Vithaya Meevootisom

Isolation, characterization and function of laccase from Trichoderma

Of fourteen natural isolates of Trichoderma, no correlation was found between substrate weight loss and phenol oxidase (PO) activity in rice straw cultures. The highest PO producer from these laccase-positive strains was subjected to UV mutagenesis in order to select high and low PO activity mutants. There was no significant difference in substrate weight loss for mutant strains with six times higher and six times lower PO activity than the parent strain. Nor did the enzyme activity result in decreased growth inhibition by inhibitory phenolic compounds. PO enzyme from the parent Trichoderma and one of its high-PO-activity mutants was subsequently purified by ethanol precipitation from liquid cultures optimized by supplementation with copper sulphate and cycloheximide. Protein staining and activity staining of disc electrophoresis gels showed that only one PO enzyme of approximately 71 000 Da was produced. The enzyme could be defined as a laccase (benzenediol: oxygen oxidoreductase E.C. 1.10.3.2) because it catalysed the oxidation of syringaldazine and p-phenylenediamine in the absence of hydrogen peroxide, and because it was inhibited by cetyltrimethylammonium because but not by cinnamic acid. No specific in-vivo function could be assigned to this enzyme.

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase

Abstract Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide).

Cloning and expression of penicillin acylase genes from overproducing strains of Escherichia coli and Bacillus megaterium

SummaryPenicillin acylase genes from Escherichia coli 194, of an overproducing mutant (194-3) of this strain, and of a similar overproducing mutant of Bacillus megaterium UN1 were cloned in E. coli DH1 on the plasmid vector pACYC184. The sizes of chromosomal DNA fragments essential for penicillin acylase production were found by Tn1000 mutagenesis and in vitro deletions to be between 2.2 and 2.5 kb in the case of both E. coli genes and between 2.3 and 2.7 kb in the case of the mutant Bacillus gene. Restriction mapping indicated substantial sequence differences between the E. coli and B. megaterium penicillin acylase genes. Enzyme production in E. coli recombinants from both overproducing mutants was found to be constitutive and higher than in the original strains. The Bacillus penicillin acylase was produced intracellularly in E. coli recombinants, which is in contrast to the normal extracellular production of this enzyme in B. megaterium. Recombinant plasmids containing penicillin acylase genes from either source were found to be unstable in the absence of selection pressure for retention of the vector.

Purification and characterization of phytase from Klebsiella pneumoniae 9-3B

Phytase, an enzyme that catalyzes the hydrolysis of phytate, was purified from Klebsiella pneumoniae 9-3B. The isolate was preferentially selected in a medium which contains phytate as a sole carbon and phosphate source. Phytic acid was utilized for growth and consequently stimulated phytase production. Phytase production was detected throughout growth and the highest phytase production was observed at the onset of stationary phase. The purification scheme including ion exchange chromatography and gel filtration resulted in a 240 and 2077 fold purification of the enzyme with 2% and 15% recovery of the total activity for liberation of inorganic phosphate and inositol, respectively. The purified phytase was a monomeric protein with an estimated molecular weight of 45kDa based on size exclusion chromatography and SDS-PAGE analyses. The phytase has an optimum pH of 4.0 and optimum temperature of 50°C. The phytase activity was slightly stimulated by Ca(2+) and EDTA and inhibited by Zn(2+) and Fe(2+). The phytase exhibited broad substrate specificity and the K(m) value for phytate was 0.04mM. The enzyme completely hydrolyzed myo-inositol hexakisphosphate (phytate) to myo-inositol and inorganic phosphate. The properties of the enzyme prove that it is a good candidate for the hydrolysis of phytate for industrial applications.

Characterization, gene cloning, and heterologous expression of β-mannanase from a thermophilic Bacillus subtilis

Bacillus subtilis BCC41051 producing a thermostable β-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular β-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pi value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60°C, respectively. The enzyme was stable over a pH range of 5.0–11.5, and at temperatures of up to 60°C for 30 min, with more than 80% of its activity retained. ManA was strongly inhibited by Hg2+ (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).

