重组Humicola insolens角质酶的高密度发酵优化 *

在采用前期已构建的重组菌E.coli BL21(DE3)/pET20b(+)-hic进行高密度发酵制备角质酶时发现,在高诱导强度发酵时,菌体浓度下降明显。同时,通过测定纯化重组角质酶的磷脂水解活性,考察验证了重组酶对宿主细胞的损伤作用。重组酶的磷脂酰乙醇胺活性为9.8U/mg(NPB水解比活力为1 047.6 U/mg),在卵黄平板出现了明显的反应圈现象。在此基础上尝试了高菌体浓度结合高诱导强度的发酵策略,以进一步提高重组酶在3L罐中的表达水平。优化后的最佳条件及结果为:OD₆₀₀为75时,恒速流加0.8g/(L ·h)的乳糖溶液,发酵24h后,酶活达到最大值4 788.0U/ml,约为摇瓶发酵酶活的28倍,与OD₆₀₀为50时、流加0.2 g/(L ·h)进行诱导的发酵策略(酶活2 233.0 U/ml)相比,提高幅度约为114.0%,发酵时间缩短40.0%。     

分散泛菌蔗糖异构酶在大肠杆菌中的表达及发酵优化

为了提高分散泛菌Pantoea dispersa UQ68J来源的蔗糖异构酶产量,研究了不同信号肽及发酵条件对蔗糖异构酶在大肠杆菌中重组表达的影响。将携带天然信号肽的蔗糖异构酶基因优化后,转入大肠杆菌Escherichia coli BL21(DE3)构建重组表达菌株——ORI菌株,摇瓶发酵总酶活和胞外酶活分别为85 U/m L、65 U/m L。从天然信号肽开始第22位氨基酸作为成熟蛋白的起始,连接Pel B或Omp A信号肽构建P22和O22菌株,其中P22菌株发酵总酶活提高至138 U/m L,是ORI菌株总酶活的1.6倍;而O22菌株发酵总酶活和ORI菌株无明显差别。采用3.0 g/L的乳糖诱导,P22菌株的蔗糖异构酶总酶活提高至168 U/m L。在3 L发酵罐中,研究甘氨酸浓度和诱导时间对蔗糖异构酶分泌的影响,当补加0.5%甘氨酸,DCW为18 g/L(OD_(600)=30)开始诱导,P22菌株的蔗糖异构酶胞外酶活最高达1 981 U/m L,同时蔗糖异构酶总酶活达到2 640 U/m L,是已报道大肠杆菌重组表达蔗糖异构酶的最高水平。     

对SDS稳定的V8(V125T)蛋白酶突变体的高效表达及性质研究

谷氨酰内切酶能特异性切割谷氨酸、天冬氨酸残基羧基端结合的肽键。将含有V8蛋白酶突变体(V125T)基因的表达质粒的重组大肠杆菌BL21(DE3),在50L发酵罐中发酵,融合蛋白为可溶性表达,可得菌湿重50g/L,相对蛋白表达量为33%。融合蛋白采用GST亲和纯化、肠激酶激活、DEAE-FF阴离子交换层析,得到纯的V8(V125T)突变体,经纯化后可获得0.998mg蛋白/g菌(湿重),比活为13.47 U/mg pro.纯化过程的酶活回收率达到了97.9%。对纯酶进行酶学性质分析,以Z-Phe-Leu-Glu-p NA作为底物测得V8(V125T)蛋白酶的Km为0.339mmol/L,Vmax为16.642μmol/min。其最适pH为8.0,在pH4.0~10.0之间较稳定;最适反应温度在45℃,12h内在4~35℃有很好的温度稳定性;25℃条件下1mmol/L的金属离子对酶具有不同程度的影响,其中Fe~(3+)的抑制作用最强;2mol/L尿素及1mmol/L EDTA对酶活性无影响,在0.1%SDS中保存12h、在0.5%SDS中4h和在1%SDS中1h,活性均能维持90%以上,在0.5%,0.1%SDS保存12h,仍能保持80%和64%的活性,与未突变的重组V8蛋白酶相比,该突变体对SDS的耐受性得到极大提高。     

