A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate):D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13 [EC] ] was changedto arginine by site-directed mutagenesis. The k_cat of the resultingmutant protein, K146R, was 500 times slower than wild-type insteady-state kinetic assays for both cleavage and condensationof fructose-1,6-bis(phosphate), while the Km for this substratewas unchanged. Analysis of the rate of formation of catalyticintermediates showed K146R was significantly different fromthe wild-type enzyme and other enzymes mutated at this site.Single-turnover experiments using acid precipitation to trapthe Schiff base intermediate on the wild-type enzyme failedto show a build-up of this intermediate on K146R. However, K146Rretained the ability to form the Schiff base intermediate asshown by the significant amounts of Schiff base intermediatetrapped with NaBH₄. In the single-turnover experiments it appearedthat the Schiff base intermediate was converted to productsmore rapidly than it was produced. This suggested a maximalrate of Schiff base formation of 0.022 s^–1, which wasclose to the value of k_cat for this enzyme. This observationis strikingly different from the wild-type enzyme in which Schiffbase formation is >100 times faster than k_cat. For K146Rit appears that steps up to and including Schiff base formationare rate limiting for the catalytic reaction. The carbanionintermediate derived from either substrate or product, and theequilibrium concentrations of covalent enzyme-substrate intermediates,were much lower on K146R than on the wild-type enzyme. The greaterbulk of the guanidino moiety may destabilize the covalent enzyme-substrateintermediates, thereby slowing the rate of Schiff base formationsuch that it becomes rate limiting. The K146R mutant enzymeis significantly more active than other enzymes mutated at thissite, perhaps because it maintains a positively charged groupat an essential position in the active site or perhaps the Argfunctionally substitutes as a general acid/base catalyst inboth Schiff base formation and in subsequent abstraction ofthe C4-hydroxyl proton.     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.     

“绿色”溶剂离子液的研究进展

近年来，离子液由于其独特的优点, 在生物催化及化学合成领域越来越受到青睐。由于缺乏蒸气压，离子液作为绿色溶剂有很大潜能。此外，离子液不像极性有机溶剂那样会使酶失活，因而简化了类似含有糖类这样极性底物的反应。在离子液中，生物催化反应表现出较高的选择性、较快的反应速率和较高的酶稳定性。但这些溶剂还存在着离子液的纯化、控制水活性和pH、高粘度以及产品分离等问题。     

固定化脱氮硫杆菌净化硫化氢气体的研究

以海藻酸钙包埋脱氮硫杆菌制成的固定化微生物颗粒填充生物固定床,用以净化低浓度H2S废气。实验研究了温度、pH值、进气浓度及流速对反应体系中的H2S脱除率的影响,测定了生物固定床中代谢物的种类及其含量。结果表明当进气口的质量浓度低于6×10-5mg/L、25～40℃、pH值在6.0～7.5范围时,生物固定床对废气中H2S的脱除率可达90%以上,在反应过程中pH值保持不变;进气口的流速对不同浓度的H2S的影响较大,当进气口H2S质量浓度为3×10-5mg/L且流速在35L/h时,脱除率高达95%以上。元素硫作为主要产物防止了生物固定床的酸化,并保证脱硫装置的稳定性。     

温敏性微凝胶对蛋白质和酶的吸附性能

用微乳液聚合方法以N-异丙基丙烯酰胺为单体合成了温敏性微凝胶聚N-异丙基丙烯酰胺(PNIPAM),研究了其对两种蛋白质和两种酶的吸附性能,测定了吸附等温线和温度对吸附量的影响。结果表明,微凝胶在低临界溶解温度(LCST)附近吸附蛋白质和酶的量有一突跃,例如在LCST前后,1 g纳米颗粒吸附的酪蛋白的质量分别为225 mg和415 mg;吸附的枯草杆菌蛋白酶的质量分别为12 000U/mg和27 500 U/mg。蛋白质和酶是通过物理吸附作用结合到PNIPAM微凝胶上,可以用调节温度的方法,来控制温敏微凝胶对蛋白质和酶的吸附与脱附。     

Theoretical examination of the mechanism of aldose-ketose isomerization

Two mechanisms for an aldose–ketose isomerization havebeen examined using high level ab initio and semiempirical molecularorbital methods. The proton transfer pathway via an enediolintermediate is shown to be favored in the absence of a metalion, while the hydride transfer pathway becomes favored in thepresence of a metal ion. Our calculations explain why the protontransfer pathway is operative in most aldose–ketose isomerizationreactions. These calculations also provide further support forthe previously proposed metal ion-mediated hydride transfermechanism for xylose isomerase.     

固定化紫草细胞生产紫草宁衍生物

用海藻胶包埋紫草细胞,在紫草色素生产培养基M_9中,常温、黑暗下培养不同时间,收集培养液并提取色素,进行紫外—可见全波长分光光度扫描和TLC分析。结果表明:固定化紫草细胞可连续分泌紫草色素,其主要成分与天然成分基本相同,产量已达到一定水平。此外对海藻胶包埋条件、固定化珠粒的稳定性以及1L固定化植物细胞反应器生产紫草色素工艺进行了讨论。     

三种不同形式的丙酸杆菌发酵的对比研究

在相同环境条件下,对游离、海藻酸钠包埋固定以及壳聚糖交联固定三种不同形式丙酸杆菌发酵的固定化颗粒性能、产丙酸量、pH值变化、残糖浓度变化等进行了对比研究.结果表明:使用壳聚糖交联固定的丙酸杆菌,分批发酵的产酸量可达14.6 g·L-1,高于游离法(11.5 g·L-1)和海藻酸钠包埋固定法(12.4 g·L-1);且在颗粒性能方面,比海藻酸钠固定颗粒有更强的耐酸性和更高的机械强度.     

Flaking and extrusion as mechanical treatments for enzyme-assisted aqueous extraction of oil from soybeans

Flaking and extruding dehulled soybeans were evaluated as a means of enhancing oil extraction efficiency during enzyme-assisted aqueous processing of soybeans. Cellulase, protease, and their combination were evaluated for effectiveness in achieving high oil extraction recovery from extruded flakes. Aqueous extraction of extruded full-fat soy flakes gave 68% recovery of the total available oil without using enzymes. A 0.5% wt/wt protease treatment after flaking and extruding dehulled soybeans increased oil extraction recovery to 88% of the total available oil. Flaking and extruding enhanced protease hydrolysis of proteins freeing more oil. Treating extruded flakes with cellulase, however, did not enhance oil extraction either alone or in combination with protease. Discrepancies in oil extraction recoveries were encountered when merely considering crude free fat because some oil became bound to denatured protein during extrusion and/or sample drying. Bound fat was unavailable for determination by using the hexane extraction method, but was accounted for by using the acid hydrolysis method for total oil determination. Oil extraction recovery from extruded soybean flakes was affected by oil determination methods, which was not the case for unextruded full-fat soy flour.     

影响酶洗涤效果因素的探讨

介绍了加酶洗涤剂的发展以及酶在洗涤剂中的应用。探讨了加酶洗涤剂中表面活性剂及其浓度、洗涤温度和洗涤时间对洗涤效果的影响，pH值对酶活性的影响。对影响酶洗涤效果的各种因素进行了阐述。并论述了酶在洗涤剂中的其他应用。