Study on the Kinetics of L-Tryptophan Adsorption by Ion Exchange Resin

通过静态吸附实验,研究了l-色氨酸在001x7型阳离子交换树脂上的等温吸附和吸附动力学特性。采用动边界模型描述l-色氨酸在该树脂上的交换行为,考察了料液浓度、树脂粒径和温度对交换过程的影响。结果表明,l-色氨酸在001x7型阳离子交换树脂上的吸附等温线符合lAngMuIr等温方程,且随PH降低,树脂的最大平衡吸附量逐渐增大;交换过程的吸附速率随l-色氨酸浓度和温度的升高而增大,但随树脂粒径的增大而减小;离子交换过程的速度控制步骤为颗粒扩散控制。交换过程的反应速率常数k0为1.199x10 5,反应级数为1.7,表观活化能EA为19.94kJ MOl 1,并得到了动力学总方程式。The isothermal adsorption and adsorption kinetics of L-tryptophan by 001×7 cation-exchange resin were studied by static experiments.The Moving Boundary Model was applied to describe the kinetics of the ion exchange process.The effects of experimental conditions including the concentration of L-tryptophan,resin particle diameter and adsorption temperature on the ion exchange process were investigated.The results show that the maximum L-tryptophan adsorption capacity of 001×7 cation-exchange resin increases with decreasing solution pH.It was found that the Langmuir equation fits the adsorption isotherm data best,and the particle diffusion is the rate-limiting step of the adsorption process.The adsorption ratio increases with the increase of L-tryptophan concentration and adsorption temperature,but decreases with the increase of the resin particle diameter.The rate constant of exchange(k0),reaction order() and the apparent activation energy of reaction(Ea) were obtained as follows: k0=1.199×10 5,Ea=19.94 kJ mol 1,and the kinetics equation was obtained too.国家自然科学基金(3107488);福建省自然科学基金(2011J01058);中央高校基本科研专项基金(2011121017

Optimization of medium components for production of antitumor agent Deacety Mycoepoxydiene by marine endophytic fungi Phomopsis sp.

从海洋真菌Phomopsis sp.分离得到的南强菌素是新发现的高抗肿瘤活性化合物,为新颖罕见的去乙酰真菌环氧二烯类化合物(Deacety Mycoepoxydiene)。以Phomopsis sp.A123经诱变后获得的818菌株为出发菌株,对其进行液体发酵培养。考察了几种常见培养基对南强菌素发酵生产的影响,确定出较适的培养基。同时以该培养基为基础培养基,探讨了碳源、氮源以及几种维生素对南强菌素积累的影响。培养条件的初步优化结果表明:最佳的培养基为PD培养基;最适碳源和氮源分别为麦芽糖及马铃薯煮汁;生物素的添加有利于南强菌素的积累,产量提高了25%。Deacety Mycoepoxydiene is a novel antitumor agent extracted from a marine endophytic fungi Phomopsis sp.A123.In order to improve the yield of Deacety Mycoepoxydiene,the liquid fermentation medium composition is optimized by a one-factor-at-a-time method for a mutant No.818,which is derived from Phomopsis sp.A123.Potato dextrose(PD) culture medium is selected as the preliminary culture medium,and the optimal carbon and nitrogen sources for liquid fermentation are investigated.The effect of several vitamins,including lipoic acid,VB12,VB1 and biotin on the accumulation of Deacety Mycoepoxydiene is also studied.The results show that the optimal carbon and nitrogen source is maltose and potato steep liquor,respectively.The yield of Deacety Mycoepoxydiene is increased by 25% while the biotin is added.国家高技术研究发展(“863”)计划海洋技术领域海洋微生物产品中试研究重点项目资

Screening of high daptomycin-producing strains by combined antibiotics resistance screening method

