短杆菌属产胆固醇氧化酶的发酵条件优化

短杆菌属(Brevibacterium sp.)是胆固醇氧化酶高产菌,对其进行底物诱导及发酵条件的优化,其最适培养基为蔗糖0.3%,酵母膏0.2%,蛋白胨0.3%,牛肉膏0.3%,K2HPO4 0.1%,MgSO4 0.05%,pH6.8。最适培养条件为接种量5%,24℃培养20h,通气量为50mL培养基/250mL三角瓶,200r/min。结果表明,胆固醇氧化酶的酶活力可达到2439U/L,比未优化前195U/L提高了12倍。在pH6.5和温度54℃条件下测得酶米氏常数Km值为7.1×10-5 mol/L。     

利用胆固醇氧化酶转化胆固醇制备胆甾-4-烯-3-酮

用酶法催化氧化胆固醇制备胆甾-4-烯-3-酮相对于化学法合成具有反应简单、成本较低等优点.作者探讨了在正辛烷作为有机相的两相反应体系中,以胆固醇氧化酶催化氧化胆固醇制备胆甾-4-烯-3-酮的方法.在胆固醇质量浓度为40g/L,反应时间40min、反应温度40℃、磷酸缓冲液-正辛烷体积比32、通氧40L/h及搅拌转速300r/min下,胆固醇转化率达到92%,经薄板层析及紫外扫描,发现转化产物为单一的胆甾-4-烯-3-酮.     

胆固醇氧化酶高产菌株的复合诱变选育

为进一步提高产酶水平,本实验采用超声波联合亚硝基胍的复合诱变方法处理胆固醇氧化酶产生菌(甾短杆菌),通过1mg/mL亚硝基胍和超声(200W,50kHz,35min)同时处理细胞悬液,获得一株红色突变株,其产酶活力从0.5U/mL提高到1.21U/mL,酶活提高了140%。证明该联合诱变手段对甾短杆菌产胆固醇氧化酶具有良好的诱变效果。     

类芽孢杆菌产胆固醇氧化酶的最适产酶条件研究

对诱变过的产胆固醇氧化酶的类芽孢杆菌(Paenibacillus sp.X534)在最适培养基进行了发酵条件的优化,确定其最适发酵条件为起始pH值为8,接种量8%,装液量16%,摇瓶转速180r/min,培养温度34℃,发酵时间50h。优化之后胆固醇氧化酶活达到约580U/L,比优化前提高了约1.5倍。     

产胆固醇氧化酶类芽孢杆菌的复合诱变育种

以已筛选出的产胆固醇氧化酶的类芽胞杆Paenibacillus sp.X5作为出发菌株,经过紫外线和60Co诱变处理,最后得到一株突变Paenibacillus sp X534,最终酶活达300U/L,其酶活为出发菌株的2倍。     

Pseudomonadaceae sp.胆固醇氧化酶产酶条件优化及其初步纯化

Pseudomonadaceae sp.可以发酵生产胆固醇氧化酶。本文对Pseudomonadaceae sp.进行产酶条件的优化及产物的初步分离。确定其最适培养基及培养条件,优化后胆固醇氧化酶的酶活力可达1691.72U/L,比优化前839.35U/L有较大提高。经过(NH4)2SO4沉淀及Sephadex G-75凝胶过滤层析初步纯化后酶比活力达24.95U/mg,纯化倍数为9.18。     

胆固醇诱导甾短杆菌发酵产胆固醇氧化酶能力的研究

利用胆固醇诱导甾短杆菌发酵来提高胆固醇氧化酶产量;通过对单因素的研究,选取影响比较显著的3个因素:胆固醇质量浓度、吐温-80添加量、超声波功率,并利用Box-Behnken试验设计和响应曲面分析法进一步研究得到结果:甾短杆菌产酶的最佳条件是当超声时间为20min时,胆固醇质量浓度为3.30g/L、吐温-80添加量为3.72mL/L、超声波功率为80W,在此条件下测得的胆固醇氧化酶产量为731.967U/L。比未使用胆固醇诱导产酶时,酶产量提高了1.17倍。     

全自动发酵罐小试生产胆固醇氧化酶发酵条件的研究

目的:为了将胆固醇氧化酶应用于低胆固醇系列食品开发和胆固醇测定中,研究优化了菌株Rhodococcus SP.R14-2发酵生产胆固醇氧化酶(COX)的工艺条件.方法:通过测定比较COX酶活力,利用全自动发酵罐小试生产COX,研究发酵温度、pH、乳化剂、通风量和搅拌速率等因素对COX产量的影响.结果:Rhodococcus SP.R14-2的生物生长量和产酶是部分偶联的,胞外COX酶的合成高峰比生物量高峰滞后约12h.发酵过程中分段控制搅拌转速和通气量分别为:发酵前10h,200r/min和4L/min;10～50h,300r/min和5L/min:50h后,250r/min和4.5L/min,COX达到1.58u/mL的最高量.结论:发酵温度、培养基pH、通气量及搅拌速度等因素影响其生物量和COX酶合成,采用分段式控制,有利于COX产量的提高.     

