Cloning and characterization of a chitinase gene <Emphasis Type="Italic">Lbchi31</Emphasis> from <Emphasis Type="Italic">Limonium bicolor</Emphasis> and identification of its biological activity

Chitinases are digestive enzymes that break down glycosidic bonds in chitin. In the current study, an endochitinase gene Lbchi31 was cloned from Limonium bicolor. The cDNA sequence of Lbchi31 was 1,107 bp in length, encoding 322 amino acid residues with a calculated molecular mass of 31.7 kDa. Clustal analysis showed that there was a highly conserved chitin-binding domains in Lbchi31 protein, containing four sulfide bridges. The Lbchi31 gene was inserted into the pPIC9 vector and transferred into yeast Pichia pastoris GS115 and KM71 for heterologous expression. The transformant harboring the Lbchi31 gene showed a clearly visible protein band with a molecular mass of more than 31 kDa in the SDS-PAGE gel, indicating that it had been translated in P. pastoris. Enzyme characterization showed that the optimal reaction condition for chitinase LbCHI31 activity was: 40°C, pH of 5.0 and 5 mmol l⁻¹ of Mn²⁺. The maximum enzyme activity was 0.88 U ml⁻¹ following exposure to the cell wall chitin of Valsa sordida. The LbCHI31 enzyme can efficiently degrade cell wall chitin of the phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, V. sordida, Septoria tritici and Phytophthora sojae, suggesting that it has the biocontrol function to fungal phytopathogen.     

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25–60 °C and pH 3.0–9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL^?1. In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.     

Cloning and Expression of a Novel Chitinase chi58 from Chaetomium cupreum in Pichia pastoris

A gene encoding a novel chitinase chi58 was cloned from the fungus Chaetomium cupreum by using inverse PCR. The DNA sequence of chi58 contains a 1,602 bp open reading frame and two introns that are 52 and 201 bp in length. Regarding our in silico analysis, chi58 is a modular enzyme composed of a family-18 catalytic domain, which is responsible for chitinase activity, and a chitin-binding domain containing several cysteines. Apparently, the function of these domains is to anchor the enzyme tightly onto the large insoluble polymeric substrate. Chi58 has a pI of 4.47 and a deduced molecular mass of 58 kDa. The optimal pH and temperature conditions were determined to be 5.8 and 45°C, respectively, when colloidal chitin was used as the substrate. SDS-PAGE and zymogram analyses indicated the presence of a single active chitinase. Cells with pPIC9K-chi58 produced an extracellular chitinase that had an activity of 39 U/ml protein. Metal ions such as Ba²⁺, Mg²⁺, K⁺, Cu²⁺, Fe³⁺, Zn²⁺, and Co²⁺ also influenced the activity of the recombinant enzyme.     

Biochemical characterization and high-level production of oxidized polyvinyl alcohol hydrolase from Sphingopyxis sp. 113P3 expressed in methylotrophic Pichia pastoris

The Sphingopyxis sp. 113P3 gene oph, encoding oxidized polyvinyl alcohol hydrolase (OPH), was optimized with the preferred codons of Pichia pastoris and ligated into the pPIC9K vector behind the α-factor signal sequence. The vector was then transfected into P. pastoris GS115 and genomic integration was confirmed. Large-scale production of recombinant protein was performed by induction with 14.4 g/L methanol at 22 °C in a 3-L bioreactor. The maximal OPH activity obtained was 68.4 U/mL, which is the highest activity reported. The optimal pH and temperature of recombinant OPH were 8.0 and 45 °C, respectively. OPH activity was stable over a pH range of 5.0–8.5, and at a maximal temperature of 45 °C. The K _cat /K _m of recombinant OPH was 598 mM^?1 s^?1, which was 4.27-fold higher than that of recombinant OPH derived from Escherichia coli. The improved catalytic efficiency of OPH expressed in recombinant P. pastoris makes it favorable for industrial applications.     

Functional expression and characterization of a chitinase from the marine archaeon Halobacterium salinarum CECT 395 in Escherichia coli

The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6–8.5) and temperature (25–45 °C) ranges. Protein activity was stimulated by the metal ions Mg⁺², K⁺, and Ca⁺² and strongly inhibited by Mn⁺². HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20 % of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc)₃, p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.     

