弗氏柠檬酸杆菌植酸酶的分离纯化及其酶学性质研究

从弗氏柠檬酸杆菌(Citrobacter freundii)中分离纯化了一种植酸酶并进行了酶学性质研究,其反应最适pH为4.0~4.5,最适温度为40℃,在37℃下以植酸钠为底物的Km值为0.85nmol/L,Vmax为0.53IU/(mg.min),具有较好的抗胰蛋白酶的能力。酶蛋白的分子量大小约为45kDa,成熟酶蛋白N端序列为QCAPEGYQLQQVLMM。     

Isolation and characterization of phytase isozymes produced by Aspergillus oryzae

A mutant strain (KL-38) of Aspergillus oryzae was obtained by UV irradiation. Phytase activity of KL-38 in molded rice (koji rice) was about 2.7-fold of that obtained from the parent strain (BP-1). Phytase activity of KL-38 in the submerged culture was similar to that of BP-1. Two types of phytase were produced from koji culture: phytase I (Phy I) was produced during incubation of both koji and submerged cultures, and phytase II (Phy II) was obtained only from koji culture. Phy II production was increased in KL-38 compared with BP-1, whereas the production of Phy I was similar for both KL-38 and BP-1. This finding indicates that A. oryzae has at least two types of phytase isozyme.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Isolation,purification and characterization of phytase from germinating mung beans

Phytase isolated from mung bean cotyledons was purified about 80-fold with a recovery of 28%. The enzyme is stable at 0°, has a pH optimum at 7·5 and optimal temperature of 57°. The energy of activation is approximately 8500 cal/mole between 37° and 57°. Inhibition by P_i has been found to be competitive, the K_i value being 0·40–0·43 × 10⁻³ M; the K_m value with phytate is 0·65 × 10⁻³ M. Divalent cations are not required for activity. Lower members of inositol phosphates are better substrates, as shown by their V_max and K_m values. When subjected to polyacrylamide gel electrophoresis two bands have been resolved; one (major) corresponds to phytase and the other (minor) to phosphatase and pyrophosphatase activity. Filtration through Biogel P-200 partially resolves phytase from phosphatase and pyrophosphatase. The molecular weight of phytase is approximately 160,000.     

Molecular Cloning of the Phytase Gene from Citrobacter braakii and its Expression in Saccharomyces cerevisiae

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppA_Ec and rAppA_Sc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppA_Sc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppA_Ec.     

Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth—a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkalinepH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such asTribolium castaneum andLocusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.     

Isolation and characterization of a new keratinolytic Bacillus licheniformis strain

A keratin-degrading bacterium strain (K-508) was isolated from partially degraded feathers and characterized. This isolate exhibited a high chicken feather-degrading activity when cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 °C. On the basis of its phenotypic characteristics (quickly moving, Gram-positive rods), the results of metabolic tests and rDNA sequence analysis, it was identified as Bacillus licheniformis. Its fermentation broth showed activity on N-Bz-l-Phe-l-Val-l-Arg-p-nitroanilide, N-Suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide, N-CBZ-Gly-Gly-l-Leu-p-nitroanilide and N-CBZ-l-Ala-l-Ala-l-Leu-p-nitroanilide as chromogenic protease substrates at near neutral pH. Both trypsin-like and chymotrypsin-like proteases were constitutively secreted by this strain.     

Alkaline phytase from Lilium longiflorum: purification and structural characterization

Phytases catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytases are of great commercial importance because their use as food and animal feed supplement has been approved by many countries to alleviate environmental and nutritional problems. Although acid phytases have been extensively studied, information regarding alkaline phytases is limited. Alkaline phytases with unique catalytic properties have been identified in plants, however, there is no report on the purification or structural properties. In this paper, we describe the purification of alkaline phytase from plant tissue. The purification was challenging because of contamination from non-specific phosphatases and acid phytases and low endogenous concentration. The purification of alkaline phytase from pollen grains of Lilium longiflorum involved selective precipitation by heat and ammonium sulfate followed by anion exchange and chromatofocusing chromatography and, finally, gel electrophoresis. Alkaline phytase was purified approximately 3000-fold with an overall recovery of 4.2%. The native molecular mass was estimated to be in the range of 118+/-7 kDa by Ferguson plot analysis and Mr of denatured protein in the range of 52-55 kDa by SDS-PAGE suggesting that the enzyme is a homodimer. Separation by 2-D gel and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis of separated proteins indicates the presence of multiple mass and charge isoforms with pI values between 7.3 and 8.3. To our knowledge, this is the first alkaline phytase to be purified from plant sources. The unique properties suggest that the enzyme has the potential to be useful as a feed and food supplement.     

