Effect of cell volume on disintegration by ultrasonics

Wide variations in resistance to ultrasonic disintegration of continuously cultivated Bacillus cereus, Saccharomyces cerevisiae and Escherichia coli could be achieved by corresponding variations in fermenter impeller speed (agitation rate). In all cases examined, the relationship between disintegration constant and impeller speed was found to be linear. Although changes in relative strengths of E. coli were observed with changes in limiting nutrient (nitrogen-limited cells being weaker than carbon-limited cells for any one impeller speed), this was compensated for by the increased volumes of nitrogen-limited cells, and the relationship between disintegration constant and mean cell volume was linear, irrespective of nutrient limitation. Comparisons with other published data confirmed the view that for any one micro-organism, irrespective of cultural conditions, the principal determinand of susceptibility to ultrasonic disintegration was mean cell volume.     

Effects of changes in agitation rate on steady-state penicillinase titres in continuously-cultivated Bacillus cereus

Increases in agitation rates at a constant dilution rate in aerobic continuous culture of Bacillus cereus furnished corresponding increases in extracellular penicillinase titres. The ratio of titre to biomass supported by the apparatus increased when the agitation rate was raised, even if the medium was diluted to half its original concentration. Potential causes of the phenomenon are discussed.     

机械搅拌反应器中挡板的结构设计

研究了内径为0．786m的搅拌釜中挡板尺寸及结构对圆盘透平桨RT和翼型桨k5及其组合在气液两相中的气体分散与混合特性的影响。对不同形式的挡板的搅拌功率、气含率及气液混合特性进行了对比分析。研究结果表明：挡板尺寸结构应根据搅拌特性需要进行优化设计;挡板系数为0．12时,组合浆的功率输入已与同一转速下的全挡板系数时的功率输入相近,它同时可改善微观混合、提高混合效率：采用开槽挡板可提高复杂快反应的选择性,混合效率提高20％～25％。     

Influence of impeller type on hydrodynamics and gas‐liquid mass‐transfer in stirred airlift bioreactor

The influence of impeller type in a mechanically stirred airlift bioreactor was analyzed in relation to the non‐Newtonian viscous fluids. The agitation was carried out through a marine impeller (axial impeller) and a paddle impeller (radial impeller) located along with the gas sparger in the region comprised by the riser. The bioreactor was sparged with air under different velocities (0.036–0.060 m s^?1). Carboxymethylcellulose 1.94% and xanthan 1.80% were used as a fluid model. The gas holdup and volumetric mass‐transfer coefficient increased in up to five and three times, respectively, when compared to a conventional airlift bioreactor; however, better results were obtained when the straight paddle impeller type was used. The results suggest that the studied bioreactor can be used successfully in viscous fluid, and it can be more efficient than conventional airlift bioreactors. The results obtained suggest the use of radial impellers. © 2015 American Institute of Chemical Engineers AIChE J, 61: 3159–3171, 2015     

Changes in the structure of flax cellulose induced by solutions of lithium,sodium, and potassium hydroxides

In the reaction of cellulose-containing flax material with aqueous solutions of LiOH, NaOH, and KOH, the degree of swelling, sorption of water and alkali, parameters of the crystal lattice formed by alkali cellulose, degree of removal of lignin, and change in the degree of polymerization are a function of the type of cation. For the sorption characteristics and crystal lattice parameters of alkali cellulose, this dependence is determined by the change in the structure of the first and second hydrate shells of the cations. Despite the important differences in the degree of swelling, crystal lattice parameters of alkali cellulose, and degree of removal of lignin, the changes in the crystal structure of the cellulose related to the transition from crystalline modification I to modification II under the effect of the three different bases are similar and take place in approximately the same concentration region.Translated from Khimicheskie Volokna, No. 6, pp. 15–19, November–December, 2004.     

Facile, reagentless and in situ release of Escherichia coli intracellular enzymes by heat-inducible autolytic vector for high-throughput screening

In an effect to broaden the application of the heat-inducible autolytic vector pUC18-cI857/p(R)-SRRz-rrnB previously developed, a new vector pUC18-cI857/p(R)(T41C)-SRRz-rrnB (pEAS-1b) was quantitatively characterized under various growth temperatures, heat induction temperatures and durations, and IPTG (isopropyl beta-d-thiogalactoside) induction times, after resolving its erratic lysis profile found previously. Escherichia coli BL21 cells harboring this vector grew well at temperatures <36 degrees C, and lysed efficiently (97.0 +/- 0.8%) just 0.5 h after heat induction at 42 degrees C for 30 min when cell growth was performed at 35 degrees C. Application of this autolytic vector either in 96-well plates, or on nitrocellulose membranes, or on agar plates led to facile, efficient and consistent release of intracellular recombinant enzymes (e.g., a lysis efficiency of 91.8 +/- 1.1% was obtained in 96-well plates). Further application in directed evolution was illustrated by improving the thermostability of amadoriase using this vector. This reagentless and in situ cell lysis method has the potentials for lysis of miniaturized samples in clinical diagnosis and bioanalytical detection, and even for lysis of cells in the microarray format.     

