恶臭假单胞菌CGMCC 1388对映选择性生物降解制备(S)-扁桃酸及其衍生物的研究

从土壤中分离得到一株（R）-扁桃酸选择性降解菌,经鉴定为恶臭假单胞菌,保存于中国普通微生物保藏中心,编号为CGMCC1388。考察了扁桃酸及其降解产物对扁桃酸脱氢酶活力的影响。研究表明,在培养基中添加少量扁桃酸、苯甲酰甲酸或苯甲酸均可显著提高其产量,以扁桃酸的诱导效果最佳。3种诱导物的最适添加质量浓度分别为4、4和2 g/L。当以消旋扁桃酸为反应底物时,该菌可高选择性降解（R）-扁桃酸,回收得到的（S）-扁桃酸对映体过量值（e.e.）高于99%,反应的对映选择率（E）达130。用静息细胞催化扁桃酸降解的最适温度和pH分别为30 ℃和6.0,最适底物浓度为60 mmol/L,以双倒数法求得Km为47 mmol/L。考察了该菌对扁桃酸苯环取代衍生物的生物转化,并以高产率制备获得高对映纯度的（S）-对羟基扁桃酸和（S）-对氯扁桃酸。     

对映选择性Geotrichum sp. GXU33脂肪酶的筛选、产酶及酶学性质研究

利用溴麝香草酚蓝作为反应指示剂,快速地筛选到产对映选择性脂肪酶菌株GXU33(Geotrichum sp.),此酶能够拆分外消旋扁桃酸甲酯产生(S)-扁桃酸.此菌株最适生长、产酶条件为橄榄油 10 g/L, 酵母粉 5 g/L, Na2HPO4·12H2O 3.5 g/L, KH2PO4 1.0 g/L, MgSO4·7H2O 0.2 g/L, pH 7.0,28℃,200 r/min.PMSF和蛋白酶K对菌株生长没有影响,PMSF显著抑制酶活,蛋白酶K具有保护酶活力的作用.该脂肪酶最适作用pH 为7.5,最适作用温度为30 ℃; Ca2 ,Mg2 ,Zn2 不同程度提高酶活性,Cu2 , Co2 ,Mn2 ,Fe2 ,Fe3 严重抑制酶活性.当以5% DMSO为助剂,消旋扁桃酸甲酯20 mg,GXU33 脂肪酶1500 U,25 mmol/L磷酸钠缓冲液(pH 7.5)加至总体积2 mL,32 ℃,100 r/min, 反应8h,得到最佳拆分效果:转化率为44.8%,(S)-扁桃酸对映过量值为83.5%.     

新型R-扁桃酸脱氢酶的基因挖掘及表达鉴定

R-扁桃酸脱氢酶在苯乙酮酸的生物合成中起着关键的作用,挖掘具有高催化活性及稳定性的新型R-扁桃酸脱氢酶具有重要的意义。为了获得理想的R-扁桃酸脱氢酶,采用了基因组挖矿技术从Lactobacillus harbinensis菌株中获得了一个新型的R-扁桃酸脱氢酶LhDMDH,重组LhDMDH的比酶活高达1264.3 U/mg,约为探针的4倍,在已报道的R-扁桃酸脱氢酶中处于领先水平。同时,考察了4个重组酶主要的酶学特性,它们的最适反应温度在25~30℃,最适反应pH在9.0~9.5。动力学参数的结果表明,LhDMDH对底物的K_(cat)值为30.28 S~(-1),明显高于其它重组酶。此外,底物谱分析的结果也表明LhDMDH在外消旋扁桃酸的手性拆分及苯乙酮酸的生物合成中更具优势。在R-扁桃酸脱氢酶基因挖掘方面取得了较为理想的结果,为进一步的改造及应用奠定了坚实的基础,也为其它酶的挖掘提供了可资借鉴的经验。     