酶水解菊芋糖浆发酵生产琥珀酸的初步研究

用产菊粉酶的一株黑曲霉菌株进行产酶发酵条件和水解条件研究,在30℃,pH 6.0,摇床转速200 r/min,发酵时间为3 d的最适产酶条件下,酶活可以达到45.9 U/mL.以总糖含量为85.2 g/L的菊芋粉为初始底物,最适酶水解条件为温度50℃,加黑曲霉培养液的量为10%(v/v),水解12 h后,水解率达到99.6%.用此酶解液在5 L搅拌发酵罐中进行琥珀酸发酵,初始还原糖浓度53.5 g/L,36 h发酵产琥珀酸43.8 g/L,琥珀酸产率0.83 g/g,糖利用率99.0%,琥珀酸生产强度1.22 g/(L·h).     

嗜热网球菌纤维二糖差向异构酶在枯草芽孢杆菌中的表达及发酵优化

通过化学方法合成嗜热网球菌(Dictyoglomus thermophilum)来源的纤维二糖差向异构酶基因ce,将其引入到载体pBSuL3-ce,构建重组质粒pBSuL3-ce并转化进枯草芽孢杆菌,发酵48h后测定胞内酶活为7. 5U/ml。酶学性质结果表明:该酶的最适pH为8. 5;最适温度为85℃,85℃的半衰期为120min。为降低发酵成本,对发酵培养基进行优化:以35g/L豆粕粉为氮源、5g/L甘油为碳源时,酶活力最高可达12. 3U/ml。依据摇瓶优化的条件在3L发酵罐中扩大培养,胞内酶活达到56U/ml,比摇瓶培养酶活提高了8倍。利用发酵所得酶制备乳果糖,在乳糖浓度为400g/L、反应温度为85℃、初始pH 8. 5、加酶量为20U/ml的条件下,乳果糖转化率可达51%。     

基因拷贝数对重组毕赤酵母产谷氨酰胺转胺酶的影响

基于毕赤酵母核糖体DNA序列 (rDNA),构建多拷贝谷氨酰胺转胺酶基因表达载体pPICZα-rDNA- mtg,并转化到表达前导肽 (Pro peptide或pro) 的宿主菌pGAP9-pro/GS115,得到共表达菌株pro/rDNA-mtg (GS115)。实时荧光定量PCR (qPCR) 分析了4株阳性表达菌株中mtg基因拷贝数,进一步研究了不同基因拷贝数对重组毕赤酵母产酶的影响及高产菌株在3 L发酵罐高密度发酵。结果表明,被检测的4株阳性表达菌株中mtg拷贝数分别为2.21、3.36、5.72和7.62 (mtg-2c、mtg-3c、mtg-6c和mtg-8c),其发酵产酶能力和蛋白质表达水平为mtg-3c>mtg-2c>mtg-6c>mtg-8c;高密度发酵较低和较高拷贝数的两株菌mtg-3c和mtg-6c,发酵上清的最高酶活和单位菌体酶活分别为3.12 U/mL、52.1 U/g湿重和2.07 U/mL、36.5 U/g湿重,其中单位菌体酶活mtg-3c是mtg-6c的1.4倍;mtg-3c纯化酶的最高酶活达到7.21 U/mL,蛋白浓度为437.2 μg /mL。通过分析拷贝数对重组毕赤酵母产酶的影响,发现mtg-3c适合pro/rDNA-mtg中pro和mtg共表达,MTG高酶活与菌株较高分泌蛋白有关。     

采用混合策略提高葡萄糖氧化酶在毕赤酵母中的表达水平

为了提高葡萄糖氧化酶 (GOD) 在毕赤酵母中的表达水平,提出了甲醇/山梨醇混合碳源诱导和共表达分子伴侣二硫键异构酶 (PDI) 和透明颤菌血红蛋白 (VHb) 两种策略。利用对照菌株X33/pPIC9k–GOD 在5 L发酵罐放大培养时,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活为456 U/mL,比只采用甲醇作为单一碳源诱导时GOD最终酶活提高了20%。利用整合伴侣蛋白菌株X33/pPIC9k-GOD/pPICZ-PDI-VHb在5 L发酵罐进行高密度发酵,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活达到716 U/mL,蛋白浓度为7.4 g/L。研究结果对提高外源蛋白在毕赤酵母中的表达有重要参考价值。     

