Isolation and properties of xyloglucanases of <Emphasis Type="Italic">Penicillium</Emphasis> sp.

Using chromatographic technique, xyloglucanase (XG) A (25 kDa, pI 3.5, 12th glycosyl hydrolase family) was isolated from the enzyme complex secreted by the mycelial fungus Penicillium canescens, and xyloglucanases XG 25 (25 kDa, pI 4.1, 12th glycosyl hydrolase family) and XG 70 (70 kDa, pI 3.5, 74th glycosyl hydrolase family) were isolated from the enzyme complex of Penicillium verruculosum. Properties of the isolated enzymes (substrate specificity, optimal ranges of pH and temperature for enzyme activity and stability, effect of metal ions on catalytic activity) were compared with the properties of xyloglucanases XG 32 of Aspergillus japonicus, XG 78 of Chrysosporium lucknowense, and XG of Trichoderma reesei. The gene xegA encoding XG A of P. canescens was isolated, and the amino acid sequence of the corresponding protein was determined.     

Characteristics of a novel secreted zinc-dependent endopeptidase of <Emphasis Type="Italic">Bacillus intermedius</Emphasis>

A novel zinc-dependent metalloendopeptidase of Bacillus intermedius (MprBi) was purified from the culture medium of a recombinant strain of Bacillus subtilis. The amino acid sequence of the homogeneous protein was determined using MALDI-TOF mass spectrometry. The sequence of the first ten residues from the N-terminus of the mature protein is ASTGSQKVTV. Physicochemical properties of the enzyme and its substrate specificity have been studied. The molecular weight of the metalloproteinase constitutes 19 kDa, the K _m and k _cat values are 0.06 mM and 1210 sec⁻¹, respectively, and the pI value is 5.4. The effect of different inhibitors and metal ions on the enzyme activity has been studied. Based on the analysis of the amino acid sequence of the active site motif and the Met-turn together with the enzyme characteristics, the novel bacterial metalloproteinase MprBi is identified as a metzincin clan adamalysin/reprolysin-like metalloprotease.     

Functional expression and purification of cytokinin dehydrogenase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (AtCKX2) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cytokinin dehydrogenase (CKX) is responsible for regulating the endogenous cytokinin content by oxidative removal of the side chain and seven distinct genes, AtCKX1 to AtCKX7, code for the enzyme in Arabidopsis thaliana. The recombinant enzyme AtCKX2 was produced in Saccharomyces cerevisiae after expressing the corresponding gene from a plasmid (pDR197) or following chromosomal integration, under either the constitutive promoter PMA1 or the inducible promoter GAL1. The recombinant protein was purified from yeast culture media using a sequence of chromatographic steps. The purified enzyme had a molecular mass of 61 kDa and a typical flavoprotein spectrum. The specific activity of the enzyme was 87.8 μkat g⁻¹, with isopentenyladenine as a substrate and 2,3-dimethoxy-5-methyl-p-benzoquinone as an electron acceptor. The pH optimum lay between 7.0 and 8.0, depending on the electron acceptor used. AtCKX2 reacts both with isoprenoid and aromatic cytokinins, the activity with isoprenoid cytokinins being two to three orders of magnitude higher. AtCKX2 prefers p-quinones and the synthetic dye 2,6-dichlorophenol indophenol as electron acceptors, although low reactivity with oxygen can also be observed. This study presents the first purification and characterization of the enzyme from Arabidopsis thaliana.     

Purification and characterization of extracellular inulinase from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the purified inulinase

The extracellular inulinase of the marine yeast Pichia guilliermondii strain 1 was purified to homogeneity resulting in a 7.2-fold increase in specific inulinase activity. The molecular mass of the purified enzyme was estimated to be 50.0 kDa. The optimal pH and temperature for the purified enzyme were 6.0 and 60°C, respectively. The enzyme was activated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Fe³⁺, Fe²⁺, Cu²⁺, and Co²⁺, but Mg²⁺, Hg²⁺, and Ag⁺ inhibited activity. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1, 10-phenanthroline. The K _m and V _max values of the purified inulinase for inulin were 21.1 mg/mL and 0.08 mg/min, respectively. A large number of monosaccharides were detected after the hydrolysis of inulin. The deduced protein sequence from the cloned P. guilliermondii strain 1 inulinase gene contained the consensus motifs R-D-P-K-V-F-W-H and W-M-N-D-P-N-G, which are conserved among the inulinases from other microorganisms.     

