产胞外青霉素酰化酶菌株的选育

以短小杆菌（B．pumilus）B－97为出发菌株,经过连续两次紫外线诱变处理,分离得一突变株B－U－29。其酶活力为4．56U／ml,较出发菌株酶活力提高113．3％。对B－U－29菌株进行连续两次亚硝酸处理,分离得一正变稳定株B—H－29,酶活力为4．93u／ml,较出发菌株酶活力提高了20％。     

巨大芽孢杆菌BP931胞外青霉素G酰化酶的产生条件

研究了巨大芽孢杆菌(Bacillus megaterium)BP 931胞外青霉素G酰化酶的产生条件。菌在由葡萄糖0.7％,氮源1号0.5％,酵母膏1.0％和苯乙酸0.8％组成的液体培养基(灭菌前pH9.0,灭菌后pH8.0)中,28℃振荡培养44h。以6-硝基-3-苯乙酰胺基苯甲酸为底物,培养滤液酶活力为9.0IU/ml。诱导物苯乙酸于培养6h后加入,酶活力可以提高到11.0IU/ml。Ca²⁺、Al³⁺、Sn³⁺、Mn²⁺和Fe²⁺离子降低酶的形成;Cu⁺和C0²⁺离子显著抑制菌生长,降低酶的形成;Zn²⁺,Cd²⁺和Hg²⁺离子完全抑制菌生长和酶形成。     

常压室温等离子体-紫外复合诱变选育β-法尼烯高产菌株

【背景】大肠杆菌(Escherichia coli)由于生长性能优良、遗传背景清楚、遗传操作手段成熟,是合成β-法尼烯的合适生产菌,但其合成β-法尼烯的产量目前仍不能满足工业化生产的需求。【目的】通过诱变筛选技术选育β-法尼烯高产突变株。【方法】采用常压室温等离子体(atmosphericand room temperature plasma,ARTP)诱变技术和紫外线照射对出发菌株大肠杆菌EC-16进行复合诱变,并以异戊烯焦磷酸耐受性为选择压力进行平板初筛,之后进行摇瓶复筛,最后进行发酵罐验证。通过连续多代培养筛选到的高产突变菌株,观察其遗传稳定性。【结果】经复合诱变选育筛选出一株β-法尼烯高产突变株E.coliHVK-9,其产量高达22.1g/L,相比出发菌株提高了168.74%。【结论】采用ARTP-紫外复合诱变,再结合异戊烯焦磷酸抗性筛选的集成方法,使得诱变菌株的正突变率大大提高,可以有效地提高诱变菌株的β-法尼烯产量。突变株HVK-9作为工业化发酵生产菌种具有较好的遗传稳定性,为β-法尼烯的工业化生产和应用奠定了良好的基础。     

胞外青霉素酰化酶产生菌的选育

利用纸片显色方法,从土壤甲诀速筛选出98株产胞外青霉素酰化酶的菌种,经复筛其中10株酶活力较高,经鉴定均属于巨大芽孢杆菌。经单株分离得46号菌,用这株菌进行了产酶条件的研究,在最适产酶条件下,酶话力比开始提高了3．6倍。在此基础上又进行了物理化学因素处理,得突变株UL-81,酶活力达720u／1 Ooml发酵液。对原株和突变株进行比较,发现UL-81菌落、细胞形态、诱导剂苯乙酸用量及添加时间等明显不同于原株。在500L罐发酵酶活达8 20u／1OOml发酵液,为开始酶活的16倍。     

活性炭吸附固定化粪产碱杆菌青霉素G酰化酶

目的：以活性炭为载体固定化粪产碱杆菌来源的青霉素G酰化酶,考察固定化酶的性质。方法：对影响酶固定化的因素优化筛选,确定有显著影响的因素：pH、离子强度、酶量、固定化时间进行L934的正交实验,获得最佳固定化条件,并对固定化酶的最适反应温度、pH及批次稳定性进行研究。结果：最佳固定化条件为：载体0.3g,酶量5mL,总反应体系为12mL,离子强度1mol/L,温度4℃,pH 7.0,固定化40h;最高固定化酶活性为135.9U/g湿载体。固定化酶性最适反应温度为55℃,最适pH为10,重复使用12次后没有活性损失。结论：活性炭吸附固定化青霉素G酰化酶的活性高,批次反应稳定,具有工业应用潜力。     