链霉菌A01-chit33CT产纳他霉素和几丁质酶协同表达发酵条件优化

生物农药由于具有良好的生态效应和安全性,因此比化学农药更受到人们的青睐,生物农药的发展契合低碳、循环、清洁绿色经济发展理念。因此,寻求利于食品安全和环境保护,同时高效控制植物病害的新型生物农药成为时下及未来研究的热点。链霉菌以产生纳他霉素等抗生素起到生防作用。链霉菌株A01-chit33CT既可以产生纳他霉素又可以高表达几丁质酶活,生防效果大大增加。为确定链霉菌A01-chit33CT产纳他霉素和几丁质酶协同表达的发酵条件,初步探索了碳氮源和发酵条件对菌株产生纳他霉素和几丁质酶的影响。结果表明,葡萄糖促进纳他霉素的产生而抑制几丁质酶的表达,因此分两阶段添加葡萄糖和几丁质粉来达到二者协同表达。研究确定最佳发酵培养基为:葡萄糖40 g/L,几丁质粉10 g/L(发酵4 d添加),黄豆粉30 g/L,大豆蛋白胨10 g/L,CaCO35 g/L,MgSO4.7H2O 0.5 g/L,K2HPO40.5 g/L。最优发酵条件为:初始pH 6.0,温度28℃,转速180 r/min。在此条件下,链霉菌A01-chit33CT产纳他霉素达1.52 g/L,同时几丁质酶活达990 U/ml,二者比优化前的水平分别提高了1.95倍和2.27倍。     

Amphibacillus xylanus谷氨酸脱氢酶基因工程菌培养条件优化

首先构建了5株表达NADH依赖型谷氨酸脱氢酶的大肠杆菌基因工程菌,获得来源于Amphibacillus xylanus的谷氨酸脱氢酶AxyGDH。其最适温度为60℃、最适p H为8.0,该条件下比酶活达到(903.1±24.6)U/mg,酶活半衰期为167h。其次,确定了表达AxyGDH的大肠杆菌基因工程菌E.coli BL21(DE3)/pET-28a(+)-AxyGDH的产酶条件:诱导剂IPTG浓度为0.7mmol/L、诱导温度为25℃;优化后的培养基组成为:甘油11.3g/L,酵母粉16.3g/L,Mg SO_4·7H_2O 0.62g/L,NaCl 0.5g/L,Na_2HPO_4·12H_2O 17.1g/L,KH_2PO_43g/L,NH_4Cl 1.5g/L。最后,在10L发酵罐中控制比生长速率为0.2h~(-1)进行补料分批发酵,所得AxyGDH的发酵酶活为(9 066±45)U/ml,是LB摇瓶发酵酶活的51.1倍,为谷氨酸脱氢酶的低成本生产奠定了基础。     

人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

将人胱硫醚β-合酶(CBS)基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta (DE3)菌株,构建了高效表达CBS的重组菌E.coli Rosetta (pGEX4T-1-CBS)。重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基。将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1%CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1mmol/L的S-腺苷甲硫氨酸(AdoMet)可使CBS单位酶活提高5.1倍,达到735U/mg。同时构建了表达CBS_1-413(删除了CBS羧基端调控域138个氨基酸残基)的重组菌E.coli Rosetta (pETDuet-1-CBS_1-413),经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965U/mg; 还表达和纯化了胱硫醚β-裂解酶(CBL),并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法。     

链霉菌G4溶菌酶的产生条件及特性

对链霉菌G4的产酶发酵条件和溶菌特性进行研究结果表明:蔗糖30 g/L、大豆蛋白胨12.5 g/L、牛肉膏2 g/L,对产酶最为有利;G4溶菌酶最适培养温度33 ℃,培养时间72 h,培养基初始pH 8.G4溶菌酶的最适作用温度和最适作用pH分别是55 ℃和6.5,多数金属离子会抑制G4溶菌酶的活性,其中Zn2+、Cu2+、Fe2+、 Pb2+几乎可以使其完全失活;对几种细菌、酵母菌的研究表明,G4溶菌酶对卵清溶菌酶不能作用的变形链球菌和金黄色葡萄球菌有很强的溶解活性.     