甘油连续生物歧化过程的过渡行为及其数学模拟

用肺炎杆菌将甘油转化为１,３－丙二醇过程对稀释速率和底物进料浓度变化的过渡行为反映了细胞生长和胞内物质代谢在不同条件下存在着显著的差异。体系的过渡响应取决于操作条件变化前后系统所处的状态,当底物浓度由限制状态向剩余状态过渡时,甘油的残余浓度不断上升,而乙醇的浓度则逐渐下降,同时生物量、１,３－丙二醇和乙酸的浓度呈现先增后降的趋势。本文用“甘油浓度变化速率”的概念修正了菌体生长模型,并对甘油歧化过程     

工业三相浆态反应器内的混合与操作

根据搅拌糟中气－液－固三相混合性能的研究，论述了工业三相浆态反应器内对气体、液体和固体的分散（混合）要求，据此对工业三相浆态反应器中叶轮的结构和搅拌浆的启动进行了讨论。     

微生物培养的振荡现象及其应用

振荡现象广泛存在于生命系统中。简述了微生物培养过程中的振荡现象,描述了其特征,介绍了振荡行为在发酵培养、废水处理、提高工程菌的稳定性等方面的应用,并指出了其今后的研究方向。     

电感耦合等离子体发射光谱法测定生物柴油中的K、Ca、Na、Mg

采用酸消解法对生物柴油样品进行前处理,使用电感耦合等离子体发射光谱仪测定生物柴油中K、Ca、Na、Mg的含量。实验结果表明该方法在0．05~2．0mg·L-1的浓度范围内呈现了良好的线性关系,相关系数均大于0．999,相对标准偏差4．54％-5．72％,加标回收率86．5％～109．0％。最终建立起了一种快速高效,能够同时分析生物柴油中K、ca、Na、Mg含量的方法。     

固-液导流简搅拌槽内流体流动和颗粒悬浮特性

在直径0．8m的导流筒搅拌槽内，对单相液体的三维速度分布、固-液两相的固体颗粒浓度分布和离底悬浮特性进行了系统的实验研究．结果表明，导流筒内外的轴向液相速度远大于径向和切向速度，导流筒外壁附近存在一个与主体轴向流动方向相反的二次流区域；搅拌槽底部结构对固体颗粒的临界离底悬浮转速（NJS）有显著的影响，浅锥底的NJS比平底的低14％以上；NJS随固相浓度的增加而增加，但当浓度超过50％时，NJS略有降低；槽内固相浓度分布的均匀性随固相浓度的增加而得到改善．本研究结果对导流筒搅拌槽的优化设计具有一定的指导意义．     

分立器件连续高速选择氨基磺酸盐镀镍工艺

介绍了用于分立器件的氨基磺酸盐镀镍工艺。探讨了添加剂和工艺条件对镍镀层性能的影响。提出了镀镍液的维护及镀液中杂质的去除。     

Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance

Both freezing tolerance and NaCI tolerance are improved whenantifreeze proteins are expressed as fusion proteins with twodomains of staphylococcal protein A (SPA) in Escherichia coli.To characterize these properties further we created a randomlymutated expression library in E.coli, based on the winter flounderantifreeze protein HPLC-8 component gene. Low-fidelity PCR productsof this gene were fused to the spa gene encoding two domainsof the SPA. The library was screened for enhanced NaCl toleranceand four clones were selected. The freezing tolerance of eachof the selected clones was enhanced to varying extents. DNAsequencing of the isolated mutants revealed that the amphiphilicproperties of the native antifreeze protein were essentiallyconserved. Furthermore, by studying the primary sequence ofthe randomly mutated clones, in comparison with the degree offreezing tolerance, we have identified clues which help in understandingthe relationship between salt and freezing tolerance.     

大肠杆菌GBP的定点突变与制备方法研究

在mglB基因上进行了E149C、 A213S、 L238S三个定点突变,并利用大肠杆菌BL21(DE3)/pET28c系统,构建了表达突变型GBP的基因工程菌BL21(DE3)/pLE3.工程菌经诱导培养后收获细胞,利用渗透休克法提取周质空间蛋白,经Ni-NTA柱纯化,从1 L培养液中可得到约3.5 mg SDS-PAGE纯度的GBP.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.     

Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein

A cDNA done of human calmodulin, isolated from liver, was subclonedinto the expression vector pKK233-2. The resulting expressionplasmid, designated pCWCaMl, produced human calmodulin in EscherichiacoliSG5. The cDNA was sequenced using novel primers designedfor use in plasmid-sequenclng protocols with pKK233-2 and pKK223-3.The expressed calmodulin was purified and subjected to NMR analysiswhich revealed a structure essentially the same as natural calmodulinisolated from human tissue. The activation of myosin light chainkinase by the genetically engineered human calmodulin and bovinebrain calmodulin was studied and found to be comparable to ahigh degree. The expressed calmodulin appears to be comparableto normal calmodulin and can be used for she-directed mutagenesisand structure/function investigations.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli

For the first time the pro-form of a recombinant cysteine proteinasehas been expressed at a high level in Escherichia coli. Thisinactive precursor can subsequently be processed to yield activeenzyme. Sufficient protein can be produced using this systemfor X-ray crystallographic structure studies of engineered proteinases.A cDNA clone encoding propapain, a precursor of the papaya proteinase,papain, was expressed in E.coli using a T7 polymerase expressionsystem. Insoluble recombinant protein was solubilized in 6 Mguanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6.A protein-glutathione mixed disulphide was formed by dilutioninto oxidized glutathione and 6 M GuHCl, also at pH 8.6. Finalrefolding and disulphide bond formation was induced by dilutioninto 3 mM cysteine at pH 8.6. Renatured propapain was processedto active papain at pH 4.0 in the presence of excess cysteine.Final processing could be inhibited by the specific cysteineproteinase inhibitors E64 and leupeptin, but not by pepstatin,PMSF or EDTA. This indicates that final processing was due toa cysteine proteinase and suggests that an autocatalytic eventis required for papain maturation.     

Expression of catalytically active hamster dihydroorotase domain in Escherichia coli: purification and characterization

Dihydroorotase is the central domain of trifunctional L-dihydroorotatesynthetase which also contains carbamyl phosphate synthetaseat the N-terminus and aspartate transcarbamylase at the C-terminus.The cDNA, corresponding to the active dihydroorotase domainas isolated after digestion of dihydroorotate synthetase withelastase, has been sub-cloned into the expression vector pCW12which was then used to transform Escherichia coli SØ1263pyrC^– lacking dihydroorotase activity. However, inductionof this recomhinant strain with IPTG produced large amountsof the dihydroorotase domain which were completely inactive.A number of cDNAs were expressed which were longer on the C-terminalside; all cDNAs expressed active dihydroorotase domain downto a minimal extension of 12 ammo adds (-Val- Pro-Pro-Gly-Tyr-GIy-Gm-Asp-Val-Arg-Lys-Trp)into the bridge region between the dihydroorotase and aspartatetranscarbamylase domains. Part of this dodecapeptide may forman amphipathk helix which in some way constrains the isolated,recombinant dihydroorotase domain to an active conformation.The recombinant hamster dihydroorotase purified from a cell-freeextract of E.coli in four steps has a turnover number of 297mol/min/(mol domain) for the conversion of L-dihydroorotateback to N-carbamyl-Laspartate with K₈ = 8.7 ± 1.5 µMfor L-dihydroorotate, a subunit molecular weight of 39 008 determinedfrom the sequence and 37 900 ± 400 when subjected toSDS–PAGE, and an isoelectric point of 5.7. Ultracentrifugalanalysis of the recombinant domain showed a single species ofs_20,w = 4.1 S and a single molecular species of M_r = 76 000corresponding to a dimer.     

Cloning, Soluble Expression and Purification of High Yield Recombinant hGMCSF in Escherichia coli

Expression of human granulocyte macrophage colony stimulating factor (hGMCSF), a cytokine of therapeutic importance, as a thioredoxin (TRX) fusion has been investigated in Escherichia coli BL21 (DE3) codon plus cells. The expression of this protein was low when cloned under the T7 promoter without any fusion tags. High yield of GMCSF was achieved (～88 mg/L of fermentation broth) in the shake flask when the gene was fused to the E. coli TRX gene. The protein was purified using a single step Ni(2+)-NTA affinity chromatography and the column bound fusion tag was removed by on-column cleavage with enterokinase. The recombinant hGMCSF was expressed as a soluble and biologically active protein in E. coli, and upon purification, the final yield was ～44 mg/L in shake flask with a specific activity of 2.3 × 10(8) U/mg. The results of Western blot and RP-HPLC analyses, along with biological activity using the TF-1 cell line, established the identity of the purified hGMCSF. In this paper, we report the highest yield of hGMCSF expressed in E. coli. The bioreactor study shows that the yield of hGMCSF could be easily scalable with a yield of ～400 mg/L, opening up new opportunities for large scale production hGMCSF in E. coli.