交联血管紧张素转化酶聚集体的制备及性质

制备了交联血管紧张素转化酶聚集体（ACE-CLEAs）,比较了ACE-CLEAs及游离ACE的酶学性质,包括最适酶促反应温度、最适pH值、Km、vmax、温度稳定性及pH稳定性等。 以酶活力回收率为参考,确定了制备ACE-CLEAs的最佳条件为：饱和度为80%的(NH4)2SO4溶液作为沉淀剂,沉淀时间0.5 h,质量分数为0.02%的戊二醛作为交联剂,交联时间1 h。 通过比较酶学性质发现,ACE-CLEAs比游离ACE具有更好的温度稳定性及pH稳定性,且与游离ACE接近的Km值表明,ACE-CLEAs对底物的亲和力与游离酶几乎相当。     

聚丙烯酸甲酯载体对柱状假丝酵母脂肪酶固定化的研究（Ⅰ）

合成了一系列不同结构的聚丙烯酸甲酯，考察了它们的固定化酵母脂肪酶催化酯水解反应的效果，得到了载体孔结构对固定化效果影响的一些规律．研究了最佳吸附条件，比较ｐＨ和离子强度对酵母脂肪酶自由酶和固定化酶催化酯水解反应活力的影响．     

聚甲基丙烯酸羟乙酯载体固定化酵母脂肪酸在有机溶剂中催化酯合成反应的研究

用悬浮聚合法合成了一系列聚甲基丙烯酸羟乙酯载体，考察了它们固定化酵母脂肪酶活力与载体的交联度和致孔剂用量之间的关系。研究了这些固定化酵母脂肪酶在有机溶剂中催化酯合成反应的活性。脂肪酶的固定化使之活力表达更为充分，对亲水性较强的有机溶剂有更强的耐受性，并能为其在有机溶剂中催化酯合成反应提供必需水。考察了ｐＨ值，底物种类对固定化酵母脂肪酶催化酯合成反应的影响。     

结合分子模拟探讨不同醚键PBS基共聚物的性能与酶催化降解

以硫二甘醇取代二甘醇,在聚(丁二酸丁二醇酯)(PBS)分子主链上分别引入硫醚和氧醚基团,得到聚(丁二酸丁二醇酯-丁二酸硫代二乙二醇酯)[P(BS-co-TDGS)]和聚(丁二酸丁二醇酯-丁二酸二乙二醇酯)[P(BS-co-DEGS)],通过热重分析(TG)和X射线衍射(XRD)测试比较了二者的结晶性能和热性能.采用南极假丝酵母脂肪酶N435(CALB)为催化剂,在水相中研究了P(BS-co-TDGS)和P(BS-co-DEGS)的降解规律及差异性.采用分子模拟方法研究了共聚物可能存在的聚集态以及N435酶与底物的结合,模拟结果验证了共聚物P(BS-co-TDGS)的结晶度下降及热稳定性降低的结论.分子对接模拟结果表明,N435酶与DEGSDEG单元的结合能更大,即含有丁二酸硫代二乙二醇酯键型底物P(BS-co-DEGS)与N435酶活性位点的对接更为稳定.     

酶促水解法拆分布洛芬乙酯的工艺研究

以布洛芬乙酯为反应底物,探索了酶促水解法拆分布洛芬的工艺.以曲拉通X-100为表面活性剂,利用南极假丝酵母脂肪酶B(CAL-B)催化拆分外消旋布洛芬乙酯,并对表面活性剂含量、有机溶剂种类和添加量、酶量、缓冲溶液pH、温度及时间等反应条件进行了优化,得到的最佳反应条件为:在pH为9.0的缓冲溶液中,添加80 mg曲拉通X-100、 50μL二氯甲烷和15 mg CAL-B,于30℃下反应48 h,在该条件下,S-布洛芬乙酯的剩余率(C)为65%,布洛芬乙酯的对映体过量值(ee_s)为94%.     

Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH₄)₂SO₄ (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U₁₀(5⁶) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.     

京尼平交联磁性壳聚糖微球的制备及其脂肪酶的固定化

采用反相悬浮法与溶胶凝胶法结合制备磁性壳聚糖微球,并以此为载体,京尼平为交联剂,脂肪酶为模型酶进行固定化,研究了酶固定化的最优条件和固定化酶的性质。结果表明,在京尼平浓度为0.6 g/L、交联温度为55 ℃、交联时间8 h,固定化酶的比活力最大,为4.31 U/g。固定化酶在25~35 ℃,pH值在8.0有最大活性,其米氏常数Km为0.26 mol/L。同时,固定化酶具有良好的热稳定性及pH稳定性,可重复利用,且能进行磁分离。     

木瓜蛋白酶交联聚体的制备及性质

采用交联酶聚体(CLEAs)技术制得了木瓜蛋白酶CLEAs, 优化了制备条件. 以纯乙醇为蛋白沉淀剂, 质量分数40%戊二醛为交联剂, 于4 ℃下对酶沉淀聚体交联16 h; 所得木瓜蛋白酶CLEAs的最适pH为6.0(游离酶最适pH=7.0), 最适温度范围由游离酶的80 ℃拓宽为50～80 ℃, 热稳定性和溶液稳定性亦明显提高; 微观形貌分析证明木瓜蛋白酶CLEAs优良的催化效能及稳定性来自于CLEAs单元所具有的高比表面积及单元内部多点共价固定的结合方式.     

