Biomedical subjects

Sheng Yang

Publications and source records attributed to Sheng Yang.

At least 19 recordsLinked to original sources

PubMed2007

Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli.

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of beta-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.

Amidohydrolases↗

PubMed2007

Ramosetron versus ondansetron in the prevention of chemotherapy-induced gastrointestinal side effects: A prospective randomized controlled study.

BACKGROUND: This study observed and compared the preventive effects of ramosetron and ondansetron on gastrointestinal side effects caused by cisplatin-containing chemotherapy. METHODS: Fifty patients with malignant tumors undergoing their first chemotherapy were randomly divided into two groups, and each group received 0.3 mg of ramosetron and 16 mg of ondansetron in a prospective crossover comparison study. RESULTS: Data were collected for analysis of the therapeutic effect in 47 cases and for adverse events in 50 cases. Both drugs showed similar results in regard to chemotherapy-induced gastrointestinal side effects, emesis and appetite loss on day 1, but by day 5, ramosetron was significantly better than ondansetron in terms of controlling appetite loss. From days 3-5, ramosetron tended to be more effective than ondansetron in its antiemetic action. The incidence of headache, fatigue and constipation was the same for both drugs. CONCLUSIONS: Ramosetron is a long-lasting and safe antiemetic agent.

Adolescent↗

PubMed2006

Increasing synthetic performance of penicillin G acylase from Bacillus megaterium by site-directed mutagenesis.

Site-directed mutagenesis based on predicted modeled structure of pencillin G acylase from Bacillus megaterium (BmPGA) was followed to increase its performance in the kinetically controlled synthesis of cephalexin with high reactant concentrations of 133 mM 7-amino-desaceto-xycephalosporanic acid (7-ADCA) and 267 mM D: -phenylglycine amide (D-PGA). We directed changes in amino acid residues to positions close to the active site that were expected to affect the catalytic performance of penicillin acylase: alpha Y144, alpha F145, and beta V24. Alpha F145 was mutated into tyrosine, alanine, and leucine. Alpha Y144 and beta V24 were mutated into arginine and phenylalanine, respectively. The S/H ratios of three mutants, BmPGAalpha144R, BmPGAbeta24F, and BmPGAbeta24F+alpha144R, were up to 1.3-3.0 times higher values. Compared to the wild-type BmPGA, BmPGAbeta24F+alpha144R showed superior potential of the synthetic performance, allowing the accumulation of up to twofold more cephalexin at significantly higher conversion rates.

Amino Acids↗

PubMed2006

NT-1, an active constituent extracted from Tiaoxin Recipe, enhances long-term potentiation of CA1 subfield in rat hippocampal slices.

Previous studies demonstrated that Tiaoxin Recipe (TXR), a traditional Chinese medicinal prescription, could alleviate cognitive dysfunction. NT-1, an active constituent isolated from this prescription under guidance of bioactive evaluation, was preliminarily proved to enhance the ability of spatial learning and memory in rats. In the present study, using hippocampal slice preparations, we investigated the mechanisms by which NT-1 improves cognitive dysfunction contributed to corticosterone (CORT). NT-1 ameliorated the inhibition of LTP by CORT more significantly than TXR did. These results suggested that enhancing LTP induction is one mechanism by which NT-1 improves spatial learning and memory impaired by CORT.

Animals↗

PubMed2006

Effects of Liuwei Dihuang decoction on ion channels and synaptic transmission in cultured hippocampal neuron of rat.

The effect of Liuwei Dihuang decoction (LW), a traditional Chinese medicine (TCM) prescription, on voltage-dependent currents and synaptic transmission were investigated in cultured hippocampal neurons of rat by whole-cell patch clamp recording technique. After application with serum from LW-treated rats, termed LW-containing serum (LWCS) for 48 h, the amplitude of delay rectifying K+ current (IK) and voltage-gated Ca2+ current (ICa) decreased. While the frequency of spontaneous excitatory post-synaptic current (sEPSC) and miniature excitatory post-synaptic current (mEPSC) increased significantly. Yet the amplitude of voltage-depended Na+ current (INa) and transient outward K+ current (IA), membrane capacitance and resistance remained unchanged. The results indicated that LWCS possessed the effect of modulating or improving neuronal and synaptic function, which possibly contribute to the cognition enhancing effect of LW.

