豆粕水解液为氮源发酵产脂肪酶的研究

研究了以豆粕水解液作为氮源,假丝酵母Candida sp．99—125发酵生产脂肪酶的过程。分析水解时间对于产酶的影响,对比豆粕水解前后作为氮源发酵时的产酶规律。在30L发酵罐中批次发酵酶活最高可达6000IU／mL,采用豆油反馈流加之后,发酵脂肪酶活力可达8500IU／mL。     

采用玉米秸秆水解糖和玉米浆发酵生产丁二酸

研究了以玉米秸秆水解糖为碳源,不同氮源条件下琥珀酸放线杆菌Actinobacillus succinogenesSF-9的丁二酸发酵产酸能力。结果表明玉米浆可以替代酵母膏作为丁二酸发酵的廉价氮源。厌氧摇瓶丁二酸发酵单因素试验,得到在初糖浓度50 g/L时,玉米浆的较佳用量为20 g/L。在5 L搅拌罐上,考察了不同初始玉米秸秆水解糖浓度对A.succinogenes SF-9发酵生产丁二酸的影响,结果显示高初始秸秆糖浓度对琥珀酸放线杆菌的生长有抑制作用。采用补料分批发酵,发酵60 h丁二酸的产量达到42.7g/L,丁二酸产率82.7%,生产强度0.81 g/(L·h)。丁二酸的产量和生产强度较分批发酵有明显提高。     

一株不需谷氨酸的产聚γ-谷氨酸菌株的筛选与鉴定

从豆制品中分离得到17株不依赖谷氨酸作为发酵底物的γ-PGA生产菌株,分别以氨盐和葡萄糖作为氮源和碳源的培养基进行好氧发酵,并测定发酵液中PGA的含量,对其中一株PGA高产菌株PGA-O-7进行了形态、生理生化和遗传学研究,结果表明PGA-O-7为枯草芽孢杆菌(Bacillus subtilis)。在以葡萄糖为碳源、硫酸铵为氮源的发酵培养基中,30℃振荡培养3d,PGA产量可达到2.8mg/mL。     

模拟青霉素分批补料发酵过程的细胞自动机模型

根据青霉素产生菌的生长机理和青霉素分批补料发酵过程的动力学特性,在Paull等建立的形态学结构动力学模型的基础上,建立了模拟青霉素分批补料发酵过程的细胞自动机模型。模型采用三维细胞自动机作为菌体生长空间,采用Moore型邻域作为细胞邻域,其演化规则根据青霉素分批补料发酵过程中菌体生长机理和简化动力学结构模型设计。模型中的每一个细胞既可代表单个产黄青霉菌体细胞,又可代表特定数量的这种菌体细胞,它具有不同的状态。对模型进行的仿真实验结果表明：模型不但能一致地复现形态学结构动力学模型所描述的青霉素分批补料发酵过程的演化特性,而且较形态学结构动力学模型更加直观地刻画了青霉素分批补料发酵过程的演化行为。最后,对所建模型在实际生产过程中的应用问题进行了分析,指出了需要进一步研究的问题。     

小麦和玉米原料生产柠檬酸工艺研究

利用小麦和玉米为生产原料生产柠檬酸。研究黑曲霉菌种对碳源的适应性、发酵过程中氮源比例,无机盐成分和添加比例,发酵最适温度的确定。优化柠檬酸发酵培养基条件,提高柠檬酸发酵代谢水平。     

泥炭固体发酵生产单细胞蛋白研究

对以泥炭为唯一碳源,固体发酵生产单细胞蛋白(SCP)进行了一系列的研究。选用酵母菌和黑曲霉进行混合发酵培养,考察影响单细胞蛋白生产的各个因素,如菌种接种量,培养基含水量,发酵时间,发酵温度,培养基外加氮源等。通过正交实验设计确定了优化的培养条件。即:菌种接种量为10%,培养基含水量为300%,28℃培养72 h,以蛋白胨为氮源。     

用石油酵母发酵产生多氧菌素

多氧菌素(Polyoxin)是低毒广谱的农用抗菌素。它的生产原料以粮食为主。为节约工业用粮,我们开展了以石油酵母代替粮食发酵生产多氧菌素的研究。本研究采用二步发酵法,先将正烷烃利用微生物生成酵母菌体和发酵液,然后用生成物作为碳氮源发酵产生多氧菌     

利用甜菜粕生产固体发酵饲料

用黑曲霉将甜菜废粕进行固体发酵,确定了适合的氮源、水份、pH、时间等发酵条件,生产出高蛋白的饲料。     

氮源对L-苏氨酸发酵的影响

以L-苏氨酸生产菌TRFC为供试菌株,研究了氮源对L-苏氨酸发酵的产量和糖酸转化率的影响。首先通过摇瓶实验确定发酵的最佳无机氮源和有机氮源分别为硫酸铵和酵母粉,进一步利用10L罐补料分批发酵确定硫酸铵和酵母粉的最佳用量,继续优化培养条件,采用发酵中后期流加硫酸铵和糖氨混合补料等措施,L-苏氨酸产量得到进一步的提高。在最优发酵条件下,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。     