Regioselective acylation of resveratrol catalyzed by lipase under microwave

The enzymatic regioselective acylation of resveratrol was achieved in organic solvents catalyzed by the lipase from Candida sp. 99–125 (CSL) under microwave irradiation. Influences of various reaction conditions have been studied. After selecting the optimum conditions [MTBE (20?ml, a_w?=?0.38), resveratrol (0.1?mmol), vinyl acetate (1.0?mmol) and CSL (100.0?mg) under microwave irradiation (35°C, 400 W)], CSL exhibited a satisfied enzyme activity (281?±?11?μmol/g/h) and yield of 75% for 4′-O-acetyl- resveratrol could be obtained in about 4?h when performing the reaction on a 25-fold-larger scale.

Schematic illustration of the enzymatic regioselective acylation of resveratrol catalyzed by the lipase from Candida sp. 99–125 (CSL) under microwave irradiation.     

青霉素酰化酶交联酶聚体的制备及热失活动力学

以0.53 g/mL硫酸铵为沉淀剂, 0.35%(体积分数)戊二醛为交联剂制得青霉素酰化酶交联酶聚体(CLEAs), 酶活收率30.1%, 其最适温度(57 ℃)比游离酶提高10 ℃, 最适pH(10.0)向碱性偏移1.7个单位. 对比游离酶及其CLEAs的热稳定性和热失活动力学模型发现, 游离青霉素酰化酶制成CLEAs后, 其热失活动力学模型由一步失活转变为连串失活, 失活反应活化能由248.8 kJ/mol增加至549.2 kJ/mol, 对CLEAs热稳定性大幅提高的原因进行了解释. CLEAs重复利用7次后, 酶活保留56%以上, 具有良好的重复利用性.     

Pseudomonas sp. Lipase Immobilized on Magnetic Porous Polymer Microspheres as an Effective and Recyclable Biocatalyst for Resolution of Allylic Alcohols

Magnetic porous polymeric microspheres containing epoxy groups were prepared by suspension polymerization (denoted as magnetic Fe₃O₄@GEM microspheres). Fe₃O₄@GEM with a specific surface area of 30.41 m²/g, average pore diameter of 17.13 nm, and pore volume of 0.13 cm³/g exhibited superparamagnetic behavior with the saturation magnetization of 7.1 emu/g. The content of epoxy groups on Fe₃O₄@GEM was 0.22 mmol/g. Pseudomonas sp. lipase (PSL) was covalently immobilized onto the Fe₃O₄@GEM microspheres through the reaction between the amino groups of the enzyme and the epoxy groups on the microspheres. PSL/Fe₃O₄@GEM exhibited enhanced enantioselectivity for the resolution of allylic alcohol to the corresponding optically active (S)‐allylic alcohol and (R)‐allylic alcohol acetate compared to free PSL. The enantiomeric excess of (S)‐l‐pheny‐2‐propen‐1‐ol for the former (98.1%) was 81.7 times that of the latter (1.2%) when the immobilized PSL was used for transesterification resolution of (R,S)‐l‐pheny‐2‐propen‐1‐ol. Furthermore, the ee_s and ee_p values were still retained at 95.2% and 95.4% after PSL/Fe₃O₄@GEM was recycled 10 times, indicating that PSL/Fe₃O₄@GEM had very good reusability. In addition, the transesterification resolution of (R,S)‐1‐(4‐methylphenyl)‐2‐propen‐1‐ol and (R,S)‐1‐(4‐bromophenyl)‐2‐propen‐1‐ol was catalyzed by PSL/Fe₃O₄@GEM, affording ideal ee_s and ee_p values of 99.3%, 97.4% and 99.6%, 98.2%, respectively. Therefore, PSL/Fe₃O₄@GEM demonstrated its potential as a highly efficient enzymatic reactor and Fe₃O₄@GEM would be very promising carriers for immobilizing enzymes in industrial application.     

Production of 4-Deoxy-L-erythro-5-Hexoseulose Uronic Acid Using Two Free and Immobilized Alginate Lyases from Falsirhodobacter sp. Alg1

Falsirhodobacter sp. alg1 expresses two alginate lyases, AlyFRA and AlyFRB, to produce the linear monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from alginate, metabolizing it to pyruvate. In this study, we prepared recombinant AlyFRA and AlyFRB and their immobilized enzymes and investigated DEH production. Purified AlyFRA and AlyFRB reacted with sodium alginate and yielded approximately 96.8% DEH. Immobilized AlyFRA and AlyFRB were prepared using each crude enzyme solution and κ-carrageenan, and immobilized enzyme reuse in batch reactions and DEH yield were examined. Thus, DEH was produced in a relatively high yield of 79.6%, even after the immobilized enzyme was reused seven times. This method can produce DEH efficiently and at a low cost and can be used to mass produce the next generation of biofuels using brown algae.