新型耐热β-1,4-木糖苷酶的重组表达及酶学性质

【目的】拟对来源于热解纤维素果汁杆菌的新型β-木糖苷酶基因(CoXyl B)进行重组表达和酶学性质研究。【方法】在大肠杆菌系统中成功表达CoXyl B基因,并通过镍柱亲和层析、强阴离子交换和凝胶层析等纯化方法获得纯酶。【结果】对CoXyl B酶学性质的研究结果显示,在以4-对硝基苯酚-β-D-木糖苷为底物时,该酶的最适反应温度为90℃,最适反应pH为6.0。在40–70℃范围内CoXyl B酶活较高且比较稳定。在pH 5.0–6.0之间,70℃孵育1 h后,CoXyl B的相对酶活仍保留80%以上。Ag~+、高浓度的SDS和PMSF对酶活力的抑制作用较显著,而高浓度Mg~(2+)、Li~+和EDTA对酶活力的激活作用较为明显。CoXyl B的k_(cat)和K_m值分别为5.0×10~(–3)s~(–1)和1.9 mmol/L。薄层层析色谱显示CoXyl B具有降解木二糖、木三糖和木四糖的能力。【结论】本研究鉴定出CoXyl B为一种新型的极端耐热木糖苷酶,CoXyl B的酶学性质研究将为其在食品热加工以及生物降解领域中的应用提供参考。     

副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei) TK1501β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86. 63kDa和675. 56U/mg。最适作用温度和pH分别为30℃和6. 5。Mg~(2+)和Ca~(2+)对β-葡糖苷酶酶活抑制作用最小,Cu~(2+)几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的K_m和V_(max)分别为1. 44mmol/L和58. 32mmol/(L·s),催化系数k_(cat)为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。     

粪便微生物宏基因组来源的热稳定性邻苯二酚1,2-双加氧酶异源表达及酶学性质

【目的】克隆倭蜂猴粪便微生物宏基因组的邻苯二酚1,2-双加氧酶基因cat PLCgl,并对该酶进行异源表达及酶学特性研究。【方法】利用宏基因组高通量测序技术获得cat PLCgl,并对其氨基酸序列进行分析。将cat PLCgl重组到载体p EASY-E2中并转化到大肠杆菌BL21(DE3)中异源表达,研究其酶学性质。【结果】cat PLCgl全长852 bp,G+C含量48%,编码283个氨基酸,理论分子量为33.56 k D。重组Cat PLCgl酶学性质分析显示最适作用p H为7.0,其中在p H 7.0–10.0范围内处理1 h后,酶活剩余90%以上;最适作用温度为40°C,在25°C和40°C条件下稳定性较好,耐受210 h酶活性几乎不变。重组酶在最适条件下的动力学参数K_m、V_(max)和k_(cat)分别为24.9μmol/L、8.3 mmol/(min·g)和13.7 s~(-1);Fe~(2+)、Hg~(2+)、Cu~(2+)、Triton X-100、SDS、Ag+强烈抑制该酶活性,而其它金属离子及有机试剂影响较小。【结论】从倭蜂猴粪便微生物宏基因组中克隆得到邻苯二酚1,2-双加氧酶基因cat PLCgl,并对重组Cat PLCgl酶学性质进行研究,该酶具有较好的热稳定性和耐碱性,在降解环境中的邻苯二酚和生产顺,顺-己二烯二酸方面具有应用潜力。     

产碱杆菌ECU0401催化拆分扁桃酸及其衍生物

从土壤中分离的1株产碱杆菌Alcaligenes sp.ECU0401具有扁桃酸脱氢酶活性,可以以扁桃酸、苯甲酰甲酸或苯甲酸为唯一C源生长,并且具有较高的脱氢酶活力。以外消旋扁桃酸为C源,采用分批补料策略培养（或反应）99h,扁桃酸累计投入量为30.4g/L,（S）-（＋）-扁桃酸被完全降解,（R）-（-）-扁桃酸回收产率为32.8%,对映体过量值（e.e.）〉99.9%。利用静息细胞作为催化剂不对称降解外消旋扁桃酸的氯代衍生物,制备获得光学活性的（R）-（-）-邻氯扁桃酸、（S）-（＋）-间氯扁桃酸和（S）-（＋）-对氯扁桃酸,光学纯度均超过99.9%e.e.。     