Halorubrum salinum sp. nov., isolated from a marine solar saltern

The halophilic archaeal strain GX71^T was isolated from the Gangxi marine solar saltern near the Weihai city of Shandong Province, China. Cells of the strain were pleomorphic and lysed in distilled water, stained Gram-negative and formed red-pigmented colonies. Strain GX71^T was able to grow at 25–45 °C (optimum 30 °C), in the presence of 1.7–4.8 M NaCl (optimum 2.6 M NaCl), with 0.005–0.7 M MgCl₂ (optimum 0.05 M MgCl₂) and at pH 5.5–9.5 (optimum pH 7.0–7.5). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 10 % (w/v). The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-3) and an unidentified lipid was also detected. The 16S rRNA gene sequence of strain GX71^T showed 94.0–97.0 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB′ gene sequence of strain GX71^T was 87.3–93.4 % similarity to current members of the genus Halorubrum. The DNA G+C content of GX71^T was 67.1 mol%. Strain GX71^T showed low DNA–DNA relatedness with Halorubrum lipolyticum CGMCC 1.5332^T, Halorubrum saccharovorum CGMCC 1.2147^T, Halorubrum kocurii CGMCC 1.7018^T and Halorubrum arcis CGMCC 1.5343^T, the most closely related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX71^T represents a novel species of the genus Halorubrum, for which the name Halorubrum salinum sp. nov. is proposed. The type strain is GX71^T (= CGMCC 1.10458^T = JCM 17093^T).     

Modified pPIC9K vector-mediated expression of a family 11 xylanase gene, Aoxyn11A, from Aspergillus oryzae in Pichia pastoris

A full-length cDNA sequence of Aoxyn11A, a mesophilic xylanase-encoding gene from Aspergillus oryzae, was obtained from total RNA, using 3′ and 5′ rapid amplification of cDNA ends methods. The cDNA sequence is 1,086 base pairs in length, containing 5′-untranslated and 3′-untranslated regions and an open reading frame encoding a 20 amino acid (aa) signal peptide, a 24 aa propeptide and a 188 aa mature peptide (designated AoXyn11A). Multiple alignments verified that AoXyn11A belongs to glycoside hydrolase family 11. Its three-dimensional structure was predicted by multiple templates–based homology modeling. In addition, an AoXyn11A-encoding cDNA gene was extracellularly expressed in Pichia pastoris GS115, mediated by the modified pPIC9K vector. One P. pastoris transformant, numbered as GSAorX4-3 and having the highest recombinant AoXyn11A (reAoXyn11A) activity of 98.0 U/ml, was chosen. The reAoXyn11A showed maximum activity at pH 5.5 and 50 °C. It was highly stable at a pH range of 4.0–8.0 and at 40 °C. Its activity was not significantly affected by metal ions that were tested or EDTA, but was strongly inhibited by Mn²⁺ and Ag⁺. The K _m and V _max of the reAoXyn11A were 1.85 mg/ml and 3,018 U/mg, respectively.     

Cold-Adapted RTX Lipase from Antarctic Pseudomonas sp. Strain AMS8: Isolation, Molecular Modeling and Heterologous Expression

A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S²⁰⁷, D²⁵⁵ and H³¹³, based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.     

Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris

The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis. Amplicons of 265 bp and 447 bp of htrA and yueF, respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with TaqI produced DNA bands of 90 bp and 175 bp with ssp. lactis, and 66 bp and 199 bp with ssp. cremoris. Similarly, restriction of PCR product of 447 bp size with AluI produced digested fragments of 125 bp and 322 bp with ssp. lactis, and 71 bp and 376 bp with ssp. cremoris. The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with TaqI and AluI, respectively.     

Molecular cytogenetic analysis of the Vigna species distributed in Korea

Vigna plants distributed in Korea were analyzed by molecular cytogenetic fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and rDNA ITS/NTS sequences. FISH revealed that variable 45S rRNA gene loci (one to four) were localized on the terminal regions of chromosomes, while two conserved 5S rRNA gene loci from all species examined, except for rice bean (single locus), were detected. FISH and GISH showed the characteristic organization of rRNA gene loci and genomic homology on the chromosomes, indicating their cytogenetic relatationships. ITS sequence revealed that there was considerable variation in length (190–207 bp in ITS1, 205–221 bp in ITS2) and nucleotide composition (7–67 bp). The 5S rRNA gene unit comprised coding region (118 bp) and extensive sequence heterogeneity (97–221 bp). Phylogenetic analysis of the ITS and NTS sequences demonstrated that the Vigna species are divided into two groups: angularis (V. angularis, V. umbellata, V. nakashimae and V. nipponensis) and unguiculata (V. unguiculata, V. sesquipedalis and V. vexillata). Sequence data also showed that mung bean was closer to the angularis group.     

