Candidasp．99-125脂肪酶催化猪油酸解合成1，3-二油酸-2-棕榈酸甘油三酯

人乳脂是一种在甘油骨架Sn-2位上富含棕榈酸（C16：0）的结构酯。经分析可知,猪油中棕榈酸主要分布在甘油酯的Sn-2位,可作为制备1,3-二油酸-2-棕榈酸甘油三酯（OPO）的原料。以Candidasp．99—125脂肪酶作催化剂,以猪油和油酸为原料,通过正交试验对无溶剂体系中酸解合成OPO的工艺条件进行研究,得到最适反应条件：猪油与油酸的质量比为1：2．0,酶用量为总底物质量的10％,反应温度40℃,反应时间4h。在该反应条件下,经酸解合成的产物三甘酯中,Sn-2C16：0的含量大于70％,占总脂肪酸中棕榈酸含量的93％以上,并合有43％以上的OPO。     

Evidence for selective hydrolysis of aliphatic copolyesters induced by lipase catalysis

High molar mass random poly(butylene succinate-co-butylene sebacate), P(BS-co-BSe), and poly(butylene succinate-co-butylene adipate), P(BS-co-BA), with different composition, were synthesized and subjected to enzymatic hydrolysis by Lipase from Mucor miehei or from Rhizopus arrhizus. The enzymatic hydrolysis of P(BS-co-BSe)s and P(BS-co-BA)s films produced a mixture of water-soluble monomers and co-oligomers that were separated and identified by on-line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Optimization of the HPLC analysis allowed the separation of isobar co-oligomers, differing only for the co-monomers sequence. Oligomers with the same monomer composition and molar mass but different sequence were identified by HPLC/ESI-MS-MS on-line analysis. The results obtained show a preferential hydrolytic cleavage induced by the lipases used. In particular, these enzymes prefer cleaving sebacic ester bonds in P(BS-co-BSe) copolymers, whereas succinic ester bonds appear to be hydrolyzed faster than adipic ester bonds in P(BS-co-BA) copolyesters. 1H NMR analysis further substantiates these findings. The primary products generated by lipase hydrolysis of polyester films underwent further degradation at longer reaction times. The HPLC/ESI-MS analysis of these mixtures at various times provided the first evidence that lipase catalysis is active also in water solution, a hydrophobic effect induced by the aliphatic units of these polyesters.     

Esterification of methyl glycoside mixtures by lipase catalysis

The acylation of a mixture of methyl -D-galacto-, methyl -D-gluco- and methyl -D-mannopyranosides by octanoic acid was catalysed by lipases from Candida antarctica, Pseudomonas cepacia, Candida rugosa OF or Mucor miehei in acetonitrile at 45°C. The methyl glycopyranosides had the same anomeric configuration as that of the softwood hemicellulose galactoglucomannan. C. rugosa OF lipase had almost no substrate specificity and P. cepacia lipase had a high substrate specificity for the esterification of methyl -D-galactopyranoside.     

几种离子液体的微波法合成及其对脂肪酶催化效果的影响

采用微波法合成9种目标离子液体,对中间体[Bmim]Br的合成条件及其离子液体对全细胞催化剂催化效果的影响进行考察.直接将产脂肪酶真菌粗状假丝酵母(Candida valida) T2细胞固定在聚氨酯颗粒中,制备固定化细胞催化剂,将其应用于合成离子液体介质中催化甲醇与大豆油酯交换反应制备生物柴油.结果表明:微波功率200 W下间隙照射100 s,中间体[Bmim]Br的收率达95.16%,有效地提高了离子液合成产率;在[Bmim]PF6离子液中固定化细胞酶催化转酯化反应30 h,大豆油的转化率达42%,反应效果较其他8种合成离子液体好;固定化细胞颗粒和[Bmim]PF6重复使用4次,其油脂转化率和酶活保持率分别达到29%和69%,表现出较好的催化反应稳定性.     

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis.