胆固醇氧化酶PsCO_4异源表达、纯化及酶学性质分析

胆固醇氧化酶专一性催化胆固醇为胆甾-4-烯-3-酮,广泛的应用于临床以及食品加工行业。本论文将来源于Pimelobacter simplex的胆固醇氧化酶PsCO_4,分别转化到大肠杆菌宿主BL21(DE3)、Rosetta(DE3)和C41(DE3)中,在不同温度(15℃、25℃、37℃)及IPTG诱导浓度(0.01mmol/L、0.1mmol/L、0.5mmol/L)下异源表达Ps CO_4。结果表明,转入Rosetta(DE3)菌株的PsCO_4蛋白,在IPTG浓度为0.1mmol/L、15℃下经18h诱导表达,PsCO_4可溶性表达量最高(0.63mg/ml)。异源表达的胆固醇氧化酶PsCO_4最适温度为30℃,最适pH为7.5。通过TLC,GC-MS检测出Ps CO_4催化胆固醇生成胆甾-4-烯-3-酮。以胆固醇和β-谷甾醇、豆甾醇和孕烯醇酮为底物,测定PsCO_4对四种底物的催化反应动力学参数,胆固醇k_(cat)/K_m为0.08s~(-1)·μM~(-1)分别高于β-谷甾醇(0.04s~(-1)·μM~(-1))、豆甾醇(0.005s~(-1)·μM~(-1))和孕烯醇酮(0.02s~(-1)·μM~(-1))。     

联苯利用节杆菌YC-RL1中2,3-二羟基联苯-1,2-双加氧酶BphC功能验证

【目的】通过节杆菌(Arthrobacter sp.)YC-RL1对多氯联苯降解过程中关键基因bph C的克隆与原核表达,鉴定其编码的2,3-二羟基联苯-1,2-双加氧酶Bph C的酶活特性与功能。【方法】以菌株YC-RL1全基因组为模板进行PCR扩增获得bph C基因,将该基因转入Escherichia coli BL21(DE3)感受态细胞后进行原核表达;利用镍柱亲和层析法对Bph C酶进行纯化并分别测定该酶在不同条件下对底物2,3-DHBP的催化特性,确定其最适反应pH、温度及不同金属离子对酶活特性的影响;进一步根据米氏方程对该酶的动力学参数进行测定与分析。【结果】通过PCR扩增获得了bphC基因,其大小为930 bp;对该基因进行原核表达,所得重组蛋白BphC携带有6个组氨酸标签,经纯化后体外仍具有活性,该酶作用于2,3-DHBP时的最适pH与温度分别为pH 7.4和30°C,且在最适条件下,Fe~(2+)、Cu~(2+)及Cd~(2+)等金属离子可明显促进其酶活作用,但多数金属离子对该酶有不同程度的抑制作用;该酶在与底物2,3-DHBP作用过程中,酶促动力学常数分别为K_m:8.67 mmol/L,V_(max):27.32μmol/s,k_(cat):15.55 s~(–1),k_(cat)/K_m:1.79 L/(mmol·s),其催化效率同有关报道中同类酶的动力学特性比较均有所提高。【结论】菌株YC-RL中的bphC基因对于多氯联苯的生物降解具有至关重要的作用,其编码的BphC是重要的芳香环裂解酶,该酶对其底物具有较高的亲和性,可在体外环境中发挥高效的酶促作用,具有良好的应用价值。     

对羟基扁桃酸合酶基因的克隆表达及催化特性

【目的】比较两种不同来源基因重组的对羟基扁桃酸合酶(HmaS),考察其在大肠杆菌中的表达效率。【方法】分别对东方拟无枝酸菌(Amycolatopsis orientalis)和天蓝色链霉菌(Streptomyces coelicolor)来源的hmas进行异源表达,经离子交换层析和凝胶过滤色谱分离纯化获得HmaS,并检测HmaS的酶活和催化特性。【结果】来源于S.coelicolor的HmaSSC2比酶活是来源于A.orientalis的3.6倍;来源于A.orientalis的HmaSAO最适反应温度为28°C,在弱碱性条件下的酶活稳定性较好;来源于S.coelicolor的HmaSSC2最适反应温度为35°C,在28-45°C内保持较高的酶活,具有良好耐热性,在pH 7.0左右酶活最高,更易在偏中性的条件下发挥功能。【结论】HmaSSC2更适用于代谢工程改造大肠杆菌发酵法生产扁桃酸。     