作者简介：张智翔，男，生于1985年，在读硕士研究生，主要从事微生物发酵，E-mail: [email protected] *通讯作者，E-mail: [email protected][中文文摘]目的利用组合抗性筛选法选育达托霉素高产菌株。方法以玫瑰孢链霉菌(Streptomyces roseosporus ATCC 11379)为出发菌株,通过在不同浓度梯度的达托霉素和链霉素复合抗性平板上进行抗性筛选。结果筛选到一株高产达托霉素的突变株D1000-S3-2,经摇瓶发酵验证达托霉素发酵单位可达59mg/L,比出发菌株提高了63.8%。结论实验证明组合抗性筛选是一种简单高效筛选方法。[英文文摘]Combined antibiotics resistance screening method was applied to screen daptomycin high-producing strain. Methods Based on the biosynthetic pathway and metabolization mechanism of daptoomycin, combined antibiotics resistance screening method was applied to screen daptomycin high-producing strain from original strain of Streptomyces roseosporus ATCC 11379. Results A mutant strain D1000-S3-2 was obtained. The yield of daptomycin was 59 mg/L in fl ask by the mutant strain, which increased 63.8%, compared to that of the parental strain. Conclusion It was demonstrated that the method provides a fast and effective way of screening S. roseosporus.基金项目：国家自然科学基金(No. 31071488

Secretive Expression and Optimized Inducement of Barnase

从解淀粉芽孢杆菌中PCR分别扩增解淀粉芽孢杆菌核糖核酸酶barnase基因及其抑制剂barstar基因,采用将barnase基因置于barstar基因保护下的克隆策略,以pET-22b(+)质粒为基础,构建大肠杆菌分泌型表达质粒.IPTG诱导表达目的蛋白后将培养基蛋白进行SDS-PAGE分析并从诱导温度、IPTG诱导浓度和诱导时间三方面初步优化诱导表达条件.Using PCR method to amplify Bacillus amyloliquefaciens ribonuclease barnase gene and its inhibitor barstar gene from Bacillus amyloliquefaciens. The secretive expression vector is constructed by the strategy of protective clone from plasmid pET-22b(+). After induced by IPTG, a 12 kD target barnase protein can be detected from medium protein by SDS-PAGE analysis. Inducing conditions of temperature, IPTG concentration and inducing time have also been optimized.福建省高等学校新世纪优秀人才支持计划资助项

超临界CO2夹带乙醇萃取裂殖壶藻油脂的工艺研究

研究了超临界CO2夹带乙醇流体从裂殖壶藻Schizochytriumsp.中提取富含DHA油脂的新工艺。基于气液相平衡性质,确定了夹带剂乙醇的添加范围,并对提取过程进行动力学分析,确定了二氧化碳的使用量。通过中心组合设计的响应面优化试验,获得了最佳提取工艺条件:温度52℃、压力38 MPa、乙醇摩尔比0.04。该条件下,微藻油脂的回收率约为88.2%,显著高于纯超临界CO2萃取的回收率（71.9%）。根据Peng-Robinson方程考察了混合流体的密度变化对油脂溶解度的影响,部分解释了操作条件对油脂回收率的影响。脂肪酸组成分析显示,新工艺得到的油脂中的DHA质量分数约为39.9%,略高于传统工艺。由此可知,超临界CO2夹带乙醇流体是一种极性范围广、溶解能力强的绿色提取介质,非常适用于中性微藻油脂的提取。浙江省基础公益研究计划(LGN19B060001)浙江省新苗人才计划(2018R404072

Asymmetric synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate with a two recombinant Escherichia coli system