Administration, Oral↗

PubMed2006

[Clinical features of 89 patients with primary non-Hodgkin's lymphoma of the tonsil].

BACKGROUND & OBJECTIVE: Head and neck lymphoma develops predominantly in the tonsil. This study was to investigate the clinical features of primary non-Hodgkin's lymphoma (NHL) of the tonsil, and to explore possible ways to improve the prognosis and quality of life of the patients after treatment. METHODS: Clinical data of 89 naive patients with NHL of the tonsil, treated from May 1990 to Jan. 2003, were retrospectively reviewed. All patients were confirmed pathologically and classified according to revised European-American Lymphoid Neoplasms and World Health Organization Classification, and staged according to the Ann Arbor classification. Stage I-II patients received radiochemotherapy-predominant treatment, whereas stage III-IV patients received chemotherapy-predominant treatment. RESULTS: Of the 89 cases, 60 (67%) were diffuse large B-cell subtype, 11 (12%) were peripheral T-cell subtype, 5 (6%) were indolent lymphoma, 1 was anaplastic large T-cell lymphoma, and 1 was T lymphoblastic lymphoma; 81 (91%) were stage I-II disease. Of the 89 patients, 58 (72%) received radiochemotherapy, 19 (21%) received radiotherapy alone, 3 received chemotherapy alone, and 1 received radiochemotherapy combined with rituximab. The 5-year overall survival rate was 80%, that of stage I-II patients was 84%. Cox regression multivariate analysis showed that the survival rate was correlated to the value of international prognostic index (IPI), and whether the patient had primary refractory or relapsed disease, but was not correlated to sex, age, pathologic subtype, B symptoms, and bulky disease. CONCLUSIONS: Most patients with NHL of the tonsil are at early stages, with good prognosis. Diffuse large B-cell lymphoma is the most common pathologic subtype. Primary refractory, relapse, and IPI>1 are independent prognostic factors.

Adolescent↗

PubMed2006

[Phase I clinical trail of intravenous pegylated recombinant human granulocyte colony-stimulating factor].

BACKGROUND & OBJECTIVE: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is effective in the prophylaxis and management of chemotherapy-induced neutropenia, but requires daily administration because of its short half-life. Pegylated rhG-CSF (PEG-rhG-CSF) is a long-acting reagent that permits less frequent injection. This study was to evaluate the safety and tolerance of PEG-rhG-CSF in Chinese patients, and to explore its efficacy of enhancing absolute neutrophil count (ANC) and CD34+ cell count in peripheral blood. METHODS: Naïve non-small lung cancer or breast cancer patients with normal bone marrow function were eligible for this open-labeled, dose-escalation trial. All patients received 2 cycles of chemotherapy of identical regimen. In cycle 1, rhG-CSF (150 microg/day) was administrated in case of febrile neutropenia or grade 4 neutropenia; in cycle 2, patients received a single injection of PEG-rhG-CSF (30 microg/kg, 60 microg/kg, 100 microg/kg, or 200 microg/kg) 48 h after administration of paclitaxel and carboplatin. RESULTS: All the 16 patients enrolled (4 in each dose group) were evaluable for safety and efficacy of PEG-rhG-CSF. Main adverse events related to PEG-rhG-CSF were musculoskeletal pain or arthralgia (13/16), fatigue (10/16), dizziness (2/16), and injection-site pain (1/16). All adverse events were mild to moderate, and most of them were reversible without treatment. PEG-rhG-CSF enhanced ANC in a dose-dependent manner to some extent, and PEG-rhG-CSF at 60 microg/kg or higher doses prevented chemotherapy-induced neutropenia with sustained effect; CD34+ cells in peripheral blood were also increased. CONCLUSIONS: PEG-rhG-CSF is well tolerated, with no serious adverse event in this trial. The recommended dose of PEG-rhG-CSF for phase II trial is 100 microg/kg because of its adequate efficacy and less adverse events than those of 200 microg/kg.