Molecular dynamics perspective on the thermal stability of mandelate racemase

Mandelate racemase from Pseudomonas putida is a promising candidate for the dynamic kinetic resolution of α-hydroxy carboxylic acids. In the present study, the thermal stability of mandelate racemase was investigated through molecular dynamics simulations in the temperature range of 303–363 K, which can guide the design of mandelate racemase with higher stability. The basic features such as radius of gyration, surface accessibility, and secondary structure content suggested the instability of mandelate racemase at high temperatures. With increase in temperature, α-helix content reduced significantly, especially the α-helices exposed to the environment. At the simulation time scale considered, intra-protein hydrogen bonds, hydrogen bonds between protein and water decreased at 363 K, while the number of salt-bridges increased. The long-distance networks remarkably changed at 363 K. A considerable number of long-lived (percentage existence time higher than 90%) hydrogen bonds and Cα contacts were lost. Root mean square fluctuation analysis revealed regions with high fluctuation, which should be helpful in the reengineering of mandelate racemase for enhanced thermal stability.     

Stabilization and activity-enhancement of mandelate racemase from Pseudomonas putida ATCC 12336 by immobilization

Mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12336 was efficiently immobilized through ionic binding onto DEAE- and TEAE 23-cellulose. The activity of the immobilized enzyme was significantly enhanced as compared to the native protein, i.e., 2.7- and 2.5-fold, respectively. DEAE-cellulose-immobilized mandelate racemase could be efficiently used in repeated batch reactions for the racemization of (R)-mandelic acid under mild conditions.     

A caveat for the use of log P values for the assessment of the biocompatibility of organic solvents

The degree of enzyme deactivation for lipases from Candida rugosa and Pseudomonas sp., hydroxynitrile lyase and mandelate racemase upon exposure to organic solvents can be correlated to their respective partition coefficients (log P values). However, three unexpected results were obtained: (1) the deactivation exerted by protic solvents, e.g., methanol, is severely underestimated; (2) little deactivation by an organic solvent cannot neccessarily be correlated to catalytic activity in this medium, and (3) in contrast to other enzymes, hydroxynitrile lyase is exceptionally stable towards deactivation by DMF.     

Characteristics of Transposon Insertion Mutants of Mandelic Acid-utilizing Pseudomonas putida Strain A10L

A soil isolate, Pseudomonas putida strain A10L that utilizes mandelate via the mandelate pathway was mutagenized by transposon Tn5-Mob insertion and a mutant 168 lacking mandelate racemase (MR) and a mutant 254 lacking benzoylformate decarboxylase (BFDC) were obtained. Expression of (S)-mandelate dehydrogenase (MDH), BFDC, NAD⁺ -dependent benzaldehyde dehydrogenase (BDH) and NADP⁺ -dependent BDH in the MR-lacking mutant was not affected by the insertion, and it was inducible similarly to the wild type strain. On the other hand, expression of MR and MDH in the BFDC-lacking mutant was low and constitutive, and NAD⁺ - and NADP⁺ -dependent BDHs were produced at a rather high level under non-induced conditions by the mutant. Genes for MR (mdlA), MDH (mdlB), and BFDC (mdlC) were indicated to be organized in an operon in the order of mdlCBA. Optical resolution to obtain (R)-mandelate, a useful synthon for pharmaceuticals, was shown to be performed with the MR-lacking mutant.     

Bacterial mandelic acid degradation pathway and its application in biotechnology

Mandelic acid and its derivatives are an important class of chemical synthetic blocks, which is widely used in drug synthesis and stereochemistry research. In nature, mandelic acid degradation pathway has been widely identified and analysed as a representative pathway of aromatic compounds degradation. The most studied mandelic acid degradation pathway from Pseudomonas putida consists of mandelate racemase, S-mandelate dehydrogenase, benzoylformate decarboxylase, benzaldehyde dehydrogenase and downstream benzoic acid degradation pathways. Because of the ability to catalyse various reactions of aromatic substrates, pathway enzymes have been widely used in biocatalysis, kinetic resolution, chiral compounds synthesis or construction of new metabolic pathways. In this paper, the physiological significance and the existing range of the mandelic acid degradation pathway were introduced first. Then each of the enzymes in the pathway is reviewed one by one, including the researches on enzymatic properties and the applications in biotechnology as well as efforts that have been made to modify the substrate specificity or improving catalytic activity by enzyme engineering to adapt different applications. The composition of the important metabolic pathway of bacterial mandelic acid degradation pathway as well as the researches and applications of pathway enzymes is summarized in this review for the first time.     