Molecular cloning and characterization of a GH11 endoxylanase from Chaetomium globosum, and its use in enzymatic pretreatment of biomass

An endo-1,4-β-xylanase gene, xylcg, was cloned from Chaetomium globosum and successfully expressed in Escherichia coli. The complete gene of 675 bp was amplified, cloned into the pET 28(a) vector, and expressed. The optimal conditions for the highest activity of the purified recombinant XylCg were observed at a temperature of 40 °C and pH of 5.5. Using oat-spelt xylan, the determined K _m, V _max, and k _cat/K _m values were 0.243 mg?ml^?1, 4,530 U?mg^?1 protein, and 7,640 ml?s^?1?mg^?1, respectively. A homology model and sequence analysis of XylCg, along with the biochemical properties, confirmed that XylCg belongs to the GH11 family. Rice straw pretreated with XylCg showed 30 % higher conversion yield than the rice straw pretreated with a commercial xylanase. Although xylanases have been characterized from fungal and bacterial sources, C. globosum XylCg is distinguished from other xylanases by its high catalytic efficiency and its effectiveness in the pretreatment of lignocellulosic biomass.     

Expression,purification, and characterization of recombinant mangrove glutamine synthetase

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg²⁺-dependent biosynthetic activity was strongly inhibited by Ni²⁺, Zn²⁺, and Al³⁺, whereas was enhanced by Co²⁺. The apparent K _m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH₄ ⁺ respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.     

Expression, purification, and activity identification of Alfimeprase in Pichia pastoris

Alfimeprase (ALF) is a truncated form of non-hemorrhagic zinc metalloproteinase fibrolase. In order to achieve a high level secretion and full activity expression of ALF, the Pichia pastoris (P. pastoris) expression system was used. ALF coding sequence fused with a 6 *histidine tag and an enterokinase recognition site at the N-terminus was cloned into the expression vector pPIC9K and then expressed in P. pastoris strains of GS115 and KM71 by methanol induction. SDS-PAGE and Western blotting analysis showed that the secreted recombinant ALF (rALF) had a molecular weight of 23.8 kDa and was bound specifically to mouse anti-His. tag monoclonal antibody. Under the optimized culture parameters of pH value, initial A(600) value, methanol daily addition concentration and induction time length, the production of rALF reached up to 510 mg/L and 465 mg/L of the GS115 and KM71 transformants, respectively. It also appeared that KM71 was producing a more pure protein than GS115 while GS115 was producing more rALF per unit volume. Through one-step affinity chromatography, the purity of rALF was as high as 96%. The fibrinolytic activity of rALF revealed by the modified fibrin plate method indicated that the protein was efficiently secreted and functionally expressed, and thrombolysis of rALF was demonstrated to be dose-dependent and time-relative. The improved expression system will facilitate further studies and industrial production of ALF.     

Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli

A highly enantioselective cis-epoxysuccinic acid hydrolase from Nocardia tartaricans was purified to electrophoretic homogeneity. The enzyme was purified 184-fold with a yield of 18.8 %. The purified cis-epoxysuccinic acid hydrolase had a monomeric molecular weight of 28 kDa, and its optimum conditions were 37 °C and pH 7–9. With sodium cis-epoxysuccinate as the substrate, Michaelis–Menten enzyme kinetics analysis gave a Km value of 35.71 mM and a Vmax of 2.65 mM min^?1. The enzyme was activated by Ni²⁺ and Al³⁺, while strongly inhibited by Fe³⁺, Fe²⁺, Cu²⁺, and Ag⁺. The cis-epoxysuccinic acid hydrolase gene was cloned, and its open reading frame sequence predicted a protein composed of 253 amino acids. A pET11a expression plasmid carrying the gene under the control of the T7 promoter was introduced into Escherichia coli, and the cis-epoxysuccinic acid hydrolase gene was successfully expressed in the recombinant strains.     

