Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability

A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.     

Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability

A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from lemon (Citrus limon) was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study. To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coliBL21(DE3). Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. The purified enzyme showed two active forms (70% monomer and 30% dimer) in equilibrium, and the specific activity was 7 456 units/mg. The activity of the dimer was 65% higher than that of the monomer. The thermal inactivation rate constant K(d) value calculated for the dimer at 90 degrees C was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min. Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS. The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure. Imidazole caused the dimer to dissociate into monomers. These studies suggested subunit interaction might be important for enzyme stability.     

Molecular cloning, characterization, and expression of a cDNA coding Copper/Zinc superoxide dismutase from black porgy

A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn-SOD) was amplified by a Polymerase Chain Reaction (PCR) based technique from cDNA synthesized from black porgy, Acanthopagrus schlegeli, mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed slightly higher identity (72.8-78.1%) with shark and swordfish Cu/Zn-SOD than with Cu/Zn-SOD from mammalian (68.1-70.7%) and plant (55.5-56.5%) sources. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. The deduced amino acid sequence lacks mitochondria targeting sequence, which suggests that the black porgy cDNA clone encodes a cytosolic Cu/Zn-SOD. The coding region of Cu/Zn-SOD from black porgy was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE. This indicates that the Cu/Zn-SOD cDNA clone can express active Cu/Zn-SOD enzyme in E. coli.     

碱茅（Puccinellia tenuifolra）Put-Cu／Zn—SOD基因的克隆及在酵母中的表达

从碱茅根cDNA文库分离得到Put－Cu／Zn—SOD全长cDNA,其序列全长为2700bp,该基因的开放读码框为长615bp,所编码的蛋白由204个氨基酸组成,其预测分子量约为20．6kD、理论等电点约为5．63。Put－Cu／Zn—SOD氨基酸序列与水稻Cu／Zn-SOD序列有较高的同源性为87％。将Put-Cu／Zn—SOD基因构建到酵母表达载体pYES2,并转化至酵母（INVSc1）。对pYES2-Put-Cu／Zn—SOD转化酵母进行盐碱、氧化胁迫实验。结果表明：重组酵母的抗盐碱、氧化能力明显高于对照（pYES2转化酵母）,结果显示了碱茅的Cu／Zn—SOD基因在酵母表达中,具有提高酵母抗盐碱和耐氧化能力。     

Characterization of the dimer-monomer equilibrium of the papaya Copper/Zinc superoxide dismutase and its equilibrium shift by a single amino acid mutation.

The coding region of the copper/zinc superoxide dismutase (Cu/Zn SOD) cDNA from papaya fruit, Carica papaya L. cv. Tainong 2, was cloned into an expression vector, pET-20b(+). The Cu/Zn SOD was expressed in Escherichia coli and purified by His-tag technique. Two active forms of the enzyme (30% dimer and 70% monomer) in equilibrium were observed. The activity of the dimeric enzyme was higher than that of the monomeric form. The thermal inactivation rate constant K(d) values calculated for the dimer and monomer at 90 degrees C were -0.0203 and -0.0216 min(-1), and the half-lives for inactivation were 41.9 and 31.8 min, respectively. This indicated that the dimeric enzyme was more stable than its monomeric form. The dimerization of the enzyme was inhibited under acidic pH (below 3.0) or imidazole buffer (above 0.5 M), whereas it was not affected under alkaline pH (above 9.0). Both activity and forms of the enzyme were not affected by 1-4% SDS. Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin or chymotrypsin. In addition, mutation of the papaya Cu/Zn SOD at position 48 from Leu to Phe (L48F) affected the association of monomer, whereas a mutant with Lys substitution (L48K) at the same position tended to dissociate into monomeric form.     