Structural and chemical trapping of flavin‐oxide intermediates reveals substrate‐directed reaction multiplicity

Though reactive flavin‐N5/C4α‐oxide intermediates can be spectroscopically profiled for some flavin‐assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition‐like complexes, the α‐ketoacid…N5‐FMN_ox complex ( I ), the FMN_ox‐N5‐aloxyl‐C′α^?‐C4α⁺ zwitterion ( II ), and the FMN‐N5‐ethenol‐N5‐C4α‐epoxide ( III ), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMN_ox an alternative reaction center can polarize to an ylide‐like mesomer in the active site. In contrast, four distinct flavin‐C4α‐oxide adducts ( IV – VII ) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer–Villiger/peroxide‐assisted decarboxylation, and epoxidation reactions. In conjunction with stopped‐flow kinetics, the multifaceted flavin‐dependent reaction continuum is physically dissected at molecular level for the first time.     

Structural basis for catalytic racemization and substrate specificity of an N-acylamino acid racemase homologue from Deinococcus radiodurans

N-acylamino acid racemase (NAAAR) catalyzes the racemization of N-acylamino acids and can be used in concert with an aminoacylase to produce enantiopure alpha-amino acids, a process that has potential industrial applications. Here we have cloned and characterized an NAAAR homologue from a radiation-resistant ancient bacterium, Deinococcus radiodurans. The expressed NAAAR racemized various substrates at an optimal temperature of 60 degrees C and had Km values of 24.8 mM and 12.3 mM for N-acetyl-D-methionine and N-acetyl-L-methionine, respectively. The crystal structure of NAAAR was solved to 1.3 A resolution using multiwavelength anomalous dispersion (MAD) methods. The structure consists of a homooctamer in which each subunit has an architecture characteristic of enolases with a capping domain and a (beta/alpha)7 beta barrel domain. The NAAAR.Mg2+ and NAAAR.N-acetyl-L-glutamine.Mg2+ structures were also determined, allowing us to define the Lys170-Asp195-Glu220-Asp245-Lys269 framework for catalyzing 1,1-proton exchange of N-acylamino acids. Four subsites enclosing the substrate are identified: catalytic site, metal-binding site, side-chain-binding region, and a flexible lid region. The high conservation of catalytic and metal-binding sites in different enolases reflects the essentiality of a common catalytic platform, allowing these enzymes to robustly abstract alpha-protons of various carboxylate substrates efficiently. The other subsites involved in substrate recognition are less conserved, suggesting that divergent evolution has led to functionally distinct enzymes.     

Protein Design through Systematic Catalytic Loop Exchange in the (β/α)8 Fold

Protein engineering by directed evolution has proven effective in achieving various functional modifications, but the well-established protocols for the introduction of variability, typically limited to random point mutations, seriously restrict the scope of the approach. In an attempt to overcome this limitation, we sought to explore variant libraries with richer diversity at regions recognized as functionally important through an exchange of natural components, thus combining design with combinatorial diversity. With this approach, we expected to maintain interactions important for protein stability while directing the introduction of variability to areas important for catalysis.Our strategy consisted in loop exchange over a (β/α)₈ fold. Phosphoribosylanthranilate isomerase was chosen as scaffold, and we investigated its tolerance to loop exchange by fusing variant libraries to the chloramphenicol acetyl transferase coding gene as an in vivo folding reporter. We replaced loops 2, 4, and 6 of phosphoribosylanthranilate isomerase with loops of varied types and sizes from enzymes sharing the same fold.To allow for a better structural fit, saturation mutagenesis was adopted at two amino acid positions preceding the exchanged loop. Our results showed that 30% to 90% of the generated mutants in the different libraries were folded. Some variants were selected for further characterization after removal of chloramphenicol acetyl transferase gene, and their stability was studied by circular dichroism and fluorescence spectroscopy. The sequences of 545 clones show that the introduction of variability at “hinges” connecting the loops with the scaffold exhibited a noticeable effect on the appearance of folded proteins. Also, we observed that each position accepted foreign loops of different sizes and sequences.We believe our work provides the basis of a general method of exchanging variably sized loops within the (β/α)₈ fold, affording a novel starting point for the screening of novel activities as well as modest diversions from an original activity.