Adult↗

PubMed2006

[Efficacy of once-per-cycle administration pegylated recombinant human granulocyte colony-stimulating factor in chemotherapy-induced neutropenia in a phase I trial].

OBJECTIVE: To explore the efficacy of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in the prophylaxis of chemotherapy-induced neutropenia. METHODS: Patients with previously untreated non-small cell lung cancer or breast cancer and with normal bone marrow function were eligible for the trial. In the phase I trial, patients were to treated with two cycles of paclitaxel/carboplatin chemotherapy every 21 days. In cycle 1, if absolute neutrophil count (ANC) dropped below 1.0 x 10(9)/ L with fever and/or ANC dropped below 0.5 x 10(9)/L, rhG-CSF 150 microg/d was administrated until WBC > or = 10 x 10(9)/L or ANC > or = 5.0 x 10(9)/L. In cycle 2, patients were to receive a single injection of PEG-rhG-CSF (30, 60, 100, or 200 microg/kg) 48 hours after chemotherapy. Pharmacodynamic analyses were performed. RESULTS: All the 16 patients enrolled (4 in each group) were eligible for efficacy evaluation. In cycle 1, 7 patients received rhG-CSF administration because of grade 4 neutropenia, while in cycle 2, there was only 1 episode of grade 4 neutropenia, which were in the 30 microg/kg group. In cycle 1, the mean ANC nadir of 1.37 x 10(9)/L occurred on day 13 in the 9 patients who received no rhG-CSF administration. In cycle 2, the mean ANC nadirs were 0.77 x 10(9)/L, 4.54 x 10(9)/L, 3.00 x 10(9)/L, and 5.56 x 10(9)/L, respectively, in 30, 60, 100, or 200 microg/kg group. The nadirs of the first two groups occurred on day 8 of cycle 2, while those of the other two groups appeared on day 7. After PEG-rhG-CSF administration, two mean ANC peaks were observed. The first one ranging from 20.87 x 10(9)/L to 33.61 x 10(9)/L in the 4 groups appeared on day 3 to day 4. In the 30 microg/ kg group, the mean ANC reached the second peak at 5.03 x 10(9)/L on day 14. The second peaks on day 10 in the other groups were 12.42 x 10(9)/L, 11.59 x 10(9)/L, and 18.92 x 10(9)/L, respectively. Pharmacodynamic analyses showed sustained serum concentrations of PEG-rhG-CSF during the period of neutropenia. CONCLUSIONS: PEG-rhG-CSF administration may decrease the incidence of grade 4 neutopenia and result in earlier and higher nadir ANCs. PEG-rhG-CSF has a potential of once-per-cycle administration. The ANC and serum concentration of PEG-rhG-CSF are consistent with neutrophil receptor-mediated clearance.

Adolescent↗

PubMed2005

Expression, purification, and immobilization of His-tagged D-amino acid oxidase of Trigonopsis variabilis in Pichia pastoris.

High-level expression of D: -amino acid oxidase (DAO) has been reported in Pichia pastoris by integrating the DAO gene under the control of the alcohol oxidase promoter (PAOX1). However, the time taken to reach peak product concentration is usually long (approximately 43 h), and cultivation requires tight regulation of methanol feeding. In this paper, we describe the expression of His-tagged DAO (HDAO) in P. pastoris using the glyceraldehydes-3-phosphate dehydrogenase promoter (PGAP). The maximal level of HDAO expression using the PGAP integrant is attained in 13 h and is equal to that obtained using the PAOX1 integrant in 43 h. We also explored the possibility of secreting HDAO in P. pastoris. In-frame fusion of Saccharomyces cerevisiae alpha-factor secretion signal under a PGAP or PAOX1 resulted in low-level secretion of active HDAO, which was not of practical use. The intracellularly expressed HDAO under PGAP was purified by agar-based affinity support and then immobilized on Amberzyme oxirane resin. The immobilized HDAO, with specific activity of 75 U g-1 (wet weight), could be recycled more than 14 times without significant loss of activity. The data suggest that intracellular production of HDAO under PGAP, followed by affinity purification and immobilization on oxirane resin, may serve as an effective process for the manufacture of immobilized DAO for industrial application.