类芽孢杆菌产胆固醇氧化酶的发酵条件优化

对诱变过的产胆固醇氧化酶的类芽孢杆菌(Paenibacillus sp X534)进行了发酵条件的优化,确定其最适培养基为:蔗糖2%,大豆粉2%,MgSO4.7H2O 0.1%,MnSO40.02%,土豆汁30%,K2HPO4.3H2O 0.4%,Tween-80 0.1%。确定最适发酵条件为:起始pH值为8,接种量8%,装液量16%,摇瓶转速180r/min,培养温度34℃,发酵时间50h。优化之后胆固醇氧化酶活达到400U/L左右,比优化前提高了约2倍。     

胆固醇氧化酶的发酵条件及酶学性质研究

Brevibacterium sp．可以发酵生产胆固醇氧化酶,实验中对该菌种进行了底物诱导及发酵条件的优化, 确定其最适培养基为(%)：蔗糖0．3,酵母膏0．2,蛋白胨0．3,牛内膏0．3,K_2PO_4 0．1,MgSO_4 0．05,pH6．8。最适培养条件为：接种量5%,24℃培养20h,通气量为50mL 培养基/250mL 三角瓶,200r/min。在最适培养基及最适条件下,胆固醇氧化酶的酶活力可达到24．01U/mg,比未优化前1．71U/mg 提高了14倍。该酶具有较强的酸碱稳定性和热稳定性,最适 pH 为6．5,最适温度为54℃。在最适 pH 和温度条件下测得该酶 km 值为7．1 ×10~(-5)mol/L。     

从乳化体系中分离纯化短杆菌产胆固醇氧化酶

添加剂W对酶的分离纯化造成很大困难,用异丙醇沉淀发酵液的同时加入(NH4)2SO4能够有效地破除乳化体系,去除添加剂W,酶收率达到90%.在pH值为8.0的条件下用SepharoseDEAEFastFlow离子交换透析酶液,比酶活从3.21U/mg提高到29.21U/mg,酶收率达70.1%.     

固定化Amycolatopsis sp.ST 2710转化洛伐他汀的研究

研究用海藻酸钠固定化ST2710的技术,确定了较好的制备工艺,并用该固定化细胞对转化洛伐他汀进行了研究。结果表明,在海藻酸钠浓度2%,CaCl2浓度为2%,固化时间为1 h,包埋量为2.5 mL菌悬液/100 mL凝胶的条件下,固定化细胞具有较好的机械强度和较高的转化率。     

Brevibacterium sp.DGCDC-82发酵罐中生产胆固醇氧化酶

用Brevibacterium sp.DGCDC-82在7L发酵罐中分批培养.分别对添加剂吐温-80、培养温度、培养基的pH、通风量和搅拌速度在胆固醇氧化酶的生产中的影响进行了研究.结果显示以上条件均对产酶有影响.在不同的发酵阶段改变发酵操作条件,发酵20 h最高酶活可达1203U/L,生产强度可达60 U/(L·h).既有效地提高了胆固醇氧化酶的产量,又防止了发酵过程中的泡沫外溢.     

拟无枝酸菌ST2710转化洛伐他汀为无锡他汀的发酵工艺的初步研究

无锡他汀是胆固醇合成途径中限速酶羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,由洛伐他汀经拟无枝酸菌(Amycolatopsis sp.ST 2710)转化产生。文中在摇瓶水平考察了底物浓度、溶氧和剪切力等因素对洛伐他汀(底物)转化为无锡他汀(产物)的影响,并在5 L发酵罐上进行了分批发酵及材料分批发酵研究。摇瓶结果表明,菌株对溶氧要求较高,对剪切力很敏感,底物适宜添加浓度为1 g/L。在5 L发酵罐间歇发酵过程中。在控制溶氧不低于35%.搅拌转速为300r/min,产物产量达到最高,为0.77 g/L左右,发酵周期为80 h,较摇瓶发酵缩短16 h。采用补料发酵工艺后,有效减缓底物抑制,产物产量达到1.83 g/L。是分批发酵的2.4倍。     