杨梅铜锌超氧化物歧化酶基因（MrCu／Zn-SOD1）的原核表达及活性鉴定

为获得高表达杨梅（Morellarubra）铜锌超氧化物歧化酶（MrCu／Zn-SOD1）的工程菌,本实验采用RT-PCR技术从杨梅果实中分离扩增了MrCu／Zn．SOD1的cDNA序列（456bp）,将该基因重组到原核表达载体pGEX-2T中,酶切、测序分析表明,重组质粒pGEX-MrCu/Zn-SOD1结构正确。重组质粒转化大肠杆菌BL21（DE3）进行诱导表达。IPTG诱导表达分子量约41kD融合蛋白GST．MrCu／Zn．SOD1。诱导表达后的菌体超声裂解液经谷胱甘肽亲和层析纯化,得到高纯度的GST—MrCu／Zn—SOD1。采用氯化硝基四氮唑蓝法和黄嘌呤氧化法分析其活性。结果表明,GST-MrCu／Zn-SOD1具有特异性SOD酶活性。     

重金属Cd、Zn、Cu和Pb复合污染对土壤生物活性的影响

通过野外土样采集及室内培养试验(25℃),研究了云南东川铜矿区土壤酶和微生物特征,以及模拟重金属Cd、Zn、Cu、Pb复合污染对土壤微生物和酶活性的影响。结果表明,矿区土壤(距矿口0~800 m)重金属污染严重,Pb、Cd、Zn、Cu全量和有效含量是对照土壤(距矿口10 000 m)的3.7~141.0倍和2.2~773.2倍;距矿口越近,土壤有机质、有效氮、有效磷和速效钾含量及土壤pH亦越低,土壤酶活性和土壤微生物数量、微生物生物量碳和氮受到的抑制程度也显著增强。与对照土壤相比,距矿口0~800 m的土壤蔗糖酶、脲酶、酸性磷酸酶、过氧化氢酶和脱氢酶活性分别降低25.5%~47.3%、22.6%~74.2%、30.9%~83.1%、16.7%~69.1%和34.6%~92.3%;细菌、放线菌和真菌数量分别较对照下降30.5%~80.1%、8.1%~49.9%和3.3%~8.3%。土壤酶中的酸性磷酸酶和过氧化氢酶,土壤微生物中的细菌对重金属污染较为敏感。恒温(25℃)培养试验中,低量的Cd、Zn、Cu、Pb复合污染刺激了土壤酶活性和细菌、真菌、放线菌、微生物生物量碳和氮的数量,但高量的Cu、Zn、Pb、Cd复合污染使土壤酶活性、细菌、真菌、放线菌、微生物生物量碳和氮均显著下降。重金属Cd、Zn、Cu、Pb之间存在着一定的协同或拮抗作用,Cd、Zn、Cu和Pb之间在微生物生物量碳和氮上表现出明显的协同效应,Pb与Cd、Zn、Cu对细菌数量的复合效应机制为拮抗效应,Cd、Zn、Cu和Pb对真菌数量和放线菌数量的复合效应机制表现为协同效应和拮抗效应并存。     

铜污染土壤微生物群落结构及酶活性研究

The microbial community structure and enzyme activities of seven paddy soils with different Cu concentrations were investigated in the vicinity of a Cu smelter in Fuyang County,Zhejiang Province in Southeast China.The microbial community structure was analyzed using the phospholipid fatty acid (PLFA) and multiplex-terminal restriction fragment length polymorphism (M-TRFLP) techniques.There was no clear dose-response relationship between Cu pollution and soil enzyme activity except for urease.Both PLFA and M-TRFLP methods showed that Cu contamination had a large effect on the soil microbial community structure.PLFA indicators of Gram-positive bacteria (16:0i,15:0i) and fungi (18:2w6,9) relatively decreased with increasing Cu concentration,whereas indicators of Gram-negative bacteria (19:0cy,16:1w7) increased.The M-TRFLP results suggested that there was a dose-dependent response between Cu pollution and bacterial community or fungal community.The fungal community was more sensitive to Cu pollution than the bacterial community.There were no significant differences in archaeal community structure between the different Cu pollution plots and archaea might be more tolerant to Cu pollution than both bacteria and fungi.     

外源铜对土壤果树系统中酶活性影响的研究

利用褐土和红富士苹果嫁接苗为供试材料 ,研究外源铜对土壤—苹果树系统中酶活性的影响。结果表明 ,低量的外源铜能使果树叶片的过氧化氢酶、多酚氧化酶、抗坏血酸氧化酶及根系过氧化氢酶的活性加强 ,而高量铜使活性大幅度降低 ,加入钙铁后在一定程度上使抑制缓解。土壤过氧化氢酶的活性与外源铜量具显着的曲线相关性 ,(y =x ( - 1 1 .2 6 0 .75x) ,r=0 .995 5 ) ,蔗糖酶活性与土壤施铜量呈Logistic函数关系 (y =2 .95 ( 1 0 .5 7exp( - 5 .0 2× 1 0 - 5x) ,r=- 0 .982 0 )。脲酶对铜过量非常敏感 ,大于 1 0 0mgkg- 1的各处理均未测出其活性     

