高密度脂蛋白（High-density Lipoprotein， HDL）是血浆中重要的脂蛋白，其主要成分为载脂蛋白AⅠ(Apoliprotein AⅠ， ApoAⅠ为了大量制备该蛋白，首先尝试利用Pichia pastoris表达系统高效表达ApoAⅠ。通过PCR扩增获得天然含人载脂蛋白ApoAⅠ的基因片断，将其插入到P.pastoris分泌型载体pPIC9K上，BglⅡ酶切线性化后电转化P.pastoris GS115，将获得的1000多个转化子依次在含不同G418浓度的YPD平板筛选高抗性转化子，得到的22个高抗性转化子经甲醇诱导，SDS-PAGE检测得到6株高表达菌。然后对其中的高表达菌株AP16的培养及诱导条件进行了优化，结果显示：接种后培养24-28h，转入诱导阶段，培养基pH值在7-7.5，菌体密度OD600=80左右，以1%甲醇诱导96h最有利于ApoAⅠ的表达，表达水平达160mg/L。14L发酵罐结果显示表达水平与摇瓶相当，均高于其它表达系统，为大批量制备奠定了基础。

用分子克隆手段获得D2氨基酸氧化酶基因后，对其在不同表达系统如大肠杆菌系统、酿酒酵母和克鲁维乳酸酵母、博伊丁假丝酵母、巴斯德毕赤氏酵母系统及动物细胞内的表达作了介绍。

以HepG2.2. 2.15细胞株为模型，以其分泌的HBsAg、HBeAg、HBV DNA及细胞存活率为观察指标，综合评价天然多肽类抗生素恩拉霉素体外抗HBV 效果。结果表明恩拉霉素对HBsAg和HBeAg的50%抑制浓度IC50分别为27μg/mL 和34μg/mL，治疗指数（TI）分别为5.9 和4.6。Southern结果显示，50μg/mL恩拉霉素对细胞内游离HBVDNA抑制率为56.8%。

Using an acyl-acyl carrier protein (ACP) as a starter unit, type Ⅱ polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensation with the two-carkon donor malonyl (ACP). In vitro experimetts have demonstrated that polketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA: ACP acyltransferase (FabD) from streptomycets. It has also been shown that holo-ACPs from a typeII PKS can catalyze self -malonylation in the presence of malonyl CoA and negate this FabD requirement. The relative roles of FabD an ACP self -malonylatin in PKS biosynthesis in vivo are still not known. Results: We have examined the ACO specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable kcat IKM values for ACPs from both type II PJS and FAS systems. Incubations of tetracenomycin ACP(TcmM) withthe Escherichia coli FAS ACP (Acp P) unexpectedly revaled that, in addition to the selfmalonylation process, TcmM can catalyze the malonylation of AcpP. The ACP is two value for the TcmM-catalyzed malonylation of S. glaucescens FAS ACP is two orders of magnitude smaller than that observed for the Fabd-CAtalyzed process. Conclusion: The abilityof APKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACP and FabD can catalyze the same reation. The differences in the acatalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP.

Entry of human immunodeficiency virus type 1 (HIV-1) requires CD4 and one of a family of related seven-transmembrane-domain coreceptors. Macrophage-tropic HIV-1 isolates are generally specific for CCR5, a receptor for the CC chemokines RANTES, MIP-1a, and MIP-1b, while T-cell line-tropic viruses tend to use CXCR4 (also known as fusin, LESTR, or HUMSTR). Like HIV-1, simian immunodeficiency virus (SIV) requires CD4 on the target cell surface; however, whether it also requires a coreceptor is not known. We report here that several genetically divergent SIV isolates, including SIVmac, SIVsmSL92a, SIVsmLib-1, and SIVcpz-GAB, can use human and rhesus CCR5 for entry. CXCR4 did not facilitate entry of any of the simian viruses tested, nor did any of the other known chemokine receptors. Moreover, SIVmac251 that had been extensively passaged in a human transformed T-cell line retained its use of CCR5. Rhesus and human CCR5 differed at only eight amino acid residues, four of which were in regions of the receptor that could be exposed, two in the amino-terminal extracellular region and two in the second extracellular loop. The human coreceptor was as active as the simian for SIV entry. In addition, HIV-1 was able to use the rhesus homologs of the human coreceptors, CCR5 and CXCR4. The SIV strains tested were specific for CCR5 regardless of whether they were able to replicate in transformed T-cell lines or macrophages and whether they were phenotypically syncytium inducing or noninducing in MT-2 cells. However, SIV replication was not restricted to cells expressing CCR5. SIV strains replicated efficiently in the human transformed lymphoid cell line CEMx174, which does not express detectable amounts of transcripts of CCR5. SIV also replicated in human peripheral blood mononuclear cells that were genetically deficient in CCR5. These findings indicated that, in addition to CCR5, SIV can use one or more unknown coreceptors that are expressed on human PBMCs and CEMx174 cells.

The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was ampliWed and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-β-D-galactoside (IPTG) was found to be 5μM. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55℃, respectively. The Km and Vmax are 17.6μM and 23.8U/mg, respectively. Therefore, the high yield and high speciWc activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.

D2氨基酸氧化酶（DAAO）在转化头孢菌素C生产72ACA和转化DL2氨基酸制备α-酮酸和L-氨基酸上起着重要的作用。采用DNA操作技术，将来源于三角酵母的DAAO基因连接至表达载体pPIC3·5K上，再将表达质粒pPIC3·5K-DAAO分别整合P. pastoris的宿主细胞KM71和GS115，经筛选获得阳性重组菌PDK13（MutS）和PD27（Mut+）。重点对两种突变菌的表达条件进行了比较。结果显示：PDK13（MutS）株比PD27（Mut+）株消耗甲醇慢、诱导时间长，但对通气量要求低、表达水平高，摇瓶活力分别达到2700和2500IUPL，14L发酵罐内活力分别达到10140和8463IUPL。初步探索了DAAO对DL2苯丙氨酸的拆分，结果显示基因工程菌表达的DAAO 具有良好的转化DL2苯丙氨酸制备苯丙酮酸和L2苯丙氨酸的能力。

用分子生物学方法获得了D-氨基酸氧化酶（DAAO）的表达质粒（pPIC3.5k-DAAO），用其电转化巴斯德毕赤酵母GS115获得重组菌，经筛选获得高表达菌株PD27，并对其高密度培养和诱导条件进行了优化。PD27工程菌经250ml摇瓶发酵得到的DAAO活力达到2500～2600IU/L，在14L发酵罐中的表达水平优于其它表达系统。

Experiments on the incorportaion of D-and L-[alanine-3-13C, 2-15N) tryptophan into the antibiotic pyrrolitrin in Pseudomonas aurefaciensconfirmed earlierconclusions abou the conversin of L-tryptophan into pyrrolnitrin. They also demonstryated that a fraction ofthe D isomer is incorporta ted without breakage of the 15 N-carbon bond, consisten with the operation of a second pathway from D-tryptophan to pyrrolnitrin. Cell-free experiments confirmed the conversion of 3-(o-aminophenyl) pyrrole into aminopyrrolnitrin but failed to detect enzymatic oxidation of the latter to pyrrolnitrin.