巨大芽孢杆菌青霉素G酰化酶基因在枯草杆菌中的高表达

用ＰＣＲ方法从巨大芽孢杆菌的基因组ＤＮＡ中扩增到青霉素Ｇ酰化酶基因,并装载到枯草杆菌质粒ｐＰＺＷ１０３中,将其转化到枯草杆菌ＤＢ１０４中进行了分泌表达,重组菌株产酶无需苯乙酸诱导。在３７℃培养２４ｈ,菌液酶活力可达６ｕ／ｍｌ。１０天的连续传代实验表明重组菌株的稳定性很高。     

青霉素酰化酶高产菌株的快速筛选方法

采用青霉素梯度琼脂平皿筛选法,利用对青霉素G高抗性表型,专一筛选大肠杆菌青霉素酰化酶高产突变株。一次涂皿可淘汰绝大部分未突变株。我们从青霉素G梯度琼脂平皿上获得528株,从中得到32株产酰化酶活性高于出发菌株的正突变株,正突变率为6.06%,最高突变幅度为96.6%。     

青霉素酰化酶高产菌株的快速筛选方法

采用青霉素梯度琼脂平皿筛选法,利用对青霉素G高抗性表型,专一筛选大肠杆菌青毒素酰化酶高产突变株。一次涂皿可淘汰绝大部分未突变株。我们从青霉素G梯度琼脂平皿上获得528株,从中得到32株产酰化酶活性高于出发菌株的正突变株,正突变率为6.06%,最高突变幅度为96.6%。     

维生素K2(MK-7)生产菌株纳豆芽孢杆菌的诱变选育及其工艺优化

维生素K2(menaquinone-7,MK-7)是一种人体必不可少的维生素,具有抗凝血、预防骨质疏松及抗肝癌等功能.全反式天然维生素K2(MK-7)的合成主要是由微生物发酵法而得.为了获得高产MK-7且传代稳定的生产菌株,以1株实验室前期分离自纳豆生产MK-7的纳豆芽孢杆菌Bacillus natto ND-1为出发...     

Effect of phenylacetic acid on the growth and production of penicillin G acylase of recombinant and host strains derived from Escherichia coli W

The industrial production strain Escherichia coli RE3(pKA18) for penicillin G acylase (PGA) bears simultaneously the pga gene on the chromosome as an inducible gene pgaⁱ, (the inductor is phenylacetic acid, PAA) and on the recombinant plasmid pKA18 as a constitutively expressed gene pga^c. Under non-selective conditions, plasmid-less strains (P⁻) appeared in 17th successive batch culture. However, the population was over taken by P⁻ cells already in fourth culture if the medium was supplemented with PAA. The rate of plasmid loss from the culture depends on the PAA concentration and on the expression of pgaⁱ, not on PGA overproduction from pga^c. PAA at inducing concentration has a negative effect on PGA expression and plasmid stability in the high-expression self-cloning system RE3(pKA18) which results in the reduction of: (1) the specific growth rate of a culture and biomass concentration, (2) the synthesis of PGA (e.g. the specific activity of the strain) and (3) the copy number of the recombinant plasmid and promotion of the plasmid loss from the culture. Segregational stability of pKA18 increases in P⁺ persisting clones and in re-transformed P⁻ clones segregated during the selection in the presence of PAA.     

巨大芽孢杆菌青霉素G酰化酶的定点突变及其动力学性质研究

为了提高青霉素G酰化酶（PGA）在酸性及有机溶剂中的稳定性,以大肠杆菌的晶体结构为模板,用软件PMODELING同源模建巨大芽孢杆菌青霉素G酰化酶的三维结构结构并且选择PGA分子表面的合适碱性氨基酸突变为丙氨酸,通过三种不同的快速PCR介导定位突变的方法,将位于PGA的α亚基21位、128位和β亚基492位、512位的赖氨酸残基分别突变为丙氨酸,获得四个突变酶Kα021A、Kα128A、Kβ492A和Kβ512A。其中Kα128A和Kβ512A保持与野生型相近的酶活力,其动力学性质如最适温度、最适pH,Km及Kcat没有明显变化;突变酶Kα021A和Kβ492A则丧失 了酶活力。上述结果表明,PGA分子表面非活性中心的赖氨酸→丙氨酸点突变使突变子的性状发生了分化,突变效应呈现出丰富的多样性。该有理设计不但可以提高酶的稳定性,而且为揭示PGA结构和功能的关系提供了一个新的研究模型。     

