与环境友好的化学-生物法制备D-精氨酸和L-瓜氨酸

L-精氨酸在水溶液中利用自身所产生的碱性溶液,用n(水杨醛):n(1-精氨酸)=0.1:1的水杨醛为催化剂,L-精氨酸可以快速消旋为DL-精氨酸.后以DL-精氨酸为底物,利用粪链球菌(streptococcus faecalis)精氨酸脱亚胺酶对L-精氨酸的专一脱亚胺作用,同时制备D-精氨酸和L-瓜氨酸,分别以92.3%、94.2%产率获得光学纯的D-精氨酸和L-瓜氨酸.     

反胶束萃取精氨酸脱亚胺酶

报道了用反胶束体系萃取精氨酸脱亚胺酶(ADI)的研究结果,为精氨酸脱亚胺酶的分离纯化提供了一种方法。在反胶束体系中采用十六烷基三甲基溴化铵(CTAB)作为表面活性剂,正辛烷和丁醇作为溶剂及助溶剂。考察了水相pH、振荡时间、离子强度、表面活性剂浓度及精氨酸脱亚胺酶质量浓度等因素对精氨酸脱亚胺酶分离纯化效果的影响。实验结果表明,当c(CTAB)=0.01 mol/L,pH=7,振荡时间15 min,体系中用于反萃取的c(NaCl)=0.75 mol/L,且起始粗酶质量浓度控制在30 g/L时,通过辛烷/丁醇/CTAB反胶束体系的萃取和反萃取,ADI酶液萃取率E达到85%,比活达到1.107 U/mg,是起始酶液的4.52倍。     

精氨酸酶转化生产D-精氨酸和L-鸟氨酸

以L-精氨酸为原料,经水杨醛催化消旋反应得到DL-精氨酸,在精氨酸酶的作用下,定向地将L-精氨酸转化为L-鸟氨酸和尿素,并经进一步分离纯化得到D-精氨酸和L-鸟氨酸盐酸盐。该文通过水杨醛对L-精氨酸进行消旋化,反应2.5 h,消旋率可达74.5%;同时,对L-精氨酸酶促反应条件进行了优化,结果表明,最佳反应条件为:37℃,pH=10,反应时间24 h,底物质量浓度40 g/L,酶质量浓度0.2 g/L,在该条件下转化率可达98.0%。经该法制备得到的产物D-精氨酸和L-鸟氨酸盐酸盐纯化后光学纯度可达99.0%。     

固定化细胞酶法拆分DL-精氨酸

以DL-精氨酸为原料,用固定化细胞酶法拆分DL-精氨酸,并对拆分条件进行了研究。结果表明:最适反应温度55℃,最适pH=6.0,c(DL-精氨酸)=0.3 mol/L,ρ(菌体)=30 g/L,拆分反应10 h,拆分率达98%以上。连续反应10批次,固定化细胞仍保留最高酶活力的75%。产物经分离纯化,D-精氨酸收率达84.0%,[α]2D5=-27.4°〔ρ(D-精氨酸)=20 g/L,5 mol/L盐酸〕。     

重组大肠杆菌苯丙氨酸解氨酶稳定性研究

考察了甘油、Mg2+和二硫苏糖醇(DTT)对重组大肠杆菌苯丙氨酸解氨酶(PAL)稳定性的影响,并用响应面法优化筛选了最佳稳定剂组合。结果表明,φ(甘油)=10%和c(Mg2+)=0.05mol/L组合可以显著提高酶稳定性,在连续三轮转化后,PAL比酶活仍然能达到62.43U/g,是对照的1.53倍,明显好于单独使用φ(甘油)=10%或Mg2+。响应面分析结果表明,甘油在提高稳定重组PAL稳定性过程中起的作用最显著,最佳稳定剂组合条件是:c(Mg2+)=0.115mol/L,ρ(DTT)=0.52g/L,φ(甘油)=10.03%。在该条件下,在连续三轮转化后,PAL比酶活基本维持在82.53U/g,是对照的2倍;5L发酵罐PAL可以达到876U/g,酶活力是对照的近4倍。     