Bioreactors↗

PubMed2005

[Quality of life of malignant patients after autologous stem cell transplantation].

OBJECTIVE: To investigate the quality of life of Chinese malignant patients who treated with the high dose chemoradiotherapy combined with autologous stem cell transplantation (ASCT). METHODS: The data of 89 patients who answer the EORTC QLQ-C30 Chinese version 3.0 after finished ASCT and disease free were analyzed using SPSS 10.0. RESULTS: The score of global health status and functional assessment were near or over 80, more than 80 percent of patient had good or very good global health status or function, the patients who were experiencing moderate or severe financial dysfunction, fatigue, dyspnea, sleeping disturbance and diarrhea were in 72.5% (65/89), 50.6% (45/89), 42.7% (38/89), 33.7% (30/89) and 32.5% (29/89) respectively. The score of dyspnea in female patients was significant higher than male patients (P = 0.024). The score of global health status (P = 0.000), physical function (P = 0.000), role function (P = 0.031) and social function (P = 0.029) became higher significantly with time from transplantation and the score of fatigue became lower (P = 0.020). The Hodgkin's lymphoma patients had higher score significantly in nausea & vomiting (P = 0.002) and dyspnea (P = 0.006) than NHL. The age at transplantation and evaluation took none effect on the score. CONCLUSION: Most patient have good global health status and function after autologous stem cell transplantation, they are more suffered from financial dysfunction, fatigue, dyspnea, sleeping disturbance and diarrhea. Females have more dyspnea symptoms, the quality of life of patient will improve gradually with the time from transplantation, the Hodgkin's lymphoma patients have more symptoms than non-Hodgkin's lymphoma patients, we do not find any effect of age at transplantation and assessment on the quality of life.

Adolescent↗

PubMed2005

Expression and purification of penicillin G acylase enzymes from four different micro-organisms, and a comparative evaluation of their synthesis/hydrolysis ratios for cephalexin.

Several genes for the enzyme penicillin G acylase, as isolated from four different micro-organisms (Alcaligenes facaelis, Escherichia coli, Kluyvera cryocrescens or Providencia rettgeri) were modified at their carboxy-termini to include His-tag fusions, then were expressed from the plasmid pET-24a(+) in E. coli JM109(DE3) cells. All fusion proteins were next purified to homogeneity in a single step by agar-based Co-IDA chromatography, and were then evaluated as catalysts for the synthesis of cephalexin by a kinetically controlled strategy. We find here that the penicillin G acylase enzyme from K. cryocrescens shows a higher intrinsic synthesis/hydrolysis ratio, when compared to three other enzymes from A. facaelis or P. rettgeri, or E. coli.

Alcaligenes faecalis↗

PubMed2005

Synthetic peptides derived from SARS coronavirus S protein with diagnostic and therapeutic potential.

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important viral structural protein. Based on bioinformatics analysis, 10 antigenic peptides derived from the S protein sequence were selected and synthesized. The antigenicity and immunoreactivity of all the peptides were tested in vivo and in vitro. Four peptides (P6, P8, P9 and P10) which contain B cell epitopes of the S protein were identified, and P8 peptide was confirmed in vivo to have a potential in serological diagnosis. By using a syncytia formation model, we tested the neutralization ability of all 10 peptides and their corresponding antibodies. It is interesting to find that P8 and P9 peptides inhibited syncytia formation, suggesting that the P8 and P9 spanning regions may provide a good target for anti-SARS-CoV drug design. Our data suggest that we have identified peptides derived from the S protein of SARS-CoV, which are useful for SARS treatment and diagnosis.