Immobilization of penicillin G acylase on macro-mesoporous silica spheres

In this study, macro-mesoporous silica spheres were prepared with a micro-device and used as the support for the immobilization of penicillin G acylase (PGA). To measure the enzymatic activity, the silica spheres with immobilized PGA were placed into a packed-bed reactor, in which the hydrolysis of penicillin G was carried out. The influences of the residence time, the initial concentration of the substrate, the accumulation of the target product 6-aminopenicillanic acid, and the enzyme loading amount on the performance of the immobilized PGA were investigated. The introduction of macropores increased the enzyme loading amount and decreased the internal mass transfer resistance, and the results showed that the enzyme loading amount reached 895 mg/g (dry support), and the apparent enzymatic activity achieved up to 1033 U/g (dry support). In addition, the immobilized PGA was found to have great stability.     

巨大芽孢杆菌青霉素G酰化酶在新型环氧载体ZH-HA上的固定化

巨大芽孢杆菌青霉素G酰化酶共价结合在新型环氧-氨基型载体ZH-HA 上,通过对酶浓度、固定化时间、pH以及缓冲液浓度等条件的考察,确定了最优固定化条件:50 mg比活力6000 U/g的巨大芽孢杆菌青霉素G酰化酶蛋白和1g ZH-HA悬浮于pH 9.01 mol/L磷酸缓冲液,室温搅拌6 h,制得固定化巨大芽孢杆菌青霉素G酰化酶,活力2126 U/g湿载体,活力回收率7.67%.比较研究了固定化酶与原酶性质,原酶最适温度45℃,最适pH为8.0.固定化酶则分别是50℃和9.0,分别比溶液酶偏移5℃、1.0个pH单位.经过40批连续水解青霉素G钾盐,固定化巨大芽孢杆菌青霉素酰化酶仍保持80%的活力,显示出良好的工作稳定性.     

Synthesis and characterization of pseudo-affinity ligand for penicillin acylase purification

The aim of this work was to test a chromatographic affinity support containing methacryloyl antipyrine (MAAP) for penicillin acylase (PA) purification by using pure penicillin acylase and crude extract. First, MAAP as a pseudo-specific ligand was synthesized by using methacryloyl chloride and 4-aminoantipyrine. Polymer beads (average size diameter: 40–120 μm) were prepared by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and MAAP. This approach for the preparation of adsorbent has several advantages over conventional preparation protocols. An expensive and time consuming step in the preparation of adsorbent is immobilization of a ligand to the adsorption matrix. In this procedure, affinity ligand MAAP acts as comonomer without further modification steps. Poly(EGDMA-MAAP) beads were characterized by FTIR, NMR and screen analysis. Elemental analysis of MAAP for nitrogen was estimated as 89.3 μmol/g. The prepared adsorbent was then used for the capture of penicillin acylase in batch system. The maximum penicillin acylase adsorption capacity of the poly(EGDMA-MAAP) beads was found to be 82.2 mg/g at pH 5.0. Chromatography with crude feedstock resulted in 23.2-fold purification and 93% recovery with 1.0 M NaOH.     

