微生物发酵生产长链二元酸的代谢调控

获得优良生产菌株之后 ,对发酵过程进行代谢调控是重要的。在条件试验时 ,除考虑培养基成分之外 ,还需要考虑烷烃浓度、培养温度、ｐＨ、种龄、通气量等。经过诱变培育出来的高产菌株 ,其ω 氧化能力比出发菌株增强了 ,而 β氧化能力减弱了 ,但β 氧化没有完全被阻断 ,所以在发酵过程中 ,必须进一步减低β 氧化酶活力 ,增强ω 氧化酶活力。由于微生物氧化正烷烃生产二元酸是胞内酶的作用 ,这就存在一个原料正烷烃如何快速转运入细胞内 ,产生的二元酸如何快速转运出细胞外的原料和产物转运速度问题 ,解决以上三个问题 ,才能在发酵过程中获得…     

利用石油发酵柠檬酸的菌体生产碱性蛋白酶

用正构烷烃发酵柠橡酸的培养液中,约有2—3%的酵母菌体产生。这些菌体一般当作废渣处理。为变废为宝,我们初步研究了用正构烷烃发酵柠檬酸后的酵母菌体发酵生产碱性蛋白酶。     

热带假丝酵母代谢烷烃过程中的β-氧化和代谢调控

热带假丝酵母 (Ｃａｎｄｉｄａｔｒｏｐｉｃａｌｉｓ)能利用烷烃作唯一碳源和能源。当以烷烃或脂肪酸为碳源时 ,在细胞内可形成大量的过氧化物酶体 (ｐｅｒｏｘｉｓｏｍｅ) ,同时诱导生成脂肪酸 β 氧化酶系 ,当以葡萄糖为碳源时 ,则极少有过氧化物酶体形成[1] ,一些Ｃ .ｔｒｏｐｉｃａｌｉｓ能氧化烷烃生成长链二元酸 (ｌｏｎｇｃｈａｉｎｄｉｃａｒｂｏｘｙｌｉｃａｃｉｄ ,ＤＣＡ)。由于这些特征 ,人们从酶学、分子生物学和实际应用等方面对这种酵母进行了深入研究 ,并阐述了Ｃ .ｔｒｏｐｉｃａｌｉｓ代谢烷烃的途径、脂肪酸β 氧化酶系…     

不同培养方法对木槿原生质体培养的影响

本文研究了不同培养方法对木槿 (Ｈｉｂｉｓｃｕｓｓｙｒ ｉａｃｕｓ)原生质体培养的影响。种子采自本校校园 ,原生质体分离按朱启忠[1] 和Ｎｏｍｕｒａ[2 ] 的方法。培养方法有 :(1)液体浅层 (培养皿中加入 3ｍｌ培养液 ) ;(2 )固液双层 (固体层用 0 .4 %的琼脂及 0 .3%琼脂糖     

长链二元酸发酵的特点及其控制

微生物发酵生产长链二元酸是以正烷烃为原料 ,它与历来以糖质为原料的发酵不同 ,有其自身的特点 :(1 )四相体系的发酵 :以糖质为原料进行微生物发酵时 ,只有菌体、空气和水三相体系 ,糖质原料易溶于水 ;而烃类发酵却是菌体、空气、烃类(油 )和水四相体系 ,石油烃类形成小油滴 ,微生物才能接触到烷烃 ,利用和氧化烷烃 ,产生二元酸。 (2 )需氧量和放热量大 :微生物发酵正烷烃生成二元酸时 ,增加了四个氧原子 ,因此需氧量大。有人经过试验和计算 ,认为烷烃发酵需氧量和放热量都是糖发酵时的 2～ 3倍。 (3)长链二元酸在水中溶解度小 :长链二元酸…     

N~ 离子注入热带假丝酵母对长链二元酸产量的影响

用热带假丝酵母（Ｃａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓ）ＳＣＢ４１２作为出发菌株,经能量５０ＫｅＶ、剂量１× １０~１１～５ ×１０~１５ ｉｏｎｓ／ｃｍ~２的Ｎ~＋离子注入诱变处理,以产生可遗传的诱变。 Ｎ~＋离子注入后,存活率与剂量呈指数衰减关系：ｌｏｇ（存活率％）＝ ８．２３－ ０．６０４ × ｌｏｇ（剂量）,在培养过程中可观察到酵母菌菌落和细胞形态均发生了变化。经筛选,获得了一株能够利用正十二烷烃发酵产生长链二元酸的高产菌热带假丝酵母ＳＣＢ６０９。在初始正十二烷烃浓度为１５％（ｖ／ｖ）下产酸量由４３．５ｇ／Ｌ上升到７３．２ｇ／Ｌ。比较两株菌发酵生长特性的差异,产酸过程有一定的变化。     