In the intestine, the hydrolysis of triglycerides by pancreatic lipase is performed only in the presence of colipase, whose function is to anchor lipase to the bile-salt-coated lipid interface. Biochemical and crystallographic data on porcine and human lipases have shown that the molecule is made of two well-delimited domains. In order to get more information on the role of the domains in catalysis and colipase binding, we performed limited proteolysis on lipase from various species and obtained different patterns of cleavage. In the case of porcine and human lipases, only the C-terminal domain (12 kDa) could be obtained after chymotryptic attack, whereas in the horse enzyme the cleavage of the Leu410-Thr411 bond gave rise to a large N-terminal (45 kDa) and a small C-terminal (4 kDa) fragment. The isolated porcine and human C-terminal domains were completely inactive towards emulsified tributyrin, though were able to bind colipase. Conversely, the horse 45 kDa fragment retained the lipase activity but failed to correctly bind colipase. This work definitely proves that catalysis and colipase binding are separate events involving topographically distinct regions of the molecule and focuses attention on the role of the C-terminal domain in colipase binding.     

Process intensification of immobilized lipase catalysis by microwave irradiation in the synthesis of 4-chloro-2-methylphenoxyacetic acid (MCPA) esters

4-Chloro-2-methylphenoxyacetic acid (MCPA) is a selective systemic herbicide which is absorbed by leaves and roots. MCPA esters are preferred due to their low water solubility and environmental friendliness. Esterification of MCPA with n-butanol was investigated as a model reaction using immobilized enzymes under the influence of microwave irradiation. Different immobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM and Lipase AYS Amano were studied under microwave irradiation amongst which Novozym 435 (immobilized Candida antarctica lipase B) was the best catalyst. Effects of various parameters were systematically studied on rates and conversion. Under microwave irradiation, the initial rates were observed to increase up to 2-fold. Under optimized conditions of 0.1 mmol MCPA and 0.3 mmol n-butanol in 15 mL 1,4-dioxane as solvent, Novozym 435 showed a conversion of 83% at 60 °C in 6 h. Based on initial rate and progress curve data, the reaction was shown to follow the Ping Pong bi–bi mechanism with inhibition by MCPA and n-butanol. Esterification of MCPA was also studied with different alcohols such as isopropyl alcohol, n-pentanol, n-hexanol, benzyl alcohol and 2-ethyl-1-hexanol.     

Biodiesel preparation from microalgae lipid by two-step lipase catalysis

To overcome the high energy-consuming process of microalgae drying, a two-step lipase catalysis technique for the preparation of biodiesel from microalgae lipid of Chlorella spp. was developed. In the first step, free fatty acids (FAAs) and triacylglycerols (TAGs) are released after cell disruption and extracted, while the TAGs were hydrolysed by free lipase in aqueous phase. In the second step, FAAs were esterified with ethanol in the catalysis of free suspended lipase. The maximum rate of hydrolysis and esterification was 93.6% and 91.3%, respectively. The effects of reaction parameters, such as reaction time, enzyme amount, water content and molar ratio of lipid to ethanol on hydrolysis or esterification, were investigated. The results indicated that two-step reaction process (hydrolyse esterify) for biodiesel production were feasible.     

Application of lipase catalysis in organic solvents for selective protection–deprotection of bioactive compounds

Lipases are useful catalysts to realise in non-conventional medium esterifications and alcoholysis of polyhydroxylated compounds with high level of regioselectivity. This paper describes some examples of regioprotection–deprotection of flavonoids and conduritols, realised in our laboratory, using Pseudomonas cepacia, Mucor miehei and Candida cylindracea (C. rugosa) lipases.     

Design and synthesis of 1,3-diarylurea derivatives as selective cyclooxygenase (COX-2) inhibitors

A group of 1,3-diarylurea derivatives, possessing a methylsulfonyl pharmacophore at the para-position of the N-1 phenyl ring, in conjunction with a N-3 substituted-phenyl ring (4-F, 4-Cl, 4-Me, 4-OMe), were designed and synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1/COX-2 isozyme inhibition structure-activity studies identified 1-(4-methylsulfonylphenyl)-3-(4-methoxyphenyl) urea (4e) as a potent COX-2 inhibitor (IC(50)=0.11 microM) with a high COX-2 selectivity index (SI=203.6) comparable to the reference drug celecoxib (COX-2 IC(50)=0.06 microM; COX-2 SI=405). The structure-activity data acquired indicate that the urea moiety constitutes a suitable scaffold to design new acyclic 1,3-diarylurea derivatives with selective COX-2 inhibitory activity.     