基于核糖体工程理论的常压室温等离子体诱变筛选多杀菌素高产菌

利用核糖体工程理论,采用常压室温等离子体(ARTP)诱变对刺糖多孢菌(Saccharopolyspora spinosa)进行诱变筛选。以刺糖多孢菌QYLZ 88912菌株为出发菌株,对其进行ARTP诱变,选取三个不同致死率的照射时间,将孢子悬液混合,以增加抗性筛选几率。然后基于核糖体工程技术理论对混合孢子悬液进行初筛,最终筛选出了1株多杀菌素高产菌Sg200Rif110St40Er90-028。该菌株同时具有磺胺胍、利福平、链霉素、红霉素的多重抗性,摇瓶发酵试验表明,发酵7d后多杀菌素浓度可达到1 516.93 mg/L,较出发菌株QYLZ 88912的产量610.75 mg/L提高了148.37%,且遗传性稳定。以得到的高产菌Sg200Rif110St40Er90-028作为出发菌株,对发酵培养基进行响应面优化实验,优化后的培养基(g/L):葡萄糖46.97、麦芽糊精35、酵母粉40.36、水浸棉籽粉32.88、碳酸钙3,多杀菌素的产量为2 361.81 mg/L,比出发菌株的产量提高了286.71%。     

阿维菌素生产菌的常压室温等离子体诱变育种及培养基优化

【目的】通过诱变筛选技术选育阿维菌素高产突变株,对其发酵培养基进行响应面优化,提高阿维菌素产量。【方法】采用常压室温等离子体(ARTP)诱变技术,结合链霉抗性和卡那霉素抗性筛选法及96深孔板高通量筛选法,筛选阿维菌素高产株。在单因素实验的基础上,应用响应面分析法对其发酵培养基进行优化,最后确定最佳培养基配方。【结果】获得一株遗传性状稳定的阿维菌素高产株K-1A6,其阿维菌素产量达到4.22 g/L,比出发菌株9-39提高了23.4%,在最佳培养基中阿维菌素产量达到5.36 g/L,较优化前提高了27.01%。【结论】通过对阿维链霉菌9-39菌株进行ARTP诱变筛选及发酵培养基优化研究能显著提高阿维菌素的产量。     

Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri.

Two isoforms of the heterodimeric enzyme penicillin G acylase (EC 3.5.1.11) from Providencia rettgeri ATCC 31052 (strain Bro1) were purified to near homogeneity. The isoforms exhibited comparable enzymatic activities but differed slightly in the molecular weight and pI of their respective alpha-subunit. The origin of this difference was traced to the partial conversion of the N-terminal Gln of the alpha-subunit to pyrrolidonecarboxylic acid (pyro-Glu). The boundaries of the mature enzyme within the translated DNA sequence of the wild-type propeptide (GenBank {"type":"entrez-nucleotide","attrs":{"text":"M86533","term_id":"150921","term_text":"M86533"}}M86533) were determined. The results conclusively identified the length of the signal peptide and the position of the spacer cleaved from the propeptide to form the active heterodimer. The molecular weights of the alpha- and beta-subunits, based on these termini, were 23.7 and 62.2 kDa, respectively. Both isoforms were crystallized independently as hexagonal bipyramids up to 0.60 mm in diameter in either space group P6(1)22 or P6(5)22 (a = b = 140.5 A and c = 209.5 A) from ammonium sulfate solutions buffered by 50 mM potassium phosphate at pH 7.5. The presence of glycerol, although not required, facilitated crystal growth. Native and heavy atom derivative data were collected to 3.0 A resolution, and the calculation of isomorphous replacement phases is under way.     

Application of surface response analysis to the optimization of penicillin acylase purification in aqueous two-phase systems

Penicillin acylase purification from an Escherichia coli crude extract using PEG 3350–sodium citrate aqueous two-phase systems (ATPS) was optimized. An experimental design was used to evaluate the influence of PEG, sodium citrate and sodium chloride on the purification parameters. A central composite design was defined centred on the previously found conditions for highest purification from an osmotic shock extract. Mathematical models for the partition coefficient of protein and enzyme, balance of protein and enzyme, yield and purification were calculated and statistically validated. Analysis of the contours of constant response as a function of PEG and sodium citrate concentrations for three different concentrations of NaCl revealed different effects of the three factors on the studied parameters. A maximum purification factor of 6.5 was predicted for PEG 3350, sodium citrate and NaCl concentrations of 15.1, 11.0 and 8.52% respectively. However, under these conditions the predicted yield was 61%. A better compromise between these two parameters can be found by superimposing the contour plots of the purification factor and yield for 10.3% NaCl. A region in the experimental space can be defined where the purification factor is always higher than 5.5 with yields exceeding 80%.