Production of fructose from inulin using mixed inulinases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

Two new effective microbial producers of inulinases were isolated from Jerusalem artichoke tubers grown in Thailand and identified as Aspergillus niger TISTR 3570 and Candida guilliermondii TISTR 5844. The inulinases produced by both these microorganisms were appropriate for hydrolysing inulin to fructose as the principal product. An initial inulin concentration of ∼100 g l⁻¹ and the enzyme concentration of 0.2 U g⁻¹ of substrate, yielded 37.5 g l⁻¹ of fructose in 20 h at 40°C when A. niger TISTR 3570 inulinase was the biocatalyst. The yield of fructose on inulin was 0.39 g g⁻¹. Under identical conditions, the yeast inulinase afforded 35.3 g l⁻¹ of fructose in 25 h. The fructose yield was 0.35 g g⁻¹ of substrate. The fructose productivities were 1.9 g l⁻¹ h⁻¹ and 1.4 g l⁻¹ h⁻¹ for the mold and yeast enzymes, respectively. After 20 h of reaction, the mold enzyme hydrolysate contained 53% fructose and more than 41% of initial inulin had been hydrolysed. Using the yeast enzymes, the hydrolysate contained nearly 38% fructose at 25 h and nearly 36% of initial inulin had been hydrolysed. The A. niger TISTR 3570 inulinases exhibited both endo-inulinase and exo-inulinase activities. In contrast, the yeast inulinases displayed mainly exo-inulinase activity. The mold and yeast crude inulinases mixed in the activity ratio of 5:1 proved superior to individual crude inulinases in hydrolysing inulin to fructose. The enzyme mixture provided a better combination of endo- and exo-inulinase activities than did the crude extracts of either the mold or the yeast individually.     

Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post‐translational processing that removes immunogenicity while retaining fibronectin binding

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn‐binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2‐DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF₂₄) and 22 kDa (CadF₂₂) mass variants. CadF variants were fully characterized by MALDI‐TOF MS and MALDI‐MS/MS. These data confirmed that CadF forms re‐folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post‐translationally. CadF₂₂ and CadF₂₄, however, were characterized as N‐terminal, membrane‐associated polypeptides resulting from cleavage between serine¹⁹⁵ and leucine¹⁹⁶, and glycine²⁰¹ and phenylalanine²⁰², respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full‐length CadF (34 kDa), CadF₂₄, and the deleted C‐terminal OmpA domain (14 kDa; CadF₁₄) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full‐length CadF retained reactivity. Binding assays showed that CadF₂₄ retained Fn‐binding capability, while CadF₁₄ did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn‐binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.     

Purification and Characterization of Class Alpha and Mu Glutathione S-Transferases From Porcine Liver

Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (M_r = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different M_r values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (M_r = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model.     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.     

Properties of hemicellulases of the enzyme complex from Trichoderma longibrachiatum

Six xylan-hydrolyzing enzymes have been isolated from the preparations Celloviridin G20x and Xybeten-Xyl, obtained earlier based on the strain 1 Trichoderma longibrachiatum (Trichoderma reesei) TW-1. The enzymes isolated were represented by three xylanases (XYLs), XYL I (20 kDa, pI 5.5), XYL II (21 kDa, pI 9.5), XYL III (30 kDa, pI 9.1); endoglucanase I (EG I), an enzyme exhibiting xylanase activity (57 kDa, pI 4.6); and two exodepolymerases, β-xylosidase (β-XYL; 80 kDa, pI 4.5) and α-L-arabinofuranosidase I (α-L-AF I; 55 kDa, pI 7.4). The substrate specificity of the enzymes isolated was determined. XYL II exhibited maximum specific xylanase activity (190 U/mg). The content of the enzymes in the preparation was assessed. Maximum contributions to the total xylanase activities of preparations Celloviridin G20x and Xybeten-Xyl were made by EG I and XYL II, respectively. Effects of temperature and pH on the enzyme activities, their stabilities under various conditions, and the kinetics of exhaustive hydrolysis of glucuronoxylan and arabinoxylan were studied. Combinations of endodepolymerases (XYL I, XYL II, XYL III, or EG I) and exodepolymerases (α-L-AF I or β-XYL) produced synergistic effects on arabinoxylan cleavage. The reverse was the case when endodepolymerases, such as XYL I or EG I, were combined with α-L-AF I.     