抗生素和干扰素

941049胶原水解物作为产黄青霉合成青霉紊的复合氮源〔英]/Leonhartsberger,5.…了J.Bioteeh-nol一1993,30(8)一299一313〔译自DBA-1993,12(22),93一12663〕 利用胶原水解物(Gelita一Bio一Tec)作为氮源测定了通过产黄青霉PS生产苯氧基甲基青霉素和生物量形成的最适条件。利用一计划的实验方案 (命名为Graeco一Latin平方技术)在振荡瓶培养物中进行。初步研究结果表明,达30%的普通复合氮源如棉籽粉能被胶原水解物替代,且青霉素产量未降低(0 .015/小时)。在中试规模试验中,利用30升振荡式发酵罐,相对空气饱和度50%、瓶压0.3巴及4个Interm…     

Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

Evidence for a compartmentation of penicillin biosynthesis in a high- and a low-producing strain of Penicillium chrysogenum

Pulse-chase experiments using [U14C]valine were done with P2 and Q176, high- and low-penicillin-producing strains of Penicillium chrysogenum. The metabolic flux of this amino acid into protein and penicillin was measured, and compartmentation of penicillin biosynthesis was assessed. Strain P2 took up 14C-valine more slowly than strain Q176, but their rates of incorporation into protein were comparable. Incorporation of 14C-valine into penicillin occurred immediately with the high-producer P2, but exhibited a lag with Q176. After 14C-valine had been removed from the medium, the specific radioactivity of penicillin continued to increase in Q176 but started to decrease immediately in P2. The specific radioactivities of 14C-valine in protein and in penicillin were significantly different in both strains: Q176 had a higher specific radioactivity of valine in penicillin than P2, whereas P2 had a higher specific radioactivity of valine in protein than Q176. Moreover, the specific radioactivity of 14C-valine in penicillin was 20-fold higher in strain Q176 than in P2. These results indicate that penicillin and protein biosynthesis use different pools of cellular valine, and that exchange of valine between the two compartments is slow in the low-producer, but rapid in the high-producer strain. Hence these results indicate a further control point of penicillin biosynthesis in P. chrysogenum.     

Growth monitoring and control in complex medium: a case study employing fed-batch penicillin fermentation and computer-aided on-line mass balancing

To broaden the practicality of on-line growth monitoring and control, its application in fedbatch penicillin fermentation using high corn steep liquor (CSL) concentration (53 g/L) is demonstrated. By employing a calculation method that considers the vagaries of CSL consumption, overall and instantaneous carbon-balancing equations are successfully used to calculate, on-line, the cell concentration and instantaneous specific growth rate in the penicillin production phase. As a consequence, these equations, together with a feedback control strategy, enable the computer control of glucose feed and maintenance of the preselected production-phase growth rate with error less than 0.002 h(-1).     

不同类型玉米籽粒的营养品质及其与籽粒质地的关系

测定普通玉米、爆裂玉米、糯玉米和甜玉米4种类型玉米籽粒不同发育时期的直链淀粉、支链淀粉、总淀粉、可溶性糖和蛋白的含量,分析这些营养物质与角质率的关系。结果表明：灌浆期间4种类型玉米的直链淀粉、支链淀粉和总淀粉含量呈上升趋势,总蛋白含量呈下降趋势,可溶性糖含量变化规律不明显。爆裂玉米的直链淀粉含量始终最高（4．7％-23．1％）,甜玉米（1．4％-4．6％）和糯玉米（2．3％-4．9％）的始终较低;甜玉米的支链淀粉含量一直最低（15．7％-35．5％）,除授粉10d以外,糯玉米的支链淀粉含量一直最高（65．5％-69．8％）;甜玉米总淀粉含量始终最低（17．1％36.1％）、总蛋白含量（15．2％-26．9％）和授粉30d后的可溶性糖含量最高（14．2％-17．6％）。高蛋白含量可能是爆裂玉米和甜玉米角质率高的原因,糯玉米的角质率低可能与支链淀粉含量高和蛋白积累少有关。     

Characteristics of Nitrate Reductase-inactivating Proteins Obtained from Corn Roots and Rice Cell Cultures