A stereo-inverting D-phenylglycine aminotransferase from Pseudomonas stutzeri ST-201: purification, characterization and application for D-phenylglycine synthesis

D-phenylglycine aminotransferase (D-PhgAT) from a newly isolated soil bacterium, Pseudomonas stutzeri ST-201, was purified to electrophoretic homogeneity and characterized. The molecular weight (M(r)) of the native enzyme was estimated to be 92,000. It is composed of two subunits identical in molecular weight (M(r)) = 47,500). The isoelectric point (pI) of the native enzyme was 5.0. The enzyme catalyzed reversible transamination specific for D-phenylglycine or D-4-hydroxyphenylglycine in which 2-oxoglutarate was an exclusive amino group acceptor and was converted into L-glutamic acid. Neither the D- nor L-isomer of phenylalanine, tyrosine, alanine, valine, leucine, isoleucine or serine could serve as a substrate. The enzyme was most active at alkaline pH with maximum activity at pH 9-10. The temperature for maximum activity was 35-45 degrees C. The apparent K(m) values for D-phenylglycine and for 2-oxoglutarate at 35 degrees C, pH 9.5 were 1.1 and 2.4 mM, respectively. The enzyme activity was strongly inhibited by typical inhibitors of pyridoxal phosphate-dependent enzymes. Possible application of this enzyme for synthesis of enantiomerically pure D-phenylglycine was demonstrated.

Assessment of a PCR technique for the detection and identification of Cryptococcus neoformans

The 18S ribosomal RNA gene of Cryptococcus neoformans was amplified by polymerase chain reaction (PCR). The primers CPL1 and CPR4 were tested for their ability to amplify DNA from 30 strains of C. neoformans and 27 specimens of cerebrospinal fluid (CSF) from patients with cryptococcal meningitis. A 343 bp product was obtained and its specificity confirmed by Southern hybridization with an internal sequence (INSR4) probe. The sensitivity was 100 fg by Southern analysis and 1 pg using the PCR. Neither human nor a variety of other fungal and bacterial strains (n = 78) gave an amplified product. This PCR method can detect as few as 5 cells ml-1 of C. neoformans in spiked-CSF following a simple processing procedure. The developed system of PCR was more sensitive than the culture method and revealed a very high specificity. The PCR was easy to perform and needed only 4 h for all processes from receiving the CSF to detection of a specific DNA band after agarose gel electrophoresis. This would provide another rapid laboratory method for the diagnosis of cryptococcal meningitis.

High expression of the penicillin G acylase gene (pac) from Bacillus megaterium UN1 in its own pac minus mutant

By marker exchange mutagenesis, Bacillus megaterium strain UN‐1 (Bm‐UN1) was used to prepare a mutant strain B. megaterium UN‐cat (Bm‐UNcat) lacking the penicillin G acylase gene (pac). The pac gene from Bm‐UN1 was subcloned into pTF6 and the resultant plasmid, pBA402, was introduced into Bm‐UNcat and Bacillus subtilis. Bm‐UNcat harbouring pBA402 produced high penicillin G acylase (PAC) activity of 13·7, 19·5 and 20·4 U ml−1 at 24, 36 and 48 h of culture, respectively. This was two‐ to fivefold higher than PAC produced by B. subtilis harbouring pBA402 and about 20‐fold higher than PAC produced by the parent strain, Bm‐UN1.

On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate.

Two microfluidic systems have been developed for specific analysis of L-glutamate in food based on substrate recycling fluorescence detection. L-glutamate dehydrogenase and a novel enzyme, D-phenylglycine aminotransferase, were covalently immobilized on (i) the surface of silicon microchips containing 32 porous flow channels of 235 mum depth and 25 mum width and (ii) polystyrene Poros beads with a particle size of 20 mum. The immobilized enzymes recycle L-glutamate by oxidation to 2-oxoglutarate followed by the transfer of an amino group from D-4-hydroxyphenylglycine to 2-oxoglutarate. The reaction was accompanied by reduction of nicotinamide adenine dinucleotide (NAD(+)) to NADH, which was monitored by fluorescence detection (epsilon(ex)=340 nm, epsilon(em)=460 nm). First, the microchip-based system, L-glutamate was detected within a range of 3.1-50.0 mM. Second, to be automatically determined, sequential injection analysis (SIA) with the bead-based system was investigated. The bead-based system was evaluated by both flow injection analysis and SIA modes, where good reproducibility for L-glutamate calibrations was obtained (relative standard deviation of 3.3% and 6.6%, respectively). In the case of SIA, the beads were introduced and removed from the microchip automatically. The immobilized beads could be stored in a 20% glycerol and 0.5 mM ethylenediaminetetraacetic acid solution maintained at a pH of 7.0 using a phosphate buffer for at least 15 days with 72% of the activity remaining. The bead-based system demonstrated high selectivity, where L-glutamate recoveries were between 91% and 108% in the presence of six other L-amino acids tested.

A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its Km and Vmax for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe2+, Fe3+, and Al3+. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).