Wen-Hwei Hsu

Production and properties of a raw-starch-degrading amylase from the thermophilic and alkaliphilic Bacillus sp. TS-23

The optimum temperature and initial medium pH for amylase production by Bacillus sp. TS‐23 were 55 °C and 8.5 respectively. Maximum amylase activity was obtained in a medium containing peptone and soluble starch as nitrogen and carbon sources. Activity staining revealed that two amylases with molecular masses of 150 and 42 kDa were produced when maltose, soluble starch or amylose was used as carbon source for growth, whereas only the 150 kDa protein was detected in the medium containing water‐ insoluble carbon sources. A raw‐starch‐degrading amylase was purified from culture supernatant of Bacillus sp. TS‐23. The molecular mass of the purified amylase was estimated at 42 kDa by electrophoresis. The enzyme had a pI of 4.2. The optimal pH and temperature for activity were 9.0 and 70 °C respectively. The thermoactivity of the purified enzyme was enhanced in the presence of 5 mM Ca2+; under this condition, enzyme activity could be measured at a temperature of 90 °C. The enzyme was strongly inhibited by Hg2+, Pb2+, Zn2+, Cu2+ and EDTA, but less affected by Ni2+ and Cd2+. The enzyme preferentially hydrolysed high‐molecular‐mass substrates with an α‐1,4‐glucosidic bond except glycogen. The raw starches were partly degraded by the purified amylase to yield predominantly oligosaccharides with degrees of polymerization 3, 4 and 5.

A gene encoding for an α‐amylase from thermophilic Bacillus sp. strain TS‐23 and its expression in Escherichia coli

An α‐amylase gene from Bacillus sp. strain TS‐23 was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli. A cell fractionation experiment revealed that approximately 60% of the amylase activity was in the periplasmic space. Analysis and activity staining of the concentrated supernatant fraction by SDS‐polyacrylamide gel electrophoresis showed an apparent protein band with a mol. wt of approximately 65 000. The amylase gene (amyA) consisted of an open reading frame of 1845 bp encoding a protein of 613 amino acids with a calculated mol. wt of 69 543. The predicted amino acid sequence showed high homology with Bacillus species, E. coli and Salmonella typhimuriumα‐amylases. Deletion of 96 amino acids from the C‐terminal portion of the amylase did not result in the loss of amylolytic activity. The truncated amylase, deletion of the first 50 amino acids from the N‐terminus, was overexpressed in E. coli system and refolded to yield an activable enzyme.

Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.

Enzymic properties of a SDS-resistant Bacillus sp. TS-23 α-amylase produced by recombinant Escherichia coli

Abstract A novel alkaline α-amylase of Bacillus sp. TS-23 was purified to homogenous state from the culture medium of recombinant Escherichia coli by ammonium sulphate precipitation and successive Sephacryl TM S-100 and PBE TM 94 chromatography. The molecular mass of the purified enzyme was estimated to be 65 kDa by electrophoresis. The pH and temperature optima for amylase activity were pH 9.0 and 60°C, respectively. The enzyme was stimulated by Mn 2+ , Co 2+ and Fe 2+ ions but was strongly inhibited by Hg 2+ and Cu 2+ and by the well-characterized inhibitors, diethylpyrocarbonate and N -bromosuccinimide. The enzyme was active in the presence of 8% sodium dodecyl sulphate (SDS). Bacillus sp. TS-23 α-amylase was stable when it was preincubated with 6% SDS for upto 1 h at 30°C, while inactivation was observed at 60°C. Under optimal condition, this enzyme was able to attack the α-1,4 linkages in soluble starch, amylose, amylopectin and glycogen to generate maltopentaose as the major end product.

Nitric Oxide Physiological Responses and Delivery Mechanisms Probed by Water-Soluble Roussin’s Red Ester and {Fe(NO)2}10 DNIC

Dinitrosyl-iron complexes (DNICs) are stable carriers for nitric oxide (NO), an important biological signaling molecule and regulator. However, the insolubility of synthetic DNICs, such as Roussins red ester (RRE), in water has impaired efforts to unravel their biological functions. Here, we report a water-soluble and structurally well-characterized RRE [Fe(mu-SC2H4COOH)(NO)2]2 (DNIC-1) and a {Fe(NO)2}(10) DNIC [(PPh2(Ph-3-SO3Na))2Fe(NO)2] (DNIC-2), their NO-induced protein regulation, and their cellular uptake mechanism using immortalized vascular endothelial cells as a model. Compared with the most common NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), the in vitro NO release assay showed that both DNICs acted as much slower yet higher stoichiometric NO-release agents with low cytotoxicity (IC50 > 1 mM). Furthermore, L-cysteine facilitated NO release from SNAP and DNIC-1, but not DNIC-2, in a dose- and time-dependent manner. EPR spectroscopic analysis showed, for the first time, that intact DNIC-1 can either diffuse or be transported into cells independently and can transform to either paramagnetic protein bound DNIC in the presence of serum or [DNIC-(Cys)2] with excess L-cysteine under serum-free conditions. Both DNICs subsequently induced NO-dependent upregulation of cellular heat shock protein 70 and in vivo protein S-nitrosylation. We conclude that both novel water-soluble DNICs have potential to release physiologically relevant quantities of NO and can be a good model for deciphering how iron-sulfur-nitrosyl compounds permeate into the cell membrane and for elucidating their physiological significance.

Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

ABSTRACT A lysine racemase (lyr) gene was isolated from a soil metagenome by functional complementation for the first time by using Escherichia coli BCRC 51734 cells as the host and d-lysine as the selection agent. The lyr gene consisted of a 1,182-bp nucleotide sequence encoding a protein of 393 amino acids with a molecular mass of about 42.7 kDa. The enzyme exhibited higher specific activity toward lysine in the l-lysine-to-d-lysine direction than in the reverse reaction.

Expression of Trigonopsis variabilis D-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants

The D-amino acid oxidase cDNA gene (daao) of Trigonopsis variabilis was prepared by reverse transcriptase-polymerase chain reaction (PCR) and cloned into Escherichia coli expression vector, pTrc99A, under the control of tac promoter. Expression of daao gene significantly affected the growth and morphology of E. coli. The highest D-amino acid oxidase (DAAO) activity was 705 U (mg of protein)(-)(1), which was about 12-fold higher than that of D-alanine-induced T. variabilis. The DAAO protein exhibited activity on native-PAGE and had a M(r)value of 39.3 kDa. We also constructed an expression plasmid, pKm-DAAO, in which kanamycin instead of ampicillin was used as the selective marker. High-performance liquid chromatography (HPLC) analysis demonstrated that cephalosporin C could be converted to 7-glutarylcephalosporanic acid by cell-free extract of E. coli harboring pKm-DAAO. Four inactive DAAO mutants were obtained by error-prone PCR. Sequence analysis of these four DAAO mutants indicated the occurrence of mutations at Val-167, Pro-291, Pro-309, and Ala-343 residues. The His(6)-tagged DAAOs were expressed in E. coli and purified by nickel ion affinity chromatography. The results showed that all DAAO mutants lost their enzymatic activities and characteristic adsorption spectra for flavoenzyme. Based on the crystal structure of a homologous protein, pig DAAO, it is suggested that these four residues may play essential structural roles in DAAO conformation, thereby influencing DAAOs catalytic activity.

Asymmetrical synthesis of L-homophenylalanine using engineered Escherichia coli aspartate aminotransferase.

Site‐directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9‐fold increase in the specific activity toward l‐lysine and 2‐oxo‐4‐phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to l‐homophenylalanine (l‐HPA) with 97% yield and more than 99.9% ee using l‐lysine as amino donor. The transamination product of l‐lysine, 2‐keto‐6‐aminocaproate, was cyclized nonenzymatically to form Δ1‐piperideine 2‐carboxylic acid in the reaction mixture. The low solubility of l‐HPA and spontaneous cyclization of 2‐keto‐6‐aminocaproate drove the reaction completely toward l‐HPA production. This is the first aminotransferase process using l‐lysine as inexpensive amino donor for the l‐HPA production to be reported.

IDENTIFICATION OF THE OPEN READING FRAME FOR THE PSEUDOMONAS PUTIDA D-HYDANTOINASE GENE AND EXPRESSION OF THE GENE IN ESCHERICHIA COLI

A DNA fragment containing the gene for D-hydantoinase was cloned from Pseudomonas putida CCRC 12857 into Escherichia coli. The cloned gene contained an open reading frame (ORF) of 1485 nucleotides encoding a protein of 53.4 kDa in which the carboxyl terminal end is longer than that previously deduced from strain DSM 84. This ORF was verified by amino acid sequencing of amino and carboxyl termini, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid sequence comparison. Deletion analysis revealed that 32 amino acids from the carboxyl terminal end were essential for D-hydantoinase activity. Tagging of six consecutive histidyl residues to the amino terminus or to the carboxyl terminus of the enzyme did not significantly affect D-hydantoinase activity. Under the control of T5lac promoter and lactose induction, the D-hydantoinase activity of transformed E. coli reached 200 U l-1 which is about 20-fold higher than that of gene donor strain.

Efficient utilization of starch by a recombinant strain of Saccharomyces cerevisiae producing glucoamylase and isoamylase

Two plasmids, designated pRTI and pTI, were constructed to allow the integration of a bacterial isoamylase gene (iso) into Saccharomyces cerevisiae G23‐8 chromosome. The integrative plasmid pRTI comprises the iso gene from Pseudomonas amyloderamosa, a portion of S. cerevisiae ribosomal DNA (rDNA), S. cerevisiae trp1 gene deficient in promoter and the bacterial vector pSP72. The structure of plasmid pTI is similar to that of pRTI, except that it lacks an rDNA segment. The Aspergillus awamori glucoamylase and P. amyloderamosa isoamylase genes were expressed in the recombinant strain of S. cerevisiae under the control of the yeast alcohol dehydrogenase gene (adh1) promoter. Southern‐blot analysis showed that these plasmids were integrated into the yeast chromosome in tandem repeat and dispersion copies. The recombinant strains could assimilate starch more efficiently than the recipient strain with a conversion rate of greater than 95%.