Conference Name:4th International Conference on Manufacturing Science and Engineering, ICMSE 2013. Conference Address: Dalian, China. Time:March 30, 2013 - March 31, 2013.Northeastern University, China; Harbin Institute of Technology; Jilin UniversityThe asymmetric reduction of ethyl 4-chloro-acetoacetate (CAAE) to ethyl (R)-4-chloro-3- hydroxybutanoate (R-CHBE) biocatalysed by the aldehyde reductase of Sporobolomyces salmonicolor expressed in E. coli M15 (pQE30-alr) in combination with regeneration of NADPH by the glucose dehydrogenase of Bacillus megaterium expressed in E. coli M15 (pQE30-gdh) was reported. The bioreduction was carried out in a two-phase reaction system with n-butyl acetate as an organic solvent. Bioconversion of 300 mmol CAAE with a final yield of 97.5% and an enantiometric excess of 99% was achieved without the addition of cofactor NADPH. ? (2013) Trans Tech Publications, Switzerland

Studies on the Conditions of β-glucanase Production by Recombinant Escherichia coli

通过对重组大肠杆菌JM109-Plf3摇瓶发酵生产β-1,3-1,4-葡聚糖酶工艺条件的研究,得出最佳培养条件为:温度39℃,摇床转速150 r/MIn,培养基装量20 Ml/250 Ml,培养基初始PH 6.7,种子培养时间16 H,接种量1%.在最佳培养条件下,发酵30 H酶活力达到最大值481.41 u/Ml.最优条件下摇瓶恒速补加氮源对酶活的提高贡献较大,且适当提高流加量对促进产酶效果更明显,酶活力可达628.30 u/Ml,为初始时的2.1倍.The fermentation conditions of recombinant Escherichia coli JM109 harboring pLF3 for extracellular β-glucanase production were investigated.The optimized culture conditions were determined as follows:temperature 39℃.shaking speed 150 r/min.culture volume 20 mL in 250 mL shake flask,initial pH 7.0,inoculating age 16 h,and inoculating quantity 1.0%.Under these conditions,a maximum β-glucanase activity of 481.41 U/mL was obtained after 30 h fermentation.Fed-batch strategies were also studied in shaking flask.Higher β-glucanase activity of 628.30 U/mL,which was almost 2.1 times higher than that obtained at the initial conditions,was achieved by feeding high concentration nitrogen sources at constant rate.厦门市科技计划项目(3502Z20055017);厦门大学新世纪优秀人才支持计划资

Study of Cultivation Conditions for Natamycin Production by Streptomyces gilvosporeus

通讯作者: [email protected][中文文摘]目前,国内纳他霉素产量低,为提高其产量,实现其工业化生产,主要对褐黄孢链霉菌ATCC 13326发酵生产纳他霉素的培养条件及前体添加策略进行研究.得出最佳培养条件为:培养温度28℃,种龄40~44 h,接种量10%,装液量10%,初始pH 7.3,发酵24 h时加入0.6%的前体丙酸钠.在此条件下摇瓶分批发酵生产纳他霉素的产量可达到6.87g/L,比未添加前体的纳他霉素产量(3.72 g/L)提高了84.7%.利用10 L发酵罐进行纳他霉素的发酵研究,在最优培养条件下,发酵罐中的纳他霉素产量可达到8.34 g/L,比摇瓶分批发酵生产纳他霉素产量(6.87 g/L)提高了21.4%.[英文摘要]In order to enhance the production of natamycin and realize industrial production,in this study,the cultivation conditions as well as the effect of precursor addition on the natamycin production by S.gilvosporeus ATCC13326 were investigated.The optimized cultivation conditions were as follows:initial pH 7.3,age of inoculum 40～44 h,inoculum level 10%,shake-flask working volume 30 mL/300 mL,temperature 28℃,cultivation time 120 h,and addition of 0.6% sodium proprionate into the broth after 24 h of cultivation.On the optimum conditions a natamycin production of 6.87g/L could be reached in the shake flask culture after 120 h cultivat ion, w hich w as 84. 7% hig her than t hat of cultiv ations without precursor addition. Even higher natamycin concentration(8.43g/L) on the o ptimal cult ivation conditions, which was 21.4% higher than that could be expected in the shake flask culture(6.87g/L).广东省教育部产学研结合项目(2008B090500218)资