Highly efficient synthesis of oligodeoxyribonucleotides using alpha-phenyl cinnamoyl group for selective amino protection.

alpha-phenyl cinnamoyl (alpha-PhCm) group has been found to be highly selective for exocylic amino function of all the three deoxynucleosides viz, 2'-deoxyadenosine, 2'-deoxyguanosine and 2'-deoxycytidine. The stereospecific nature of the group confers stability to the N-protected derivatives of 2'-deoxyadenosine and 2'-deoxyguanosine towards acids thereby minimising depurination. The easy preparation and introduction of the group, stability of the protected monomers, milder conditions for deprotection resulting in negligible side products during synthesis and above all hydrophobicity of the group are the additional advantages.     

Highly selective chemical modification of cruciform loops by diethyl pyrocarbonate.

Diethyl pyrocarbonate reacts with the single-stranded loops of cruciform structures with great selectivity. Adenine bases are carbethoxylated, as a result of which the backbone may be cleaved with piperidine, and the level of chemical modification at each base may be determined. We have studied the ColE1 and (A-T)34 cruciforms of pColIR315 and pXG540. In each case we observe maximal modification at the most central adenosine of the loop, and an overall pattern of modification corresponding to a total loop size of about six bases. The results may be interpreted in terms of a model in which the loop has a defined tertiary structure. No modification was detected at either cruciform four-way junction, suggesting that this region is fully base-paired.     

Cocrystallization of random copolymers of omega-pentadecalactone and epsilon-caprolactone synthesized by lipase catalysis

Random copolymers were prepared by Candida antarctica lipase B (Novozyme-435) catalyzed copolymerization of omega-pentadecalactone (PDL) with epsilon-caprolactone (CL). Over the whole composition range PDL-CL copolymers are highly crystalline (melting enthalpy by differential scanning calorimetry, above 100 J/g; crystallinity degree by wide-angle X-ray scattering, WAXS, 60-70%). The copolymers melt at temperatures that linearly decrease with composition from that of poly(omega-pentadecalactone) (PPDL; 97 degrees C) to that of poly(epsilon-caprolactone) (PCL; 59 degrees C). The WAXS profiles of PCL and PPDL homopolymers are very similar, except for the presence in PPDL of the (001) reflection at 2theta = 4.58 degrees that corresponds to a 19.3 angstroms periodicity in the chain direction. In PDL-CL copolymers the intensity of this reflection decreases with increasing content of CL units and vanishes at 50 mol % CL, as a result of randomization of the ester group alignment and loss of chain periodicity. PDL-CL copolymers crystallize in a lattice that gradually changes from that of one homopolymer to that of the other, owing to comonomer isomorphous substitution. Cocrystallization of comonomer units is also shown by a random PDL-CL copolymer obtained in a polymerization/transesterification reaction catalyzed by C. antarctica lipase B (Novozyme-435) starting from preformed PCL and PDL monomer.     

Conformational changes induced by binding UDP-2F-galactose to alpha-1,3 galactosyltransferase- implications for catalysis

Alpha-1,3 galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-galactose to beta-linked galactosides with retention of its alpha configuration. Although several complexes of alpha3GT with inhibitors and substrates have been reported, no structure has been determined of a complex containing intact UDP-galactose. We describe the structure of a complex containing an inhibitory analogue of UDP-galactose, UDP-2F-galactose, in a complex with the Arg365Lys mutant of alpha3GT. The inhibitor is bound in a distorted, bent configuration and comparison with the structure of the apo form of this mutant shows that the interaction induces structural changes in the enzyme, implying a role for ground state destabilization in catalysis. In addition to a general reduction in flexibility in the enzyme indicated by a large reduction in crystallographic B-factors, two loops, one centred around Trp195 and one encompassing the C-terminal 11 residues undergo large structural changes in complexes with UDP and UDP derivatives. The distorted configuration of the bound UDP-2F-galactose in its complex is stabilized, in part, by interactions with residues that are part of or near the flexible loops. Mutagenesis and truncation studies indicate that two highly conserved basic amino acid residues in the C-terminal region, Lys359 and Arg365 are important for catalysis, probably reflecting their roles in these ligand-mediated conformational changes. A second Mn(2+) cofactor has been identified in the catalytic site of a complex of the Arg365Lys with UDP, in a location that suggests it could play a role in facilitating UDP release, consistent with kinetic studies that show alpha3GT activity depends on the binding of two manganese ions. Conformational changes in the C-terminal 11 residues require an initial reorganization of the Trp195 loop and are linked to enzyme progress through the catalytic cycle, including donor substrate distortion, cleavage of the UDP-galactose bond, galactose transfer, and UDP release.     