不同金属辅基和酶剂量对超氧化物歧化酶抗氧化、促氧化作用的影响

研究含不同类型金属辅基(Mn或Cu,Zn)的超氧化物歧化酶(SOD)的抗氧化/促氧化作用特征,以及酶剂量对其抗氧化/促氧化作用的影响。用化学发光法分析超氧化物歧化酶对由Fenton体系产生的羟基自由基(.OH)导致DNA氧化损伤的影响。进一步利用.OH所致质粒DNA氧化后在琼脂糖凝胶电泳中的构型改变为实验模型,比较不同类型金属辅基和剂量对SOD抗氧化/促氧化活性的影响。Mn-SOD对.OH引起的DNA氧化损伤没有显著影响。然而,Cu,Zn-SOD在较高浓度(>200 U/ml)下,表现出强烈的促氧化效应,能够加剧DNA氧化损伤。研究结果表明,金属辅基对SOD的抗氧化或促氧化效应有着重要的影响,这可能与金属辅基在Fenton反应中的氧化还原特性相关。高浓度的Cu,Zn-SOD对.OH所致DNA氧化损伤存在显著的促氧化效应。高浓度的Mn辅基离子不能与Fenton体系中H2O2直接作用,并且不能促进羟基自由基的形成。     

铜对小白菜的毒性效应及其生态健康指标

以小白菜地上部分及根系重量、叶绿素值(SPAD值)以及与抗逆性有关的丙二醛(MDA)、超氧化物歧化酶活性(SOD)、脯氨酸含量等作为毒性指标,研究了04～00.mg/kg外源铜对青紫泥小白菜生长的影响,并应用“生态剂量”概念计算青紫泥栽培小白菜的铜污染安全指标及食品卫生指标。结果表明,低浓度铜(200.mg/kg)则造成小白菜减产,400.mg/kg的外源铜使小白菜减产84%;铜污染使根系重量下降表现出与地上部分同样的趋势。SPAD值随铜污染浓度的增加而呈线性下降;铜污染浓度低于100.mg/kg有促进根体积、根长度、根表面积增加的趋势,但超过200.mg/kg的铜浓度则显著降低根长、根体积和根表面积。在与重金属污染引起的抗逆性有关的生理生化指标中,脯氨酸最为敏感,铜浓度小于50.mg/kg或大于200.mg/kg都能显著影响脯氨酸含量及SOD活性;与脯氨酸的变化一样,高浓度铜增加了MDA含量;青紫泥铜污染的临界指标为171.mg/kg。     

Interactions between biomass production and ethylene biosynthesis in copper‐treated rice

Rice (Oryza sativa L.) plants were grown over a 30‐day‐period in nutrient solutions containing increasing copper (Cu) concentrations (0.002, 0.01, 0.05, 0.25, 1.25, and 6.25 mg/L). It was observed that in both root and leaf tissues the total activity of l‐aminocyclopropane‐l‐carboxylate synthase decreased at concentrations above 0.05 mg/L Cu treatment, whereas the total activity of the ethylene forming enzyme slightly increased until the 1.25 mg/L Cu treatment. In the root and leaf tissues, the 1‐aminocyclopropane‐l‐carboxylic acid concentrations decreased after the 0.05 mg/L and 0.01 mg/L Cu treatments, respectively, whereas the ethylene production decreased in both tissues after the 0.05 mg/L Cu treatment. It is proposed that excess Cu in both root and leaf tissues decrease the conversion of S‐adenosylmethionine to 1‐aminocyclopropane‐l‐carboxylic acid through the inhibition of the total l‐aminocyclopropane‐l‐carboxylate synthase activity. The concomitant effect of this inhibition on adventitious root formation and leaf senescence is evaluated.     