Amino Acid Sequence↗

PubMed2005

Interleukin-1beta enhances NMDA receptor-mediated current but inhibits excitatory synaptic transmission.

Interleukin (IL)-1beta is often characterized as the prototypic proinflammatory cytokine but is involved in various pathophysiological conditions in the central nervous system (CNS). A whole-cell recording technique was used to observe its effect on N-methyl-D-aspartate (NMDA)-evoked currents and spontaneous synaptic activity in cultured rat hippocampal neurons. The results showed that the frequencies but not the amplitudes of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC) were decreased by 10 or 100 ng/ml IL-1beta. IL-1beta at these concentrations also increased the NMDA receptor-mediated current. In addition, 10 ng/ml IL-1beta significantly increased the amplitude of the voltage-dependent Ca2+ current (I(Ca)). The increase in I(Ca) following treatment of cultures with IL-1beta resulted mainly from an increase in L-type current. These data suggest that IL-1beta modulates hippocampus-related functions via its effect on synaptic activity and Ca2+ signaling in neurons.

Animals↗

PubMed2005

Folding of the SARS coronavirus spike glycoprotein immunological fragment (SARS_S1b): thermodynamic and kinetic investigation correlating with three-dimensional structural modeling.

Spike glycoprotein of SARS coronavirus (S protein) plays a pivotal role in SARS coronavirus (SARS_CoV) infection. The immunological fragment of the S protein (Ala251-His641, SARS_S1b) is believed to be essential for SARS_CoV entering the host cell through S protein-ACE-2 interaction. We have quantitatively characterized the thermally induced and GuHCl-induced unfolding features of SARS_S1b using circular dichroism (CD), tryptophan fluorescence, and stopped-flow spectral techniques. For the thermally induced unfolding at pH 7.4, the apparent activation energy (E(app)) and transition midpoint temperature (Tm) were determined to be 16.3 +/- 0.2 kcal/mol and 52.5 +/- 0.4 degrees C, respectively. The CD spectra are not dependent on temperature, suggesting that the secondary structure of SARS_S1b has a relatively high thermal stability. GuHCl strongly affected SARS_S1b structure. Both the CD and fluorescent spectra resulted in consistent values of the transition middle concentration of the denaturant (Cm, ranging from 2.30 to 2.45 M) and the standard free energy change (deltaG(o), ranging from 2.1 to 2.5 kcal/mol) for the SARS_S1b unfolding reaction. Moreover, the kinetic features of the chemical unfolding and refolding of SARS_S1b were also characterized using a stopped-flow CD spectral technique. The obvious unfolding reaction rates and relaxation times were determined at various GuHCl concentrations, and the Cm value was obtained, which is very close to the data that resulted from CD and fluorescent spectral determinations. Secondary and three-dimensional structural predictions by homology modeling indicated that SARS_S1b folded as a globular-like structure by beta-sheets and loops; two of the four tryptophans are located on the protein surface, which is in agreement with the tryptophan fluorescence result. The three-dimensional model was also used to explain the recently published experimental results of S1-ACE-2 binding and immunizations.

Amino Acid Sequence↗

PubMed2005

D-serine enhances impaired long-term potentiation in CA1 subfield of hippocampal slices from aged senescence-accelerated mouse prone/8.

The molecular and cellular mechanisms underlying the cognitive deficiency of senescence-accelerated mouse prone/8 (SAMP8) have been attributed to many pathological changes in neurons. Recently, increasing evidence has shown that astrocytes, by mean of d-serine, involve in the process of synaptic transmission. Here we reported that the long-term potentiation (LTP) in CA1 area of hippocampal slices prepared from 2-, 6- and 12-month-old SAMP8 significantly decreased with age. Meanwhile, the LTP in the slices of 6- and 12-month-old mice markedly decreased below that of the age-matched normal strain SAMR1. Supplement with exogenous d-serine, a main product of astrocytes and a coagonist at the "glycin-binding" site of N-methyl-d-aspartate (NMDA) receptors, not only directly enhanced the deficient LTP but also rescued the abolished LTP by d-amino acid oxidase (DAAO) in slices from 12-month-old SAMP8. This ameliorative effect of d-serine was inhibited by either AP-V or 5,7-dichlorokynurenic acid (DCKA). These results suggest that absence of d-serine or dysfunction of the astrocytes possibly was one of mechanisms underlying the decrease of NMDA receptor-dependent LTP and cognition in aged SAMP8.