Translation of bacteriophage T4 mRNA into active lysozyme by a wheat germ cell-free system.

T4 bacteriophage mRNA for lysozyme was extracted from T4 phage infected $\text{E. coli}$ cells, partially purified by column chromatography, and translated in a heterologous cell-free protein synthesizing system prepared from wheat germ. The translation product was confirmed by SDS polyacrylamide gel electrophoresis and enzymatic activity — bacteriolysis as tested with $\text{Micrococcus luteus}$. The specific activity of the enzyme prepared was 660 U/mg.     

Enhanced cellulase production through random mutagenesis of Talaromyces pinophilus OPC4-1 and fermentation optimization

Reducing cellulase cost remains a major challenge for lignocellulose to fuel and chemical industries. In this study, mutants of a novel wild-type cellulolytic fungal strain Talaromyces pinophilus OPC4-1 were developed by consecutive UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG) and ethylmethane sulfonate (EMS) treatment. A potential mutant EMM was obtained and displayed enhanced cellulase production. Using Solka Floc cellulose as the substrate, through fed-batch fermentation, mutant strain T. pinophilus EMM generated crude enzymes with an FPase activity of 27.0 IU/mL and yield of 900 IU/g substrate. When corncob powder was used, strain EMM produced crude enzymes with an FPase activity of 7.3 IU/mL and yield of 243.3 IU/g substrate. In addition, EMM crude enzymes contained 29.2 and 16.3 IU/mL β-glucosidase on Solka Floc cellulose and corncob power, respectively. The crude enzymes consequently displayed strong biomass hydrolysis performance. For corncob hydrolysis, without supplement of any commercial enzymes, glucose yields of 591.7 and 548.6 mg/g biomass were obtained using enzymes produced from Solka Floc cellulose and corncob powder, respectively. It was 553.9 mg/g biomass using the commercial enzyme mixture of Celluclast 1.5 L and Novozyme 188. Strain T. pinophilus EMM was therefore a potential fungus for on-site enzyme production in biorefinery processes.     

Use of magnetic poly(glycidyl methacrylate) monosize beads for the purification of lysozyme in batch system

The hydrophobic affinity ligand L-tryptophan immobilized magnetic poly(glycidyl methacrylate) [m-poly(GMA)] beads in monosize form (1.6 microm in diameter) were used for the affinity purification of lysozyme from chicken egg white. The m-poly(GMA) beads were prepared by dispersion polymerization in the presence of Fe3O4 nano-powder. The epoxy groups of the m-poly(GMA) beads were converted into amino groups with 1,6 diaminohexane (i.e., spacer arm). l-tryptophan was then covalently immobilized on spacer arm attached m-poly(GMA) beads. Elemental analysis of immobilised L-tryptophan for nitrogen was estimated as 42.5 micromol/g polymer. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration and temperature). Maximum lysozyme adsorption amount of m-poly(GMA) and m-poly(GMA)-L-tryptophan beads were 1.78 and 259.6 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, m-poly(GMA)-L-tryptophan beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. It was found to be successful in achieving purification of lysozyme in a high yield of 76% with a purification fold of 71 in a single step. The specific activity of the eluted lysozyme (62,580 U/mg) was higher than that obtained with a commercially available pure lysozyme (Sigma (60,000 U/mg).     