Effects of lead pollution on different soil enzyme activities

We studied the effects of Pb pollution on soil dehydrogenase and phosphatase activity. Samples of four soils (Saxe, Podestà, Porto Teulada, and Sa Xia Manna) were collected from various locations in southwestern Sardinia, Italy. The soils, which differ mainly in heavy metal contents of pedologic origin (Cu, Zn, Cd, and Pb), were treated with Pb (0, 100, 500, 1000, and 5000 g Pb g^-1 soil) and incubated in the laboratory. Samples of the incubated soils were collected periodically (0, 1, 2, 4, 8, and 16 weeks) and the enzymes were measured. Soil dehydrogenase activity was influenced by both the Pb additions and variations in soil moisture content. Only the addition of 5000 g Pb g^-1 soil led to a significant decrease in dehydrogenase activity compared to the controls, while the other doses of Pb did not always result in a clear reduction in enzyme activity. Drying the soil led to a considerable reduction in dehydrogenase activity, sometimes so far as to render the differences found between the various treatments not statistically significant. Soil phosphate activity was also influenced by the Pb additions, but the effect of the variation in soil moisture content was less than that found for the dehydrogenase. After the 2nd week of incubation, the phosphate activity in the Podestà and Saxe soils had decreased proportionally to the increase in Pb content. At the end of the incubation period, in the Porto Teulada and Sa Xia Manna soils, a net reduction in phosphatase activity versus controls was found only at the highest Pb concentration. Although both enzyme activities were influenced by the Pb additions, the phosphate activity was less sensitive to variations in the soil moisture content and may thus be a more suitable indicator for soil pollution by Pb.     

Gene cloning and characterization of a novel recombinant antifungal chitinase from papaya (Carica papaya)

A chitinase cDNA clone (CpCHI, 1002 bp) was isolated from papaya fruit, which encoded a 275 amino acid protein containing a 28 amino acid signal peptide in the N-terminal end. The predicted molecular mass of the mature protein was 26.2 kDa, and its pI value was 6.32. On the basis of its amino acid sequence homology with other plant chitinases, it was classified as a class IV chitinase. An active recombinant CpCHI enzyme was overexpressed in Escherichia coli. The purified recombinant papaya chitinase showed an optimal reaction temperature at 30 degrees C and a broad optimal pH ranging from 5.0 to 9.0. The recombinant enzyme was quite stable, retaining >64% activity for 3 weeks at 30 degrees C. The spore germination of Alternaria brassicicola could be completely inhibited by a 76 nM level of recombinant CpCHI. Recombinant CpCHI also showed antibacterial activity in which 50% of E. coli was inhibited by a 2.5 microM concentration of the enzyme.     

Monothiol glutaredoxin cDNA from Taiwanofungus camphorata: a novel CGFS-type glutaredoxin possessing glutathione reductase activity

Glutaredoxins (Grxs) play important roles in the redox system via reduced glutathione as a reductant. A TcmonoGrx cDNA (1039 bp, EU158772) encoding a putative monothiol Grx was cloned from Taiwanofungus camphorata (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved among the reported monothiol Grxs. Two 3-D homology structures of the TcmonoGrx based on known structures of human Grx3 (pdb: 2DIY_A) and Mus musculus Grx3 (pdb: 1WIK_A) have been created. To characterize the TcmonoGrx protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli C41(DE3). The recombinant His6-tagged TcmonoGrx was overexpressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed a predominant band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited glutathione reductase (GR) activity via dithionitrobenzoate (DTNB) assay. The Michaelis constant (K(M)) values for GSSG and NADPH were 0.064 and 0.041 mM, respectively. The enzyme's half-life of deactivation at 60 °C was 10.5 min, and its thermal inactivation rate constant (k(d)) was 5.37 × 10(-2) min(-1). The enzyme was active under a broad pH range from 6 to 8. The enzyme retained 50% activity after trypsin digestion at 37 °C for 40 min. Both mutants C(40)→S(40) and C(165)→S(165) lost 40-50% GR activity, whereas the mutant S(168)→C(168) showed a 20% increase in its GR activity.     