硝酸银沉淀L-精氨酸的研究

以硝酸银为沉淀剂,用稀硝酸和热氢氧化钡调节pH值,沉淀溶液中的L-精氨酸。考察沉淀温度、pH值、硝酸银与L-精氨酸的摩尔比、L-精氨酸的初始质量浓度、和L-赖氨酸对硝酸银沉淀L-精氨酸的影响。结果表明,随着温度升高沉淀率略有下降;pH值在10.0以上时沉淀率明显增大,pH值至11.0时沉淀率达到最大,继续增大pH值沉淀率基本不再变化;硝酸银与L-精氨酸的摩尔比达到2.0时,沉淀率可达90.0%以上;L-精氨酸的初始浓度应大于10.0 g/L,沉淀率才可达90.0%以上;钠离子浓度对沉淀率基本无影响,但是当铵离子浓度或L-赖氨酸与L-精氨酸的质量比增大时,沉淀率迅速下降。最后,用2.0 mol/L盐酸溶解沉淀,过滤,滤液用高效毛细管电泳检测,并与L-精氨酸标准峰比较,结果表明沉淀中的L-精氨酸可初步分离出来。     

精氨酸脱亚胺酶发酵条件研究

对一种新型肿瘤增殖抑制酶类一精氨酸脱亚胺酶的发酵生产工艺进行了研究。运用单因子扫描方法,考察了不同碳源、氮源等影响因子对粪肠球菌（Enterococcus faecalis）NJ402产精氨酸脱亚胺酶的影响。优选出其较佳培养基组分为（g／L）;蔗糖15,蛋白胨5,酵母膏5,牛肉膏2．5,NaCl3,KH2PO42,MgSO40．01,MnSO40．0025,L-精氨酸15;接种量以4％～5％为宜,最佳培养温度是37℃,最适起始pH为7．5。100L发酵罐实验表明,发酵到10h时,菌量与比酶活量均为最大,分别为40mg／mL和2．57U／mL。     

苯丙氨酸脱氨酶发酵工艺及其酶学性质研究

选育出一株具有较高苯丙氨酸脱氨酶活性的菌株巨大芽孢杆菌AS1.127-NJU10。考察了该菌的发酵产酶条件,结果表明:蔗糖为最佳碳源、酵母浸膏和NH4C l组成最佳氮源,其质量浓度分别为:ρ(蔗糖)=20 g/L,ρ(酵母浸膏)=2 g/L和ρ(NH4C l)=10 g/L;发酵培养基最适pH=6.5,培养温度为37℃;诱导物ρ(L-苯丙氨酸)=1 g/L时,酶活最高达1 070 U。同时对苯丙氨酸脱氨酶的性质进行了研究,结果表明:该酶最适pH=5.8,最适温度为40℃,反应液中添加φ(吐温-80)=0.2%和c(K+)=10-5mol/L能明显提高酶活。     

732离子交换树脂从胱氨酸母液提取L-精氨酸的研究

实验考察了温度、pH值、氯化铵和氯化钠浓度对 732阳离子交换树脂吸附L -精氨酸的影响 ,并测定了 2 5℃时 732离子交换树脂吸附L -精氨酸的吸附等温线。结果表明 :温度变化对吸附率影响较小 ;pH值增大 ,吸附率下降 ;溶液中氯化铵或氯化钠浓度增大 ,吸附率迅速下降 ,且当氯化铵或氯化钠物质的量浓度达到1.0mol·L-1时 ,L -精氨酸难于被吸附 ;2 5℃时 ,最大饱和吸附量约为 117g·kg-1树脂。根据实验结果开发了从胱氨酸母液提取L -精氨酸的工艺 ,提取率达到 80 %以上。     

酶法分离制备γ-氨基丁酸和L-天冬氨酸

以L-谷氨酸(L-G lu)和L-天冬氨酸(L-Asp)两种混合酸性氨基酸〔m(L-G lu)∶m(L-Asp)=1∶1〕为原料,利用大肠杆菌菌体内脱羧酶对L-谷氨酸的专一脱羧作用,酶法分离制备了γ-氨基丁酸和L-天冬氨酸。考察了转化体系温度、pH等影响L-谷氨酸脱羧酶活力的主要因素,实验表明,最佳工艺条件为:温度35℃,转化体系pH=5.0,ρ(菌体)=6 g/L,ρ(Tween80)=0.15 g/L,菌龄16 h,ρ(底物)=60 g/L。L-谷氨酸脱羧酶在最适转化条件下比酶活高达15 036 U。1 g湿菌体可重复使用3次共转化L-谷氨酸和L-天冬氨酸混合物30 g,其中L-谷氨酸完全转化为γ-氨基丁酸。γ-氨基丁酸及L-天冬氨酸的总收率可分别达到理论收率的88%和90%。     

Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223

Experiments were carried out to investigate the effects of various factors on the activity and conformation of recombinant leucine aminopeptidase of Bacillus kaustophilus CCRC 11223 (BkLAP) and potential utilization of BkLAP in the hydrolysis of anchovy protein. Optimal temperature and pH of BkLAP were 70 °C and 8.0 in potassium-phosphate buffer, respectively, and the activity was strongly stimulated by Ni(2+), followed by Mn(2+) and Co(2+). Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of BkLAP to different extents and further induce the changes in enzymatic activity. The secondary structure of BkLAP was slightly modified by Ni(2+) at the concentration of 1×10(-4) M, however, significant changes on the secondary structures of the enzyme were observed when Hg(2+) was added to the concentration of 1×10(-4) M. The potential application of BkLAP was evaluated through combination with the commercial or endogenous enzyme to hydrolysis the anchovy protein. Results showed that combining the BkLAP with other enzymes could significantly increase the degree of hydrolysis and amino acid component of hydrolysate. In this regard, BkLAP is a potential enzyme that can be used in the protein hydrolysate industry.     

Tyrosine-derived quinone cofactors

Copper amine oxidases (CAOs) and lysyl oxidase (LOX) both contain Cu(2+) and quinone cofactors that are derived from a tyrosine residue in the active site. In CAOs, the cofactor is 2,4,5-trihydroxyphenylalanine quinone (TPQ), and in LOX it is lysine tyrosyl quinone (LTQ). The mechanism of oxidative deamination by CAOs is well understood, but there is a controversy surrounding the role of Cu(2+) in cofactor reoxidation. The chemistry of LTQ in LOX, by contrast, has not been as extensively studied. This Account discusses the strategies that CAOs have evolved to control the mobility of TPQ to optimize activity. In addition, some recent studies on CAOs whose active-site Cu(2+) has been replaced with Co(2+) or Ni(2+) are summarized. Finally, there is a discussion on the properties of a model compound of LTQ and their relevance to the chemistry of LOX.     

贫燃条件下丙烯选择还原NO铜基SO_4~(2-)改性钛交联蒙脱催化剂的制备与性能

采用水热法制备钛交联蒙脱土,SO2-4改性后作为载体,担载上铜,制成铜基SO2-4改性钛交联蒙脱土(Cu Ti PILM/SO2-4)催化剂,用C3H6作还原剂,考察了贫燃条件下不同空速、不同氧浓度以及H2O对催化剂活性的影响。结果表明,SO2-4用量w(SO2-4)为30%、Cu2+担载量w(Cu2+)为5%、500℃焙烧制得的Cu Ti PILM/SO2-4催化活性高,反应温度为250℃时,能使NO转化率高达70 6%,在水蒸气存在下,NO最大转化率仅下降7 0%。     

固定化黄孢原毛平革菌生产乙二醛氧化酶的研究

聚氨酯泡沫固定化黄孢原毛平革菌限碳培养能有效地生产乙二醛氧化酶,乙二醛氧化酶的底物丙酮醛、乙二醛等不能诱导乙二醛氧化酶的合成,藜芦醇、苯甲醇能诱导乙二醛氧化酶的合成。Mn2+浓度为2.96×10-5mol/L时活力最高,Fe2+、Cu2+对产量影响不大,空气中能合成乙二醛氧化酶,但效果不如通氧。在较优的培养条件下,乙二醛氧化酶活力为29.1U/L。     

Ag／TiO2光催化氧化还原反应脱除水体中无机氮

采用光催化还原法制备了高活性的载银二氧化钛光催化剂，并用XRF、TEM、XRD及XPS、UV-Vis方法对催化剂进行了表征．考察了负载舨的含量、催化剂制备时间、搅拌气体的种类以及Fe^2＋的添加等条件对该催化剂光催化水体脱氮活性的影响．结果表明：载银量1．0％时去除效果最佳；制备催化剂时光照时间不充分会使催化剂失去还原效力；氮气保护下催化剂反应活性更高；Fe^2＋的加入利于光催化反应；2h光催化含氨氮和亚硝基氮的水样。总脱氮率达到了63．9％．     