Molecular cloning of the ribosomal P-proteins MgP1, MgP2, MgP0, and superoxide dismutase (SOD) in the mussel Mytilus galloprovincialis and analysis of MgP0 at stress conditions

The stalk, a characteristic structure of the large ribosomal subunit, is directly involved in the interaction with the soluble factors during translation. In the Mediterranean mussel Mytilus galloprovincialis, the stalk consists of one 32 kDa protein, MgP0, and two smaller, 12 kDa acidic proteins, MgP1 and MgP2, of pI 3.0 and 4.0, respectively, as revealed by analysis of purified ribosomes with electrophoresis and Western blot with a specific monoclonal antibody. Treatment of the ribosomes with alkaline phosphatase showed movement of the bands corresponding to the acidic MgP1 and MgP2 proteins to more basic pH after isoelectrofocusing, implying phosphorylation. The cDNA molecules of M. galloprovincialis ribosomal proteins MgP0, MgP1 and MgP2 and superoxide dismutase (MgSOD) were isolated from a cDNA library or constructed by RT-PCR, cloned in expression vectors and expressed in Escherichia coli. The recombinant proteins were purified with immobilized metal ion affinity chromatography (IMAC) and identified with immunoblotting. Exposure of mussels at cadmium and sorbitol and analysis of gill tissue extracts showed over expression of MgP0 protein.     

High expression of recombinant <Emphasis Type="Italic">Streptomyces</Emphasis> sp. S38 xylanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by codon optimization and analysis of its biochemical properties

In recent years, the biotechnological use of xylanases has grown remarkably. To efficiently produce xylanase for food processing and other industry, a codon-optimized recombinant xylanase gene from Streptomyces sp. S38 was synthesized and extracellularly expressed in Pichia pastoris under the control of AOX1 promoter. SDS-PAGE and activity assay demonstrated that the molecular mass of the recombinant xylanase was estimated to be 25 kDa, the optimum pH and optimum temperature were 5.5 and 50°C, respectively. In shake flask culture, the specific activity of the xylanase activity was 5098.28 U/mg. The K _m and V _max values of recombinant xylanase were 11.0 mg/ml and 10000 μmol min⁻¹ mg⁻¹, respectively. In the presence of metal ions such as Ca²⁺, Cu²⁺, Cr³⁺ and K⁺, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of Hg²⁺. This is the first report on the expression properties of a recombinant xylanase gene from the Streptomyces sp. S38 using Pichia pastoris. The attractive biochemical properties of the recombinant xylanase suggest that it may be a useful candidate for variety of commercial applications.     

<Emphasis Type="Italic">APHO1</Emphasis> from the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis> encodes an acid phosphatase of broad substrate specificity

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36–39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60°C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.     

Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (<Emphasis Type="Italic">Octopus vulgaris </Emphasis>Cuvier, 1797)

The NAD⁺ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K_m for both NAD⁺ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity V_max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.     

Enzymological properties of endo-(1–4)-β-glucanase Eg12p of <Emphasis Type="Italic">Penicillium canescens</Emphasis> and characteristics of structural gene <Emphasis Type="Italic">egl2</Emphasis>

Gene egl2 of secreted endo-(1–4)-β-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding β-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60°C, respectively, exhibited specific activity of 33 IU, and had K _m and V _max in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 μmol/sec per mg, respectively.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

A chitinase with antifungal activity from the mung bean

A chitinase with antifungal activity was isolated from mung bean (Phaseolus mungo) seeds. The procedure entailed aqueous extraction, (NH₄)₂SO₄ precipitation, ion-exchange chromatography on CM-Sepharose, high-performance liquid chromatography (HPLC) on Poros HS-20, and gel filtration on Sephadex G-75. The protein exhibited a molecular mass of 30.8 kDa in SDS–polyacrylamide gel electrophoresis. Its pI was 6.3 as determined by isoelectric focusing. The specific activity of the chitinase was estimated to be 3.81 U/mg. The enzyme expressed its optimum activity at pH 5.4 and was stable from 40 to 50 °C. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Mycosphaerella arachidicola, Pythium aphanidermatum, and Sclerotium rolfsii.     