Age Factors↗

PubMed2005

Autologous transplantation of porcine myogenic precursor cells in skeletal muscle.

Myoblast transplantation is a potential therapy for severe muscle trauma, myopathies and heart infarct. Success with this therapy relies on the ability to obtain cell preparations enriched in myogenic precursor cells and on their survival after transplantation. To define myoblast transplantation strategies applicable to patients, we used a large animal model, the pig. Muscle dissociation procedures adapted to porcine tissue gave high yields of cells containing at least 80% myogenic precursor cells. Autologous transplantation of 3[H]-thymidine labeled porcine myogenic precursor cells indicated 60% survival at day 1 followed by a decay to 10% at day 5 post-injection. Nuclei of myogenic precursor cells transduced with a lentivirus encoding the nls-lacZ reporter gene were present in host myotubes 8 days post-transplantation, indicating that injected myogenic precursor cells contribute to muscle regeneration. This work suggests that pig is an adequate large animal model for exploring myogenic precursor cells transplantation strategies applicable in patients.

Animals↗

PubMed2005

SO-3, a new O-superfamily conopeptide derived from Conus striatus, selectively inhibits N-type calcium currents in cultured hippocampal neurons.

Whole-cell currents in cultured hippocampal neurons were recorded to investigate the effects of SO-3, a new O-superfamily conopeptide derived from Conus striatus, on voltage-sensitive channels. SO-3 had no effect on voltage-sensitive sodium currents, delayed rectifier potassium currents, and transient outward potassium currents. Similar to the selective N-type calcium channel blocker omega-conotoxin MVIIA (MVIIA), SO-3 could concentration-dependently inhibit the high voltage-activated (HVA) calcium currents (I(Ca)). MVIIA(3 microM), 10 microM nimodipine, and 0.5 microM omega-agatoxin IVA (Aga) could selectively block the N-, L-, and P/Q-type I(Ca), which contributed approximately 32, approximately 38, and approximately 21% of the HVA currents in hippocampal neurons, respectively. About 31% of the total HVA currents were inhibited by 3 microM SO-3. SO-3 (3 microM) and 3 microM MVIIA inhibited the overlapping components of HVA currents, whereas no overlapping component was inhibited by 3 microM SO-3 and 10 microM nimodipine, or by 3 microM SO-3 and 0.5 microM Aga. Also, 3 microM SO-3 had no effect on R-type currents. SO-3 had less inhibitory effects on non-N-type HVA currents than MVIIA at higher concentrations (30 and 100 microM). The inhibitory effects of SO-3 and MVIIA on HVA currents were almost fully reversible. However, the recovery from block by MVIIA was more rapid than recovery from block by SO-3. It is concluded that SO-3 is a new omega-conotoxin selectively targeting N-type voltage-sensitive calcium channels. Considering the significance of N-type calcium channels for pain transduction, SO-3 may have therapeutic potential as a novel analgesic agent.

Amino Acid Sequence↗

PubMed2005

Improving the activity and stability of GL-7-ACA acylase CA130 by site-directed mutagenesis.

In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151alphaF and Q50betaN, showed two- to threefold-increased catalytic efficiency (k(cat)/K(m)) compared to wild-type CA130. Their K(m) values were decreased by ca. 50%, and the k(cat) values increased to 14.4 and 16.9 s(-1), respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121betaA and K198betaA had a 30 to 58% longer half-life than wild-type CA130, and K198betaA and D286betaA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50betaN/K198betaA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.

Cephalosporins↗