Isolation of laccase gene from Bacillus subtilis and analysis of its physicochemical properties

The present study reports the cloning and sequencing of lac2 from Bacillus subtilis. The gene is composed of 1542 bp and encodes a 514-amino acid protein. The gene has 86% homology with a published laccase with GeneID 936023. The lac2 gene was deposited in GenBank as a new nucleotide sequence. This new sequence was cloned into the multiple cloning site of pPIC9K to generate pPIC9K-lac2, which was then transformed into Pichia pastoris GS115 via electroporation. The recombinant GS115 (pPIC9K-lac2) was grown initially in BMGY medium and transferred to BMMY to induce gene expression for 48 h. The recombinant Lac2 protein shows laccase activity with α-naphthol and guaiacol as substrates. The optimal pH is between 3.2 and 4.7, and the optimal temperature is 25 °C for enzyme reaction.     

Ogataea kanchanaburiensis sp. nov. and Ogataea wangdongensis sp. nov., two novel methylotrophic yeast species from phylloplane in Thailand

Three strains (KM03^T, KM13 ^T and KM15) representing two novel methylotrophic yeast species were isolated from the external surface of plant leaves, which were collected from Kanchanaburi province, Thailand, by three-consecutive enrichments in methanol broth. Strain KM03^T was isolated from phylloplane of a mango tree (Mangifera indica) and two strains, KM13^T and KM15, were obtained from phylloplane of different wine grapes (Vitis vinifera). The sequences of the D1/D2 region of the large subunit (LSU) rRNA gene of the two strains (KM13^T and KM15) were identical and differed markedly from that of strain KM03^T. In terms of pairwise sequence similarity of the D1/D2 region the closest species to the strains KM13^T and KM15 were Candida suzukii (CBS 9253^T) and Candida nitratophila (CBS 2027^T) but with 2.1 % nucleotide substitutions. Strain KM03^T differed from Ogataea wickerhamii (CBS 4307^T), its closest relative, by 2.3 % nucleotide substitutions. Phylogenetic analysis based on the D1/D2 sequences placed the three strains in the Ogataea clade. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analyses of the D1/D2 and the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene (nrRNA) operon, the three strains represent two novel Ogataea species although formation of ascospores was not observed. Ogataea kanchanaburiensis sp. nov. is proposed for strain KM03^T (=BCC 47626^T = NBRC 108603^T = CBS 12673^T). Two strains, KM13^T and KM15, were assigned to Ogataea wangdongensis sp. nov. (type strain KM13^T = BCC 42664^T = NBRC 107778^T = CBS 12674^T). GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 and the ITS regions of O. kanchanaburiensis KM03^T are AB734090 and AB734093, respectively, of O. wangdongensis KM13^T are AB734091 and AB734094, respectively, and of O. wangdongensis KM15 are AB734092 and AB734095, respectively.     

Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies

This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9 K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350 U/mL under the optimal culture conditions of a 500 mL shaking flask containing 20 mL culture medium with the addition of 1.2 % (w/v) methanol, cultivation for 144 h at pH 7.0 and 27 °C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600 U/mL, 6,000 U/mL and 4,800 U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000 U/mL after 130 h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500 U/mL after 145 h incubation when methanol was used as the sole carbon source.     

Molecular cloning, characterization and expression of PmRsr1, a Ras-related gene from yeast form of Penicillium marneffei

GTP-binding proteins such as Ras act as molecular switches in a large number of signal pathways. In this report, we isolated and characterized a novel Ras small monomeric GTPase Rsr1 gene, designated PmRsr1, from yeast-form Penicillium marneffei. The full-length PmRsr1 cDNA sequence is 1,866 bp in size, and contains an open reading frame of 642 bp encoding 213 amino acids. The predicted molecular mass of PmRsr1 is 24.41 kDa with an estimated theoretical isoelectric point of 9.21. The deduced amino acid sequence of PmRsr1 shows 87% identity with that of Aspergillus fumigatus and A. clavatus. Eight exons and seven introns are identified within the 2,102 bp PmRsr1 genomic DNA sequence of P. marneffei. The open reading frame was subcloned into the pcDNA6-myc-His B expression vector, and the recombinant plasmid was transfected into Vero cell line. The expressed fusion protein was analyzed by SDS-PAGE and western blotting. Differential expression of the PmRsr1 was demonstrated by real-time RT-PCR. The expression of PmRsr1 was the highest in the yeast phase comparing with that in the mycelia and conidia phases.     

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S

A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe³⁺ had an active effect on the enzyme obviously.