial acid hydrolysis of the alkali-stable glycopeptides, and most of the carbohydrate in brain glycoproteins appears to be linked by N-acetylglucosaminylasparagine linkages. The results of the treatment of the sulphated mucopolysaccharides from bovine brain with alkaline-borohydride indicate that the polysaccharide chains in chondroitin sulphate and heparan sulphate are linked exclusively to serine.     

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.     

Antioxidants enhanced production of destruxin E from cultivation of Metarhizium anisopliae

The effect of antioxidants on the production of an important cyclohexadepsipeptide congener destruxin E (dtx E) was investigated using the entomopathogenic fungus Metarhizium anisopliae F061. In shaker flask cultivations, 0.015% of menadione-enhanced dtx E production of 220.4 mg/l compared to the control cultivation 90.2 mg/l, which was illustrated by stimulation of dtx E biosynthesis through two electron reduction DT-diaphorase processes in cultivation of M. anisopliae. In 5-l stirred-tank bioreactor cultivation with menadione addition and of control pH 4.0, a yield of 454.6 mg/l of dtx E was obtained after 7 days, and was nearly 30 and 15-fold higher than that from no pH control, and controlled pH 2.0 cultivations, respectively. Further cultivation in a 20-l airlift bioreactor, at pH 4.0, dtx E obtained on the 9th day was 406.0 mg/l, which was much higher than the standard cultivation of no pH control yield 203.3 mg/l on the 11th day. Thus, the present study provides useful information for enhancing dtx E production in cultivation.     

12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors.

A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures. Digitoxin was used as the substrate for biotransformation. Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor. A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method. Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l. Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively. About 80% of the digoxin produced was found in the bathing medium.     

Effects of okadaic acid on agonist-stimulated PGI2 production by rat liver cells (the C-9 cell line).

Preincubation of rat liver cells (the C-9 cell line) with okadaic acid (0.6 microM), a known inhibitor of protein-serine/threonine phosphate phosphatases 2A and 1, for 30 min amplified 6-keto-PGF1 alpha production stimulated by thapsigargin, thrombin, platelet activating factor (PAF), 12-O-tetradecanoylphorbol-13-acetate (TPA), the Ca2+ ionophore A-23187 and lysine-vasopressin (Lys.ADH) but not that stimulated by exogenous arachidonic acid. The amplification occurred within minutes after addition of the stimulators. The effect of preincubation was time dependent. Preincubation of the cells with okadaic acid (0.6 microM) for longer than 30 min decreased this amplification. The results suggest that inhibition of protein-serine/threonine phosphate phosphatase(s) can both positively and negatively regulate deesterification of phospholipids although the negative regulation may reflect a toxic response. Microcystin LR and nodularin, inhibitors of protein-serine/threonine phosphate phosphatases 2A and 1 in vitro, did not amplify 6-keto-PGF1 alpha production by PAF when incubated with intact cells.     

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.     

琼氏不动杆菌FM 208850产果胶酶的发酵条件优化

目的:优化琼氏不动杆菌FM208850发酵生产果胶酶的培养基组成及发酵条件,以提高其产量。方法:在研究碳源、氮源、无机盐的种类及量的单因素实验基础上,选取香蕉皮、牛肉膏、NaCl和CaCl2做4因素3水平的正交试验。结果:FM208850产果胶酶的最适培养基组成为:香蕉皮2%,牛肉膏0.25%,NaCl 0.2%,CaCl20.3%;发酵条件为:初始pH7.0、32℃、2%接种量,培养12h。最优条件下酶活可达160.7U/mL。结论:琼氏不动杆菌FM208850作为新型果胶酶产生菌及罗布麻脱胶菌种,利用香蕉皮作为其发酵产果胶酶的碳源和诱导物是可行的,为其综合利用开辟了新思路。     

Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems

Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for clinical production of recombinant proteins. The transfection with polyethylenimine (PEI) was shown here to be scalable using both stirred-tank bioreactors of 3- and 150-l and novel agitated cultivation vessels (50 ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2 ml to 80 l. The maximum transient recombinant antibody yield was 22 mg/l, the highest ever reported for a multiliter transfection in CHO. The transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell line.