Assessment of microbial activity and bacterial community composition in the rhizosphere of a copper accumulator and a non-accumulator

Soil microorganisms may play an important role in the uptake of heavy metals from soils. However, assessments of bacterial activity and community composition in the rhizosphere of accumulators have been largely ignored. We studied potential effects of a copper (Cu)-accumulator, Elsholtzia splendens, and a non-Cu-accumulator plant, Trifolium repens, on soil microbial activity and community composition with increasing Cu addition. The results showed that concentrations of Cu in the shoots of E. splendens were 2.1, 2.2 and 2.4 times those of T. repens under the treatment of different Cu concentrations. Soil microbial biomass and phosphatase activity in the rhizosphere of E. splendens were higher than those of T.repens. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprint analysis revealed that addition of Cu decreased the number of bands in bare soil and soil with T. repens. However, there was a significant increase in the number of bands in soil with E. splendens incorporated with either 200 or 500 mg kg⁻¹ Cu. The abundances of five phylogenetic groups related most closely to -, β-, γ-proteobacteria, Gram-positive bacteria and CFB group, respectively, were determined in the rhizosphere of plants. Some specific clone such as E13 (metal-contaminated soil clone K20-64) was found in the rhizosphere of E. splendens. Results indicated that E. splendens, as a Cu-accumulator, played an important role in governing soil microbial activity and bacterial community composition in the rhizosphere in response to Cu stress.     

棉花肉桂醇脱氢酶基因GhCAD3的克隆及原核表达

肉桂醇脱氢酶(CAD)是木质素生物合成过程中的一个关键酶类。本研究从棉花中克隆了一个CAD基因,命名为GhCAD3(GenBank登录号为FJ376601)。GhCAD3全长1573 bp,具有1个1080 bp的开放阅读框,5′非编码区为35 bp,3′非编码区为458 bp,编码359个氨基酸,预测分子量约为39.116kD,等电点为7.48。氨基酸同源性分析发现,GhCAD3与其他CAD一致性为64.13%。为了进一步研究GhCAD3基因的功能,构建了该基因的原核表达载体pET-28a-CAD3,经酶切鉴定后转化到大肠杆菌BL21(DE3)中。SDS-PAGE电泳分析表明,最佳诱导表达条件为0.5 mmol/L IPTG在37℃下诱导7 h。     

铜与草甘膦单一污染和复合污染对水稻土酶活性的影响

通过实验室培养试验,研究了铜与草甘膦单一污染和复合污染对水稻土中淀粉酶、脲酶、磷酸酶和过氧化氢酶活性的影响。结果表明,铜与草甘膦单一和复合污染对土壤中4种酶活性的影响效果明显不同。当铜单一污染时,抑制淀粉酶、脲酶、磷酸酶的活性,对过氧化氢酶活性的影响是低浓度激活高浓度抑制;而草甘膦单一污染时,对4种酶活性的影响是：激活淀粉酶和脲酶,抑制过氧化氢酶,对磷酸酶活性的影响则是低浓度激活高浓度抑制。铜和草甘膦复合污染,显著改变了铜或草甘磷单一污染对土壤酶的毒性效应。即复合污染对过氧化氢酶的毒性大于单一污染;对淀粉酶、脲酶和磷酸酶的毒性,小于铜单一污染,但大于草甘磷。方差分析结果表明,不同铜浓度间和不同草甘膦浓度间4种酶活性差异均达到极显著水平（P〈0．01）;铜和草甘膦互作浓度间,淀粉酶和脲酶活性差异分别达到了显著水平和极显著水平,其他2种酶活性差异不显著。     

十溴联苯醚对两种土壤酶活性的影响

以十溴联苯醚(BDE-209)作为外加污染源,测定不同浓度的污染物(1、10、50、200、500 mg/kg)在1~115天处理期对水稻土和菜田土脲酶、蔗糖酶、酸性磷酸酶活性的影响。结果表明,不同浓度BDE-209处理对水稻土和菜田土中3种土壤酶活性的影响不同:在水稻土中,第1天时BDE-209处理即对土壤蔗糖酶、脲酶活性产生了较为显著的促进作用,对酸性磷酸酶活性产生了显著的抑制作用,而在菜田土中,这种现象的出现是在处理后的第14天;在1~50 mg/kg浓度围内,BDE-209处理对水稻土和菜田土酶活性的促进/抑制作用基本随着BDE-209浓度的增大而增强,而在200、500 mg/kg浓度处理下,作用不明显;在水稻土中各处理脲酶与酸性磷酸酶活性动态变化相关性显著,在菜田土中各处理磷酸酶和蔗糖酶活性动态变化相关性显著。因此,BDE-209对不同土壤酶的作用因土壤类型不同而有所差异,应根据不同土壤选择恰当的土壤酶作为土壤BDE-209污染程度的指标。     

Expression and characterization of sweet potato invertase in Pichia pastoris

An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.