Highly selective hydrolysis of kinins by recombinant prolylcarboxypeptidase

We have previously cloned a cDNA encoding human prolylcarboxypeptidase (PRCP) and expressed the cDNA in the Schneider 2 (S2) drosophila cell line. Here, we further characterized this recombinant enzyme. Investigations were performed to determine whether recombinant PRCP (rPRCP) metabolizes kinins (BK 1-9 and BK 1-8). The metabolites of these kinins were identified by LC/MS. rPRCP metabolized BK 1-8 to BK 1-7, whereas rPRCP was ineffective in metabolizing BK 1-9. The hydrolysis of BK 1-8 by rPRCP was dose- and time-dependent. A homology model of PRCP was developed based upon the sequence of dipeptidyl-peptidase 7 (DPP7, PDB ID: 3JYH), and providentially, the structure of PRCP (PDB ID: 3N2Z) was characterized during the course of our investigation. Docking studies of bradykinin oligopeptides were performed both from the homology model, and from the crystal structure of PRCP. These docking studies may provide a better understanding of the contribution of specific residues involved in substrate selectivity of human PRCP.     

1,3-Butadiene oxidation by human myeloperoxidase. Role of chloride ion in catalysis of divergent pathways.

1,3-Butadiene was oxidized by human myeloperoxidase in the absence of KCl to yield butadiene monoxide (BM) and crotonaldehyde (CA), but at KCl concentrations higher than 50 mM, 1-chloro-2-hydroxy-3-butene (CHB) was the major metabolite detected; metabolite formation was dependent on incubation time, pH, KCl, 1,3-butadiene, and H2O2 concentrations. The data are best explained by 1,3-butadiene being oxidized by myeloperoxidase by two different mechanisms. First, oxygen transfer from the hemoprotein would occur to either C-1 or C-4 of 1,3-butadiene to form an intermediate which may cyclize to form BM or undergo a hydrogen shift to form 3-butenal, an unstable precursor of CA. Further evidence for this mechanism was provided by the inability to detect methyl vinyl ketone, a possible product of an oxygen transfer reaction to C-2 or C-3 of 1,3-butadiene, and by the finding that CA was not simply a decomposition product of BM under assay conditions. In the second mechanism, however, chloride ion is oxidized by myeloperoxidase to HOCl which reacts with 1,3-butadiene to yield CHB. Further evidence for this mechanism was provided by the finding that CHB was readily formed when 1,3-butadiene was added to the filtrate of a myeloperoxidase/H2O2/KCl incubation and when 1,3-butadiene was allowed to react with authentic HOCl. In addition, CHB was not detected when BM or CA was incubated with myeloperoxidase, H2O2, and KCl for up to 60 min, or when 1,3-butadiene and KCl were incubated with chloroperoxidase and H2O2 or with mouse liver microsomes and NADPH, enzyme systems which catalyze 1,3-butadiene oxidation to BM and CA, but unlike myeloperoxidase, do not catalyze chloride ion oxidation to HOCl. These results provide clear evidence for novel olefinic oxidation reactions by myeloperoxidase.     

Highly sensitive and rapid detection of antibody catalysis by luminescent bacteria

A highly sensitive, inexpensive, and facile bioluminescent assay for the detection of catalytic antibodies has been developed. This assay may be used for the early detection of antibody catalysis. The efficiency of this technique was exemplified by the use of the luminescent bacterium VhM42 for monitoring an antibody-catalyzed retroaldol fragmentation reaction with aldolase antibodies 38C2 and 24H6.