Expression of Candida antarctica lipase B in Pichia pastoris and Studying of Enzyme Properties

为提高南极假丝酵母脂肪酶b(CAndIdA AnTArCTICAlIPASE b,CAlb)的表达量,根据毕赤酵母(PICHIA PASTOrIS)密码子偏好性设计合成CAlb基因,插入表达载体PPIC9k构建重组质粒PPIC9k-CAlb.重组质粒转化毕赤酵母gS115,经过g418抗性筛选得到多拷贝转化子.摇瓶发酵120 H后上清液酶活力达到46 u/Ml,通过金属螯合层析纯化发酵液将CAlb纯化了5.23倍,比活力达到856.7 u/Mg,去糖基化实验显示重组CAlb比野生型CAlb大5 ku.实验考察了不同反应温度、PH值和金属离子对重组CAlb活性和稳定性的影响,发现重组CAlb最适反应温度和PH分别为30℃和6.5,在PH 5.0--8.0之间以及40℃以下有较好稳定性,金属离子(10 MMOl/l)CA2+,zn2+,Mn2+有助于CAlb酶活力的提高,而Cu2+,Ag+,fE3+强烈抑制酶催化反应.To improve expression efficiency of recombinant Candida antarctica lipase B(CALB) in methylotrophic yeast Pichia pastoris GS115,the DNA sequence encoding CALB was designed and synthesized based on the codon bias of P.pastoris.The new CALB gene was cloned into pPIC9K,yielding pPIC9K-CALB,and transformed into P.pastoris GS115.In shake-flask cultivation,the enzyme activity was 46 U/mL after induction by methanol for 120 h.The CALB was purified by metal chelate chromatography by 5.23 folds.Deglycosylation showed the recombinant CALB was 5 ku bigger than wild type CALB.The optimal pH and temperature of this recombinant enzyme were pH 6.5 and 30°C,respectively.CALB was relatively stable at temperatures below 40 ℃ within a pH range from 5.0 to 8.0.Ca2+,Zn2+,Mn2+ conduced to enzyme activity,while Cu2+,Ag+,Fe3+ inhibited it.广东省教育部产学研结合项目(2008B090500218

Codon Optimization of Human Proinsulin and Expression in Pichia pastoris

通信作者：[email protected][中文文摘]依据毕赤酵母偏好密码子,设计合成长效人胰岛素原(human proinsulin,HPI)基因序列,所合成的HPI基因全长为200bp,并在其N端添加信号肽(EEAEAEAEPK)以提高目的蛋白表达.将改造后基因克隆到pPIC9K载体中,构建分泌表达型载体pPIC9K-HPI,转化到毕赤酵母(Pichia pastoris)GS115中.用遗传霉素G418梯度筛选获得高拷贝菌株,在甲醇诱导下表达分子质量为7.87ku的HPI,利用金属螯合层析纯化蛋白质,胰蛋白酶酶切后利用人胰岛素试剂盒测得生物活性,HPI摇瓶最高产量可达35.4mg/L.该研究为获得大量长效胰岛素基因工程产品奠定研究基础.[英文摘要]According to the preferential codon of Pichia pastoris,the cDNA of human proinsulin(HPI) with the full length of 200 bp was designed and synthesized.It contains the singnal peptide(EEAEAEAEPK) in N terminal in order to increase the protein expression level.The HPI was cloned into shuttle vector pPIC9K to construct recombinant expression plasmid pPIC9K-HPI.It was then transformed into the Pichia pastoris GS115 by electrotransformation.A transformant with a high copy number of HPI gene was obtained by G418 concentration gradient screening,and then started to express HPI protein after induction with methanol in shake flasks for 120h.The HPI was digested by trypsin at ４℃ for 12h after being purified by metal chelate chromatography．the HPI reached ３５.４ｍｇ／Ｌ，and showed the biological activity detected by ELISA kit．国家自然科学基金项目(3107488); 福建省自然科学基金项目(2011J01058