小鼠肝脏树突状细胞前体分离培养及影响其成熟因素的初步探讨

本次首次建立了从小鼠肝脏分离、培养、扩增肝树突状细胞（ｈｅｐａｔｉｃｄｅｎｄｒｉｔｉｃｃｅｌ,ＨＤＣ）前体的方法。采用经门静脉插管胶原酶二步循环灌流法及Ｐｅｒｃｏｌ不连续密度梯度离心法分离非肝细胞,得到含ＨＤＣ前体组分,将其分三组培养：第一组加入重组小鼠粒细胞／巨噬细胞集落刺激因子（ＭｒＧＭＣＳＦ）,待培养９天后,将细胞转移至鼠尾胶原上继续培养。第二组加入ＭｒＧＭＣＳＦ培养,但细胞不转移。第三组为对照组,仅加完全培养液。结果显示：第一、二组细胞于培养４天可见有许多细胞集落形成,第一组细胞转移至鼠尾胶原上培养１天后,细胞集落减少,而培养液中ＤＣ密度明显增加,光镜和扫描、透射电镜观察证明培养ＨＤＣ纯度达９５％,第二组则无上述细胞释放现象。实验结果提示,ＨＤＣ前体在体外受细胞因子刺激能大量扩增,但必须在胶原存在下才能分化成熟     

重组大肠埃希菌发酵工艺的影响因素及策略

重组大肠埃希菌的高密度发酵是现代发酵工程研究的一个热点 ,也是基因工程产品大规模生产的重要技术。采用高密度培养技术 (Ｈｉｇｈｃｅｌｌ ｄｅｎｓｉｔｙｃｕｌｔｒｅ,ＨＣＤＣ) ,也就是高密度发酵技术 ,提高菌体的发酵密度 ,最终提高产物的比生产率 ,不仅可减少培养体积、优化下游分离提取 ,还可以缩短生产周期、降低生产成本 ,从而极大地提高在市场上的竞争力。这一目的的实现 ,除了重组菌本身的表达性质外 ,还必须赋予重组菌生长和产物表达的最适环境条件 ,包括适宜的培养基组成、宿主菌的选择、补料的调控、代谢副产物的限制、比生…     

电子嗅在线反馈控制毕赤酵母糖化酶发酵过程中甲醇浓度新方法的应用

探索了电子嗅传感仪直接通过发酵尾气进行发酵液中甲醇浓度在线检测的方法,建立了毕赤酵母表达糖化酶过程中甲醇浓度的自动化反馈补料控制模型,可准确实现发酵过程中甲醇浓度的精确控制;研究表明,当利用电子嗅将培养液中甲醇浓度稳定控制在(890±35)ppm水平下,发酵诱导培养到128h时目的蛋白糖化酶酶活达到了8 153U/ml,与甲醇浓度控制在(350±26)ppm时的发酵水平相比提升了48.8%。该方法具有无需前处理、与发酵液非接触、快速和准确性的优点,为提升工程酵母在工业发酵培养过程工艺的优化控制具有重要的指导作用。     

钙离子和水杨酸诱导灵芝多糖和三萜的合成

灵芝多糖和三萜是灵芝主要的药理活性物质。如何提高液体发酵中灵芝多糖和三萜的含量是目前研究的热点。本研究以J‐7/AL‐2菌株为例,在灵芝的液体发酵中加入10mmol/L钙离子诱导,多糖含量比对照提高34%,三萜含量提高64%。在发酵液中加入150μmol/L水杨酸(SA)诱导,多糖含量提高57.54%,三萜含量提高37.19%。在灵芝的液体发酵第12天加入10mmol/L钙离子和150μmol/L水杨酸,三萜含量提高46.9%。在不同诱导条件下,三萜的高效液相图谱(HPLC)显示钙离子与对照的差异性比较大。灵芝三萜关键酶基因的表达也有所不同。水杨酸提高法尼基焦磷酸合酶(FPS)、鲨烯合酶(SQS)和羊毛兹醇合酶(LS)基因的表达量,而钙离子和钙离子与水杨酸共同处理提高3‐羟基‐3‐甲基戊二酰辅酶A合酶(HMGS)、3‐羟基‐3‐甲基戊二酰辅酶A转录水平还原酶(HMGR)、焦磷酸甲羟戊酸脱羧酶(MVD)、法尼基焦磷酸合酶(FPS)、鲨烯合酶(SQS)和羊毛兹醇合酶(LS)6个三萜关键酶基因的表达量。在3种处理下,都是SQS的基因表达量提高最多,推测SQS基因是灵芝三萜合成过程中重要的基因。     

