Long-Liu Lin

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.

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.

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%.

Engineering of a truncated α-amylase of Bacillus sp. strain TS-23 for the simultaneous improvement of thermal and oxidative stabilities

BACDeltaNC/Delta RS is a thermostable variant derived from the truncated alpha-amylase (BAC Delta NC) of alkaliphilic Bacillus sp. strain TS-23. With the aim of enhancing its resistance towards chemical oxidation, Met231 of BAC Delta NC/Delta RS was replaced by leucine to create BAC Delta NC/Delta RS/M231L. The functional significance of the 31 C-terminal residues of BAC Delta NC/Delta RS/M231L was also explored by site-directed mutagenesis of the 483 th codon in the gene to stop codon (TAA), thereon the engineered enzyme was named BAC Delta NC/Delta RS/M231L/Delta C31. BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 were very similar to BAC Delta NC in terms of specific activity, kinetic parameters, pH-activity profile, and the hydrolysis of raw starch; however, the engineered enzymes showed an increased half-life at 70 degrees C. The intrinsic fluorescence and circular dichroism spectra were nearly identical for wild-type and engineered enzymes, but they exhibited a different sensitivity towards GdnHCl-induced denaturation. This implicates that the rigidity of the enzyme has been changed as the consequence of mutations. Performance of the engineered enzymes was evaluated in the presence of commonly used detergent compounds and some detergents from the local markets. A high compatibility and performance of both BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 may be desirable for their practical uses in the detergent industry.

The N-terminal signal sequence and the last 98 amino acids are not essential for the secretion of Bacillus sp. ts-23 α-amylase in Escherichia coli

A truncated Bacillus sp. TS-23 α-amylase gene lacking 96 and 294 bp at its 5′ and 3′ end respectively was prepared by polymerase chain reaction and cloned into Escherichia coli expression vector, pQE-30, under the control of T5 promoter. SDS-PAGE and activity staining analyses showed that the His6-tagged amylase had a molecular mass of approximately 54 kDa. Isopropyl-β-d-thiogalactopyranoside (IPTG) induction of E. coli M15 cells bearing the recombinant plasmid resulted in the extracellular production of active amylase. Western blot analysis also revealed that the truncated amylase was present in the periplasmic space and culture medium.

High-level expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli using lactose as inducer

The use of lactose as inducer for the expression of Trigonopsis variabilis d-amino acid oxidase gene (daao) was investigated in Escherichia coli regulated by T7 or T5 promoter. The daao gene was prepared by reverse transcriptase-polymerase chain reaction and cloned into pET21b and pQE-30 to yield pET-DAAO and pQE-DAAO, respectively. The His6-tagged DAAO was expressed in E. coli and had a Mr value of approximately 39.3 kDa. In lactose-induced E. coli BL21 (DE3) (pET-DAAO), the expressed DAAO could comprise up to 15% of total soluble proteins and a productivity of 23.4 U ml−1 was obtained.

Construction of an amylolytic yeast by multiple integration of the Aspergillus awamori glucoamylase gene into a Saccharomyces cerevisiae chromosome

Abstract A plasmid containing the glucoamylase cDNA gene ( glu ) from Aspergillus awamori , a portion of Saccharomyces cerevisiae ribosomal DNA, S. cerevisiae ura 3 gene deficient in promoter, and bacterial cloning vector pSP72 was constructed for the integration of an A. awamori glu gene into the genome of S. cerevisiae . Several transformants were able to form halos around colonies on the plate containing 2% soluble starch. This indicated the secretion of glucoamylase by the yeasts. Glucoamylase production by transformant G23-8 reached 162 U l −1 . Southern hybridization analysis revealed that the plasmid was integrated into the host chromosome in tandem repeat and dispersion copies. It was estimated that the integrants carried up to 140 glu gene copies per genome. The integrants were mitotically stable for 50 generations without selective pressure.

Covalent Immobilization of Bacillus licheniformis γ-Glutamyl Transpeptidase on Aldehyde-Functionalized Magnetic Nanoparticles

This work presents the synthesis and use of surface-modified iron oxide nanoparticles for the covalent immobilization of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT). Magnetic nanoparticles were prepared by an alkaline solution of divalent and trivalent iron ions, and they were subsequently treated with 3-aminopropyltriethoxysilane (APES) to obtain the aminosilane-coated nanoparticles. The functional group on the particle surface and the amino group of BlGGT was then cross-linked using glutaraldehyde as the coupling reagent. The loading capacity of the prepared nanoparticles for BlGGT was 34.2 mg/g support, corresponding to 52.4% recovery of the initial activity. Monographs of transmission electron microscopy revealed that the synthesized nanoparticles had a mean diameter of 15.1 ± 3.7 nm, and the covalent cross-linking of the enzyme did not significantly change their particle size. Fourier transform infrared spectroscopy confirmed the immobilization of BlGGT on the magnetic nanoparticles. The chemical and kinetic behaviors of immobilized BlGGT are mostly consistent with those of the free enzyme. The immobilized enzyme could be recycled ten times with 36.2% retention of the initial activity and had a comparable stability respective to free enzyme during the storage period of 30 days. Collectively, the straightforward synthesis of aldehyde-functionalized nanoparticles and the efficiency of enzyme immobilization offer wide perspectives for the practical use of surface-bound BlGGT.

Recovery of isoamylase from Pseudomonas amyloderamosa by adsorption-elution on raw starch

The isoamylase from Pseudomonas amyloderamosa WU2130 was adsorbed onto raw starch, and the adsorbed enzyme was eluted by maltose in 50 mm acetate buffer. The adsorption of isoamylase to adsorbent was affected by the sources of raw starch, temperature, and pH, whereas the temperature and elution time had no obvious effects on the elution of adsorbed isoamylase. Raw starch could be used repeatedly on the adsorption-elution cycle with good reproducibility. About 52.5% of isoamylase was recovered, and the eluted enzyme had a specific activity of 72,828 U mg−1 protein with a purification of 13.3-fold. These results suggest that the raw starch adsorption-elution technique has a great potential in recovery of isoamylase from culture broths.