Biochemical properties of isoprene synthase in poplar (<Emphasis Type="Italic">Populus</Emphasis> × <Emphasis Type="Italic">canescens</Emphasis>)

Isoprene synthase (ISPS) catalyzes the elimination of pyrophosphate from dimethylallyl diphosphate (DMADP) forming isoprene, a volatile hydrocarbon emitted from many plant species to the atmosphere. In the present work, immunological techniques were applied to study and localize ISPS in poplar leaves (Populus × canescens). Immunogold labeling using polyclonal antibodies generated against His-tagged recombinant ISPS protein detected ca. 44% of ISPS in the stroma of the chloroplasts and ca. 56% of gold particles attached to the stromal-facing side of the thylakoid membranes. ISPS isolated from leaves exhibited the same biochemical properties as the recombinant ISPS without the plastid-targeting peptide heterologous expressed in E. coli, whereas an additional C- or N-terminal His-tag changed the biochemical features of the recombinant enzyme with regard to temperature, pH, and substrate dependence. In comparison to the closely related class of monoterpene synthases from angiosperms and ISPS of oaks, the most striking feature of the poplar ISPS is a cooperative substrate dependence which is characteristic to enzymes with positive substrate activation. The detection of four immunoreactive bands in poplar leaf extracts with isoelectric points from 5.0 to 5.5 and a native molecular weight of ca. 51 kDa give reason for future studies on post-translational modifications of ISPS.     

Cloning,Isolation, and Properties of a New Homologous Exoarabinase from the <Emphasis Type="Italic">Penicillium canescens</Emphasis> Fungus

A novel exo-arabinase (GH93, exo-ABN) enzyme produced by the ascomycete Penicillium canescens has been studied. Cloning of the abn1 gene coding for exo-ABN into the recipient P. canescens strain RN3-11-7 yielded recombinant producing strains characterized by a high yield of extracellular exo- ABN production (20–30% of the total amount of extracellular protein). Chromatographic purification yielded a homogenous exo-ABN with a molecular weight of 47 kDa, as shown by SDS-PAGE. The enzyme showed high specific activity towards linear arabinan (117 U/mg) and low specific activity towards branched arabinan and arabinoxylan (4–5 U/mg) and para-nitrophenyl-α-L-arabinofuranoside (0.3 U/mg), whereas arabinogalactan and para-nitrophenyl-α-L-arabinopyranoside, the substrates that contained the pyranose form of arabinose, were not hydrolyzed. Arabinohexaose was the major product of linear arabinan hydrolysis. Exo-ABN had a pH optimum at 5.0 and a temperature optimum at 60°C. The enzyme was stable in a broad pH range (4.0–7.0) and upon heating to 50°C during 180 min. Extensive hydrolysis of linear and branched arabinans by exo- and endo-arabinase mixtures, arabinofuranosidase, and arabinofuran-arabinoxylan hydrolase has been performed. The degree of substrate conversion amounted to 67 and 83% of the maximal possible value, respectively.     

Overexpression of acid protease of <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and characterization of the recombinant acid protease for skimmed milk clotting

The gene encoding an acid protease natively produced by Saccharomycopsis fibuligera was cloned and overexpressed in Yarrowia lipolytica and the resultant recombinant acid protease was purified and characterized. The molecular mass of the purified enzyme was estimated as 94.8 kDa by gel filtration chromatography. The optimal pH and temperature of the purified acid protease were 3.5 and 33°C, respectively, and the enzyme was very stable over a pH range of 1.0 ∼ 3.0. The recombinant acid protease was activated by Zn²⁺, but was inhibited by Hg²⁺, Fe²⁺, Fe³⁺, and Mg²⁺, EDTA, EGTA, iodoacetic acid, and pepstatin. The purified recombinant acid protease from the positive transformant 71 had high milk clotting activity, suggesting that it may be used as a rennet substitute in the cheese industry.