热带假丝酵母转化烷烃过程中P450酶活的研究

a-、ω-长链二元酸(α-、ω-Long Chin Dicarboxylic Acid,DCA)是一种重要的化工原料,是合成工程塑料、香料、耐寒性增塑剂、涂料和液晶等物质的主要原料.目前,人们主要通过热带假丝酵母(Candidatropicalis)代谢烷烃来生产从DCA11到DCA18等不同碳链长的二元酸[1,2].多年来在各种微生物,尤其是假丝酵母的烷烃氧化途径方面有大量的研究[3,4].在假丝酵母转化烷烃生成长链二元酸的代谢过程中[5-7],烷烃被吸引进入细胞后,首先经过细胞色素P450酶(Cy-tochrome P450)氧化生成a-一元醇,再进一步被氧化生成a-一元酸,引过程称为a-氧化.     

热带假丝酵母多倍体变种的细胞色素P450和尿素在烷烃代谢中的作用

建立了可行的细胞色素 P450测定方法。考察了以烷烃为单一碳源的酵母细胞色素P450的一氧化碳差示光谱,峰值约为455nm。观察了烷烃培养的酵母细胞色素 P450在生长期中的消长。比较了十四醇、十四醇添加苯巴比妥、以及正十四烷等三种不同培养条件下酵母细胞色素P450的含量和发酵产物的成份。结果表明,细胞色素 P450为烷烃转化成二元酸所必需。烷烃为单一碳源培养酵母时,培养基中过量尿素(0.2%以上)促进烷烃利用和酵母生长,降低细胞色素 P450生成和二元酸的积累。根据上述实验结果和本研究室以前报道,提出了烷烃代谢调控模式。     

Effects and mechanisms of H(2)O(2) on production of dicarboxylic acid.

The system of producing long chain dicarboxylic acid (DCA) by Candida tropicalis is an aerobic and viscous fermentation system. A method to overcome the gas-liquid transport resistance and to increase oxygen supply is by adding hydrogen peroxide (H(2)O(2)) to the fermentation system. Here we report that the H(2)O(2) not only can enhance the oxygen supply but also change the metabolism by inducing cytochrome P450, the key enzyme of a, o-oxidation. When C. tropicalis was cultivated in a 3-L bioreactor using the combination of aeration and H(2)O(2) feeding, DCA production rates increased by about 10% after a short period of decrease at the beginning. Furthermore, the experiments showed that the maximum activities of P450 could be induced at 2 mM H(2)O(2), and the inducible mechanisms are also discussed. Moreover, we suggest that alkane might be oxidized through the "peroxide shunt pathway" when H(2)O(2) is present. By adding H(2)O(2), the DCA yield in a 22-L bioreactor could increase by 25.3% and reach 153.9 g/L.     

Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

Eight experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher (P less than 0.01) cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose (Solka Floc), or 6.0% arenaceous flour. NADPH cytochrome c reductase activity was not affected by treatment. Chicks fed 12.0% wood molasses had a higher (P less than 0.06) cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced (P less than 0.05) cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic (bacitracin:neomycin sulfate, 2:1), had a higher (P less than 0.01) cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower (P less than 0.01) intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower (P less than 0.05) rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.     

Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications

The alkane-assimilating yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates such as n-alkanes, fatty acids, fats and oils for which it has specific metabolic pathways. An overview of the oxidative degradation pathways for alkanes and triglycerides in Y. lipolytica is given, with new insights arising from the recent genome sequencing of this yeast. This includes the interaction of hydrophobic substrates with yeast cells, their uptake and transport, the primary alkane oxidation to the corresponding fatty alcohols and then by different enzymes to fatty acids, and the subsequent degradation in peroxisomal beta-oxidation or storage into lipid bodies. Several enzymes involved in hydrophobic substrate utilisation belong to multigene families, such as lipases/esterases (LIP genes), cytochromes P450 (ALK genes) and peroxisomal acyl-CoA oxidases (POX genes). Examples are presented demonstrating that wild-type and genetically engineered strains of Y. lipolytica can be used for alkane and fatty-acid bioconversion, such as aroma production, for production of SCP and SCO, for citric acid production, in bioremediation, in fine chemistry, for steroid biotransformation, and in food industry. These examples demonstrate distinct advantages of Y. lipolytica for their use in bioconversion reactions of biotechnologically interesting hydrophobic substrates.     