Nitrate reductase (NR)-inactivating proteins from corn roots (Wf-9 × 38-11) and rice cell suspension cultures were tested against a partially purified NR obtained from corn leaves (W64A × W182E). The corn protein was purified 921-fold and the rice protein, 1,660-fold using standard purification procedures. Approximate molecular weight values were 75,000 for the corn protein, and 150,000 for the rice protein as determined by Sephadex G-100 gel filtration. The Sephadex-treated proteins were characterized by electrophoresis on polyacrylamide gels. With a running pH of 9.4 the corn protein remained at the origin whereas the rice protein migrated with an R_F value of 0.49. With a running pH of 4.0 the corn protein migrated with an R_F value of 0.25. With the corn protein the activities of NR inactivation and hydrolysis of azocasein were detected in the same protein band. The rice protein, however, had no associated protease activity. From sodium dodecyl sulfate gel electrophoresis, there was one major protein band with an estimated molecular weight of 66,000 in corn protein. In rice protein four bands were observed with estimated molecular weights of 73,000, 66,000, 62,500, and 58,500, respectively.     

Effect of Growth Rate on the Synthesis of Penicillin by Penicillium chrysogenum in Batch and Chemostat Cultures

The kinetics of penicillin production by Penicillium chrysogenum Wis 54-1255 in a glucose-limited chemostat and in batch cultures are reported. The specific production rate of penicillin, q_pen (units per milligram of dry weight per hour) was independent of specific growth rate over the range 0.014 to 0.086 hr^-1. Growth was stopped by restricting the glucose supply to the “maintenance ration,” that is, the glucose requirement of the organism at zero growth rate with all other nutrients in excess. Under such conditions, the organism dry weight remained constant, but the q_pen fell approximately linearly to zero at a rate inversely related to the previous growth rate. Glucose supplied in excess of the maintenance ration inhibited the decay of q_pen. At a critical growth rate between 0.009 and 0.014 hr^-1, the decay was completely inhibited. Quantitative expressions for the q_pen of growing and nongrowing cultures were derived and used to predict the steady-state concentrations of penicillin accumulating in one- and two-stage continuous processes. A rational explanation of the kinetics of penicillin accumulation in batch cultures is given, relating the rate of penicillin synthesis to growth rate. It is concluded that an important role of corn steep liquor (CSL), a heterogeneous carbon and nitrogen source commonly used in penicillin production media, is the provision of substrates which allow a high concentration of mold to be reached before the growth rate falls below the critical value. CSL had no significant effect on q_pen.     

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.     

Requirement of de novo synthesis of the OdhI protein in penicillin-induced glutamate production by Corynebacterium glutamicum

We found that penicillin-induced glutamate production by Corynebacterium glutamicum is inhibited when a de novo protein synthesis inhibitor, chloramphenicol, is added simultaneously with penicillin. When chloramphenicol was added 4 h after penicillin addition, glutamate production was essentially unaffected. ³H-Leucine incorporation experiments revealed that protein synthesis continued for 1 h after penicillin addition and then gradually decreased. These results suggest that de novo protein synthesis within 4 h of penicillin treatment is required for the induction of glutamate production. To identify the protein(s) necessary for penicillin-induced glutamate production, proteome analysis of penicillin-treated C. glutamicum cells was performed with two-dimensional gel electrophoresis. Of more than 500 proteins detected, the amount of 13 proteins, including OdhI (an inhibitory protein for 2-oxoglutarate dehydrogenase complex), significantly increased upon penicillin treatment. Artificial overexpression of the odhI gene resulted in the decreased specific activity of the 2-oxoglutarate dehydrogenase complex and increased glutamate production without any triggers. These results suggest that the de novo synthesis of OdhI is the necessary factor for penicillin-induced glutamate overproduction by C. glutamicum. Moreover, continuous glutamate production was achieved by overexpression of odhI without any triggers. Thus, the odhI-overexpressing strain of C. glutamicum can be useful for efficient glutamate production.     

Effects of induction starting time and Ca2+ on expression of active penicillin G acylase in Escherichia coli

Formation of inclusion bodies is an important obstacle to the production of active recombinant protein in Escherichia coli. Thus, soluble expression of penicillin G acylase from Kluyvera citrophila was investigated in BL21(DE3). In this study, the yield of active enzyme was significantly enhanced by the composition of the medium and induction opportunity. When 0.5 mmol/L IPTG was added to complex medium at 15 h after incubation, the volumetric and specific activities of penicillin G acylase both achieved the highest values, respectively. However, aggravation of intracellular proteolysis and decline of enzyme expression were also observed if induction occurred too much later. Ca2+ ion was another critical factor in cell growth and protein expression. When 24 mmol/L Ca2+ ion was adding to the medium at the beginning of fermentation, a greater than 2-fold increase in cell density and a 7-fold increase in volumetric activity of penicillin G acylase were reached. Nevertheless, no significant benefit for recombination protein expression was found when excess Ca2+ was added after induction time. This study demonstrates that the induction starting time and Ca2+ ion are two critical factors for the expression of active penicillin G acylase.