根霉转化坎利酮的培养基优化

以坎利酮转化率为指标,优化了根霉转化坎利酮的营养需求。通过摇瓶培养和高效液相色谱分析等技术研究了不同碳源、氮源、无机盐以及一些重要因素的浓度对转化率的影响。通过L9(34)正交试验对培养基组合进行优化,优化后的培养条件为:葡萄糖30 g/L,玉米浆25 g/L,酵母膏12 g/L,KH2PO41.5 g/L。在优化后的条件下,坎利酮的转化率为87.68%,较优化前提高9.06%。     

Accumulation of ectoine in the halotolerant Brevibacterium sp. JCM 6894

Resting cells of Fusarium moniliforme strain MS31 produced (R)-1-phenylpropanol from propylbenzene. The components of the medium and the reaction conditions were adjusted to increase the specific activity of the hydroxylating enzyme involved. Glucose and sodium nitrate were selected as carbon and nitrogen sources, respectively. The substrate, propylbenzene, inhibited fungal growth and the activity of the enzyme. Acetoin added to the medium increased both growth and activity of the enzyme, and hydroxylation of propylbenzene increased by 1.4-fold. Maximum bioconversion of propylbenzene by resting cells of the fungus was at 25-30 degrees C and pH 7.0 with cells at concentration of 40 mg (dry) per milliliter of reaction mixture. Conversion was accelerated as soon as propylbenzene was added; slowing 2 h later. In the end, F. moniliforme strain MS31 produced (R)-1-phenylpropanol with an enantiomeric excess of 98% at the concentration of 16 mM (2.2 mg.ml(-1)).     

响应面分析应用于短杆菌产酶工艺的优化

本文研究了用响应面分析法(RSM)优化短杆菌产胆固醇氧化酶的工艺，发现胆固醇添加量及超声处理时间是影响短杆菌产酶最重要的因素。在模拟优化的条件下：胆固醇为4．076g／L，吐温-80为0．2932％(v／v)，照射时间为22．361min，COD最大产量1．483U／mL。     

Purification and characterization of extracellular proteinase produced by Brevibacterium linens ATCC 9172

The micro-organism Brevibacterium linens ATCC 9172 produced five extracellular proteinases, as shown by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) with copolymerized gelatine. One of these was purified to homogeneity by ion-exchange chromatography and native preparative PAGE. The optimum pH and temperature for the proteinase were 8.0 and 50°C, respectively. The enzyme remained stable over a pH range from 6 to 10. The molecular weight estimated by SDS–PAGE was 56 kDa. Serine proteinase inhibitors 3,4-dichloroisocoumarin (3,4-DCI) and phenylmethylsulphonylfluoride (PMSF) inhibited, while Mg²⁺ and Ca²⁺ ions activated the proteinase.     

Enhanced glutamic acid production of Brevibacterium sp. with temperature shift-up cultivation

For cells of Brevibacterium sp. under conditions of biotin limitation, the efflux of metabolites through the cell membrane was affected by temperature. After the temperature shift-up from 30 degrees C to 38 degrees C, both the specific production rate of glutamic acid and the excretion of intracellular materials, such as glucose-6-phosphate and nucleotides, were increased simultaneously. For the production of glutamic acid, not only the yield but also the specific production rate was increased by the temperature shift-up.     

Uptake and utilization of ectoine by halotolerant Brevibacterium sp. JCM 6894 subjected to osmotic downshock

The concentration changes of the cyclic amino acid ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyridine carboxylic acid) in Brevibacterium sp. JCM 6894 cells subjected to an osmotic downshock were investigated. When the cells grown in the presence of 2 M NaCl were suspended in deionized water, they immediately released about 60% of the ectoine synthesized intracellularly. During the subsequent incubation, we observed that both the extra- and intracellular concentrations of ectoine were reduced almost linearly with the incubation time. When ectoine was provided externally to the downshocked cells, a similar reduction in both intra- and extracellular ectoine concentrations was recognized. In addition, we observed an increase in ectoine accumulation at about 10 h of incubation, which indicates that ectoine was taken up by such downshocked cells in the absence of external Na+. Furthermore, the downshocked cells showed higher levels of survival, respiration, and growth in the presence of ectoine than in its absence. The ability to take ectoine up was induced in the cells grown in the presence of >0.25 M NaCl for >12 h. Thus, we conclude that even under the lower osmotic condition ectoine might be taken up and subsequently utilized by strain JCM 6894 subjected to the osmotic downshock, indicating that the uptake of ectoine by such cells occurred for the survival and growth of the cell itself rather than for cellular osmoregulation.