6 research outputs found
The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity
The Bacillus pumilus gene encoding acetyl xylan esterase tare) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) from B. pumilus. The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B. subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima. These four proteins are of similar sire and represent a new family of esterases having a broad substrate specificity. The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C
Glycosylation and pH stability of penicillin G acylase from providencia rettgeri produced in Pichia pastoris
Penicilin G acilaza (PAC) je jedan od najÅ”ire koriÅ”Äenih enzima u industrijskoj sintezi polusintetskih antibiotika. U ovom radu dobijeni nivo ekspresije PAC gena iz Providencia rettgeri u ekspresionom sistemu Pichia pastoris iznosio je 2.7 U/ml. Rekombinantni enzim je preÄiÅ”Äen i odreÄen je njegov glikozilacioni status. NaÄeno je da osim Å”to su obe subjedinice enzima (Ī± i Ī²) N-glikozilovane, Ī² subjedinica sadrži joÅ” i O-glikane. TakoÄe je ustanovljeno da je rekombinantna PACP. rett. stabilna u Å”irokom pH opsegu Å”to ju je, zajedno sa predhodno ustanovljenom visokom termostabilnoÅ”Äu, uÄinilo izuzetno privlaÄnim biokatalizatorom sa industrijske taÄke glediÅ”ta.Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (Ī± and Ī²) of the enzyme were N-glycosylated, while the Ī²-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view
DOI:10.2298/ABS0701001S EFFECT OF THE KANAMYCIN RESISTANCE MARKER ON STABILITY OF 2m-BASED EXPRESSION PLASMIDS
Abstract ā In this paper we describe the effect of the kanamycin resistance gene (Km r) on 2Ī¼m-based plasmid maintenance in Saccharomyces cerevisiae. The influence of this marker gene on the loss of the stable model-vectors proved to be constant, as well as independent of carbon source and culture growth rates. In strains for GAL UAS ā driven heterologous protein production introduction of Km r resulted in curing of the yeast episomal plasmid (YEp) from the population in a small number of generations. Application of selective pressure on the strain producing recombinant penicillin G amidase (rPGA) did not provide the expected increase of protein yield. The influence of genetic elements for heterologous protein production on vector stability was examined, and the most destabilizing factors prove to be the presence and expression of the foreign gene
GLYCOSYLATION AND PH STABILITY OF PENICILLIN G ACYLASE FROM PROVIDENCIA RETTGERI PRODUCED IN PICHIA PASTORIS
Abstract ā Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (Ī± and Ī²) of the enzyme were N-glycosylated, while the Ī²-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view
Expression and characterization of human interferon-beta I in the methylotrophic yeast Pichia pastoris
We describe the heterologous expression of a human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon-beta1 (rHulFN-beta1) was secreted from shake-flask-grown P pastoris cells into the medium using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence at the level of (1-3) x 10(5) i.u. (international units)/ml (6-12 mg/litre). An rHulFN-beta1 with an N-terminal sequence identical with that of native HulFN-beta1 was purified and the specific activity was determined (2-3 x 10(7) i.u./mg). It was found that the secreted recombinant protein was partially N-glycosylated
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 applicability
A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri (rPAC(P).(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 PEN gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/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 rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P. pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.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 gt 3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of P-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process