Cytochromes P450 and drug resistance

Cytochromes P450 are the key enzymes for activating and inactivating many drugs, in particular anticancer drugs. Therefore, individual expression levels of cytochromes P450 may play a crucial role in drug safety and drug efficacy. Overexpression of cytochrome P450 may yield rapid turnover and elimination of drugs before the target site was reached and any pharmacological effect is observed. Therefore, it may be vital to know the individual cytochrome P450 status in order to select the appropriate drug before drug resistance occurs. Expression levels and activity of cytochromes P450 depend on many different factors. These factors include tissue and organ specific expression, sex- and age-dependent expression, genetic differences yielding polymorphic forms, competitive inhibition or induction of cytochromes P450 due to multiple drug interaction, nutrition and diet. Genetically engineered test cells defined for cytochromes P450 are available for studying drugs for metabolic activation and for identifying the metabolically competent cytochrome P450 isoform.     

Age- and sex-related expression of cytochromes p450f and P450g in rat liver

We have previously shown that rat hepatic cytochromes P450f, P450g, P450h, and P450i possess a high degree of immunochemical and, presumably, structural relatedness. Polyclonal antibodies directed against cytochromes P450f and P450g were made monospecific by immunoabsorption against the cross-reactive proteins. The specificity of the immunoabsorbed antibodies was established by using Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays (ELISA), and immunoblots. Since factors regulating the expression of cytochromes P450f and P450g are unknown, a competitive ELISA employing the monospecific antibodies was developed to quantitate each of these isozymes in hepatic microsomes from control and treated rats. The results obtained showed that expression of cytochrome P450f is developmentally regulated in both male and female rat liver. Cytochrome P450f levels rise from less than 1% in young animals to approximately 7 and 14% of total cytochrome P450 in adult male and female rats, respectively. Cytochrome P450g is sex-specific since it is expressed only in male rat liver where it also is developmentally regulated. Levels of cytochrome P450g rise from less than 1% in 3-week-old male rats to an average value of 17% of total cytochrome P450 in 6-week-old adult animals. However, there appear to be at least two subpopulations of adult male Long Evans rats, one of which expresses low levels (less than 1%) of cytochrome P450g and the other high levels (greater than or equal to 10%). This expression appears to be independent of serum testosterone levels. Treatment of immature and adult male rats with 20 xenobiotics that are known inducers of certain cytochrome P450 isozymes revealed that cytochromes P450f and P450g are relatively refractory to induction, although Kepone appears to be a weak inducer of cytochrome P450f.     

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.     

Laboratory evolution of a soluble,self-sufficient,highly active alkane hydroxylase

We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.     

A proteolytically sensitive region common to several rat liver cytochromes P450: effect of cleavage on substrate binding.

Limited proteolysis of rat liver microsomes was used to probe the topography and structure of cytochrome P450 bound to the endoplasmic reticulum. Three cytochromes P450 from two families were examined. Monoclonal antibodies to cytochrome P450 forms 1A1, 2B1, and 2E1 were used to immunopurify these proteolyzed cytochromes P450 from microsomes from rats treated with 3-methylcholanthrene, phenobarbital, and acetone, respectively. Electrophoretic and immunoblot analysis of tryptic fragments revealed a highly sensitive cleavage site in all three cytochromes P450. N-Terminal sequencing was performed on the fragments after transfer onto poly(vinylidene difluoride) membranes and showed that this preferential cleavage site is at amino acid position 298 of P450 1A1, position 277 of P450 2B1, and position 278 of P450 2E1. Multiple sequence alignment revealed that these positions are at the amino terminal of a highly conserved region of these cytochromes P450. The important functional role implied by primary sequence conservation along with the proteolytic sensitivity at its amino terminal suggests that this region is a protein domain. Comparison with the known structure of the bacterial cytochrome P450cam predicts that this proteolytically sensitive site is within an interhelical turn region connected to the distal helix that partially encompasses the heme-containing active site. Substrate binding to the cleaved cytochromes P450 was examined in order to determine whether the newly added conformational freedom near the cleavage site functionally altered these cytochromes P450. Cleavage of P450 2B1 abolished benzphetamine binding, which indicates that the cleavage site contains an important structural determinant for binding this substrate. However, cleavage did not affect benzo[a]pyrene binding to P450 1A1.