离子及表面活性剂对甜高粱秆渣酶解的影响

为了提高纤维素酶水解经高温液态水处理后的甜高粱秆渣的效率,探讨了多种阴离子、阳离子以及吐温80(Tween 80)对纤维素酶活力的影响,并初步探讨了Tween 80影响甜高粱秆渣酶解的机制。酶激活试验表明,Br^-、I^-、NO₃^-、Ca²⁺、Mg²⁺和Co²⁺对纤维素酶有激活作用,但对甜高粱秆渣的水解效率提高不明显。添加Tween 80发现,随着浓度的增加,它对纤维素酶的抑制作用增强,而Tween 80添加量为0.175 ml·(g甜高粱秆渣)^-1时,甜高粱秆渣的酶解效率由16.6%提高到37.9%。吸附试验表明,甜高粱秆渣对纤维素酶和Tween 80的吸附达到一定限度后不再上升,Tween 80能显著降低甜高粱秆渣对纤维素酶的吸附。红外光谱分析发现,木质素对Tween 80的吸附要强于它对纤维素酶的吸附。     

PVP-PdCl2-CrCl3/GLM-PEG400催化芳香卤化物的脱卤研究

用聚乙烯吡咯烷酮(PVP)配合双金属Pd2+、Cr3+后,再负载于平均相对分子质量为400的聚乙二醇(PEG-400)与高岭土(GLM)组成的载体上,制成双金属双负载催化剂PVP-PdCl2-CrCl3/GLM-PEG400,以甲酸钠为氢转移试剂,催化难溶于水的芳香卤化物水相脱卤,转化率可达97.36%,且催化剂易于分离,在重复使用5次后转化率仍在60%以上。     

塑料电镀前处理的离子型活化

介绍了各种离子型活化工艺,如ABS-Pd^2 活化、pd^2 -HD-1活化、银离子型活化和钯离子型活化等。ABS-Pd^2 活化是Pd^2 和ABS经粗化后的表面活性基团直接络合,Pd^2 -HD-1活化是以有机碱HD-1为络合剂,银离子型活化一般以氨为络合剂,钯离子型活化以Clˉ为络合剂。提出了银离子型和钯离子型2种活化液的配制方法、活化机理。同时从活化机理、稳定性等方面比较了胶体钯活化液和离子型活化液。     

The importance of peripheral sequences in determining the metal selectivity of an in vitro-selected Co(2+) -dependent DNAzyme

DNAzymes are catalytically active DNA molecules that use metal cofactors for their enzymatic functions. While a growing number of DNAzymes with diverse functions and metal selectivities have been reported, the relationships between metal ion selectivity, conserved sequences and structures responsible for selectivity remain to be elucidated. To address this issue, we report biochemical assays of a family of previously reported in vitro selected DNAzymes. This family includes the clone 11 DNAzyme, which was isolated by positive and negative selection, and the clone 18 DNAzyme, which was isolated by positive selection alone. The clone 11 DNAzyme has a higher selectivity for Co(2+) over Pb(2+) compared with clone 18. The reasons for this difference are explored here through phylogenetic comparison, mutational analysis and stepwise truncation. A novel DNAzyme truncation method incorporated a nick in the middle of the DNAzyme to allow for truncation close to the nicked site while preserving peripheral sequences at both ends of the DNAzyme. The results demonstrate that peripheral sequences within the substrate binding arms, most notably the stem loop, loop II, are sufficient to restore its selectivity for Co(2+) over Pb(2+) to levels observed in clone 11. A comparison of these sequences' secondary structures and Co(2+) selectivities suggested that metastable structures affect metal ion selectivity. The Co(2+) selectivity of the clone 11 DNAzyme showed that the metal ion binding and selectivities of small, in vitro selected DNAzymes may be more complex than previously appreciated, and that clone 11 may be more similar to larger ribozymes than to other small DNAzymes in its structural complexity and behavior. These factors should be taken into account when metal-ion selectivity is required in rationally designed DNAzymes and DNAzyme-based biosensors.