产木聚糖酶白地霉培养特性及部分纯化的酶学特性

本文对白地霉Ref1的培养特性、产酶条件和酶学特性进行了初步研究。结果表明:该菌为低温型菌株,其最佳生长条件为pH6、20℃和酵母膏作为氮源;最佳产酶条件为pH3-7、15℃及以酵母膏氮源;条件优化后产酶可达118.7U/mL,可溶蛋白含量可达到60μg/mL,酶溶液的比活可达到1250U/mg蛋白质;该木聚糖酶的最适反应温度和pH分别为50℃和5,金属离子Mg2+、Na+和8mmol/L的Fe2+、Cu2+、Zn2+等对木聚糖酶的活性有抑制作用,而Ca2+、4mmol/L的Fe2+、Cu2+、Zn2+和8mmol/L的Mn2+等对该酶反应则有促进作用;该木聚糖酶在保温2h后在15-40℃范围内能保持80%以上的酶活性,在50℃时能保持68%的酶活性;用lineweaver-Burk作图法(双倒数作图法)求得该酶的最大反应速度Vmax和Km值分别为163.38mmol/mg/min和0.75mg/mL。     

Lysozyme purification with dye-affinity beads under magnetic field

Magnetic poly(2-hydroxyethyl methacrylate) mPHEMA beads carrying Cibacron Blue F3GA were prepared by suspension polymerization of HEMA in the presence of Fe3O4 nano-powder. Average size of spherical beads was 80-120 microm. The beads had a specific surface area of 56.0m(2)/g. The characteristic functional groups of dye-attached mPHEMA beads were analyzed by Fourier transform infrared spectrometer (FTIR) and Raman spectrometer. mPHEMA with a swelling ratio of 68% and carrying 28.5 micromol CibacronBlueF3GA/g were used for the purification of lysozyme. Adsorption studies were performed under different conditions in a magnetically stabilized fluidized bed (i.e., pH, protein concentration, flow-rate, temperature, and ionic strength). Lysozyme adsorption capacity of mPHEMA and mPHEMA/Cibacron Blue F3GA beads were 0.8 mg/g and 342 mg/g, respectively. It was observed that after 20 adsorption-desorption cycle, mPHEMA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 87.4% with recovery about 79.6%. The specific activity of the desorbed lysozyme was high as 41.586 U/mg.     

里氏木霉液体发酵产纤维素酶的研究

在摇瓶试验基础上,采用里氏木霉(Trichoderma reesei)HC-415菌株进行5L自控罐产纤维素酶深层发酵试验。在通气量为 0.2—0.6vvm、搅拌速度为 400r/min、发酵液pH控制在5.8—6.1的条件下,发酵液的羧甲基纤维素(CMC)酶酶活最高为325.0mg糖/ml,滤纸糖酶(FPA)酶活最高达17.9mg糖/ml。发酵周期为108h。所得冻干纤维素酶粉CMC酶活最高3111IU/g,FPA最高135IU/g ,对发酵液得率平均6.7g/L。酶活总收率CMC酶活平均78.2％,FPA酶活平均73.5％。     

Isolation and properties of malate dehydrogenase from meso- and thermophilic bacteria

A scheme of purification of malate dehydrogenase from Macromonas bipunctata strain D-405 and Vulcanithermus medioatlanticus DSM 14978T was developed. This scheme was used to obtain electrophoretically homogeneous enzyme preparations of the mesophilic bacterium M. bipunctata (specific activity, 26.9 +/- 0.8 U/mg protein; yield, 10.9%) and the thermophilic bacterium V. medioatlanticus (specific activity, 5.0 +/- 0.2 U/mg protein; yield, 19.2%). Using these high-purity enzymatic preparations, the physicochemical and regulatory properties of malate dehydrogenase were studied and the differences in kinetic characteristics and thermal stability of the preparations were determined.     

Immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571 using modified sodium alginate beads

An experimental design was carried out to evaluate the effect of the concentrations of sodium alginate, glutaraldehyde and activated coal on the immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571. The experimental condition of 20?g/L of sodium alginate, 50?mL/L of glutaraldehyde and 30?g/L of activated coal led to the highest specific activity (2,063.5?U/mg of protein), corresponding to an enhancement of about 26 times compared to the activity of the free enzyme (79.1?U/mg of protein). The effect of pH and temperature on the immobilized enzyme activity was also evaluated, showing optimal activities at pH of 5.5 and 55?°C. The study of storage of immobilized inulinase in different temperatures showed that the extract kept its initial activity after 43?days of storage at 40 and 50?°C and after 138?days of storage either at 4 or 25?°C.