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Pac Gene (pac + gene)
Selected AbstractsExpression of penicillin G acylase from the cloned pac gene of Escherichia coli ATCC11105FEBS JOURNAL, Issue 5 2001Effects of pacR, temperature The structural gene pac in Eschericia coli ATCC11105 encodes penicillin G acylase (PGA). Within the pac gene, there is a regulatory gene pacR, which is transcribed in the opposite direction. Site-directed mutagenesis was performed at base 1045 of pac by replacing a T with a C. This substitution did not alter the amino-acid sequence of PGA, but changed the translation start codon of pacR from AUG to GUG. The expression of the mutant pacR decreased dramatically and the lacZ transcriptional fusion analysis showed that GUG was an extremely poor initiation codon for pacR. The pacR mutation caused PGA expression to be constitutive rather than inductive in two strains (E. coli A56, DH10B). The pac inducer phenylacetic acid (PAA) gave significant induction of PGA production at a concentration of 0.2% in wild type, but PAA at this concentration inhibited both cell growth and PGA production in the pacR mutated strains. The temperature-dependent expression character of pac is preserved in the pacR translation-initiation mutant and the optimum temperature of PGA production was 22 °C in both wild type and mutant. At a higher temperature of 37 °C, the PGA precursor polypeptide could not be matured into subunits and formed inclusion bodies, as revealed by western blot analysis. Our investigations confirmed the hypothesis of pacR-mediated PAA induction for PGA expression and clarified the inhibitory effect of high temperature upon the post-translational processing of the PGA precursor polypeptide. [source] High-level extracellular production of penicillin acylase by genetic engineering of Escherichia coliJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 10 2001Wen-Jer Lin Abstract The extracellular production of penicillin acylase (PAC) in genetically engineered Escherichia coli by coexpression of the brp gene encoding bacteriocin release protein (BRP) and the pac gene was demonstrated. Cell physiology was affected while PAC was released into the medium, depending on the strategy for brp expression. The performance for the production and release of PAC was optimized by taking several culture parameters, including host, inducer (mitomycin C) concentration, and induction timing for brp expression, into consideration. The effect of PAC release on inclusion body formation was also investigated. It was observed that the amount of inclusion bodies was significantly affected by brp expression. A reason for the limitation of PAC production and a strategy for resolving this problem are proposed. © 2001 Society of Chemical Industry [source] High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicabilityBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2006Lidija Senerovic Abstract A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPACP.rett)of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPACP.rett activity of approximately 40% (3.8 U/mL vs. 2.7U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPACP.rett purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPACP.rett expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPACP.rett. The enzyme wasimmobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPACP.rett performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g dry based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPACP.rett was finally evaluated by studying the kinetically controlled synthesis of ,-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process. © 2005 Wiley Periodicals, Inc. [source] Cytoplasmic Overexpression, Folding, and Processing of Penicillin Acylase Precursor in EscherichiacoliBIOTECHNOLOGY PROGRESS, Issue 5 2005Yali Xu Penicillin acylase (PAC) precursor, proPAC, was overproduced in a soluble or insoluble form in the cytoplasm of Escherichia coli through the expression of the leader-less pac gene (ll-pac) devoid of the coding region for the signal peptide of PAC. Also, a portion of the overexpressed proPAC was further processed to form mature PAC, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E.coli. The cultivation performance for ll-pac expression was limited by several factors, including (1) misfolding of proPAC, resulting in the aggregation of insoluble proPAC as inclusion bodies, (2) intracellular proteolysis, leading to the degradation of the overexpressed gene products, and (3) inefficient PAC maturation, limiting the formation of active PAC. The effect of coexpression of various cytoplasmic chaperones, including trigger factor, GroEL/ES, DnaK/J-GrpE, and their combinations, on ll-pac expression was investigated. Intracellular proteolysis of the overexpressed gene products could be prevented by coexpression of GroEL/ES. On the other hand, coexpression of trigger factor appeared to be able to facilitate the folding of soluble proPAC and to improve PAC maturation. The roles of trigger factor and GroEL/ES could be coordinated to significantly improve ll-pac expression performance. DnaK/J-GrpE had an effect for solublization of proPAC and perhaps, similar to trigger factor, for improving PAC maturation. The ll-pac expression performance was also significantly improved through the simultaneous coexpression of DnaK/J-GrpE and GroEL/ES. The results of the study suggest that the folding and/or processing of proPAC could be a major issue limiting the overproduction of PAC in E. coli and the bottleneck could be eliminated through the coexpression of appropriate chaperone(s). [source] | |||||||||||||