来源于柠檬酸杆菌的高比活植酸酶基因在毕赤酵母中的高效表达

柠檬酸杆菌(Citrobacterbraakii)来源的植酸酶是目前报道的比活最高的植酸酶。按照毕赤酵母(Pichiapastoris)对密码子的选择偏向性,对来源于柠檬酸杆菌的高比活植酸酶基因AppA进行了密码子优化改造。改造后的基因AppA(m)按正确的阅读框架融合到毕赤酵母表达载体pPIC9的α-因子信号肽编码序列3′端,通过电击转化得到重组转化子。通过PCR验证,AppA(m)已整合在酵母染色体上。SDS-PAGE分析和表达产物的研究表明,植酸酶得到了高效分泌表达,在5L发酵罐中植酸酶蛋白表达量达到3·2mg/mL发酵液,发酵效价达到每毫升发酵液1·4×107IU以上,高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

Purification and characterization of a thermostable and acid-stable phytase from Pichia anomala

Phytase of Pichia anomala was purified to near homogeneity by a two-step process of acetone precipitation followed by anion exchange chromatography using DEAE-Sephadex. The enzyme had a molecular weight of 64 kDa. It was optimally active at 60 °C and pH 4.0. This enzyme was found to be highly thermostable and acid-stable, with a half life of 7 and 8 days at 60 °C and pH 4.0 respectively. At 80 °C, the half life of phytase could be increased from 5 to 30 min by the addition of materials such as sucrose, lactose and arabinose (10% w/v). The enzyme exhibited a broad substrate specificity, since it acted on p-nitrophenyl phosphate, ATP, ADP, glucose-6-phosphate besides phytic acid. The K _m value for phytic acid was 0.20 mM and V _max was 6.34 mol/mg protein/min. There was no requirement of metal ions for activity. SDS was observed to be highly inhibitory to phytase activity. Sodium azide, DTT, -mercaptoethanol, EDTA, toluene, glycerol, PMSF, iodo-acetate and N-bromosuccinimide did not show inhibitory activity. The enzyme was inhibited by 2,3-butanedione, indicating the involvement of arginine residues in catalysis. Phytase activity was not inhibited in the presence of inorganic phosphate upto 10 mM. The shelf life of the enzyme was 6 months at 4 °C and there was no loss in the activity on lyophilization. Very few studies have been done on purification of yeast phytases. This is the first report on purification and characterization of phytase from P. anomala. The enzyme is unique in being thermostable, acid-stable, exhibiting broad substrate specificity and in not requiring metal ions for its activity. The yeast biomass containing phytase appears to be suitable for supplementing animal feeds to improve the availability of phosphorus from phytates.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

Biochemical properties of a thermostable phytase from Neurospora crassa

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.     

水貂源高产蛋白酶枯草芽孢杆菌的分离筛选及酶学性质

【背景】开发、筛选优良益生菌菌种是当下畜牧业的研究热点,益生菌的潜在功能也被广为挖掘。【目的】分离、筛选具有良好耐受性且高产胞外蛋白酶的菌株,研究其生物学特性和酶学性质,为后续微生物蛋白酶的制备和微生态制剂的开发提供菌种资源。【方法】采集健康水貂新鲜粪便,配制酪蛋白培养基初筛和优化Folin-酚法复筛,对筛选菌进行生物学特性研究,获得产蛋白酶能力较强、耐受性较优的菌株,并进行常规鉴定和分子生物学鉴定,最后对蛋白酶酶学性质进行研究。【结果】筛选得到一株高产蛋白酶、耐受性较优的芽孢杆菌,经鉴定为枯草芽孢杆菌(Bacillussubtilis),编号为3。在初始发酵培养基条件下,酶学性质研究结果表明,该蛋白酶的最适反应温度为70℃,最适反应pH值为9.0,最佳金属离子激活剂为K+,Cu2+和Fe2+对酶活力有明显的抑制作用,在20%浓度的有机溶剂作用时蛋白酶未变性失活。【结论】从水貂粪便中分离获得一株具有良好的生物学特性、酶学性质和碱性蛋白酶活性的枯草芽孢杆菌,为该菌株在实际生产应用中提供了基础保障。     

Isolation and characterization of peroxisomes from diatoms

Peroxisomes were isolated by gradient centrifugation from two different diatoms: Nitzschia laevis (subgroup of Pennales) and Thalassiosira fluviatilis (subgroup of Centrales). In neither of these organelles could catalase or any H₂O₂-forming oxidase be demonstrated. The glycolate-oxidizing enzyme present in the peroxisomes is a dehydrogenase capable of oxidizing l-lactate as well. The peroxisomes also contain the glyoxysomal markers isocitrate lyase and malate synthase. However, enzymes of the fatty-acid -oxidation pathway are located exclusively in the mitochondria. The mitochondria additionally possess glutamate-glyoxylate aminotransferase and a glycolate dehydrogenase which differs from the peroxisomal glycolate dehydrogenase since it preferably utilizes d-lactate as an alternative substrate. Hydroxypyruvate reductase and glyoxylate carboligase were not found in the cells of either diatom. By culturing Nitzschia laevis it could be demonstrated that decreasing the CO₂ concentration in the aeration mixture from 2% to 0.03% and increasing the irradiance from 40 to 250 mol quanta · m^–2 · s^–1 resulted in an increase of all peroxisomal enzyme activities. In addition, enzyme activities of the -oxidation pathway were increased. However, mitochondrial glycolate dehydrogenase and aminotransferase did not alter their activities under these conditions. Summarizing all results, it is postulated that there are two different pathways for the metabolism of glycolate in the diatoms.This work was supported by the Deutsche Forschungsgemeinschaft.     

Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria

Citrobacter freundii, Paracoccus denitrificans and Pseudomonas stutzeri were grown either singly or in mixed culture in anaerobic nitrate or nitrite limited chemostats with formate and/or succinate as electron donors and carbon sources. C. freundii reduced nitrate or nitrite stoichiometrically to ammonia. Maximum molar growth yields for nitrate (nitrite) were 15.3 (9.9) g/mol for C. freundii on formate with succinate as carbon source, 15.3 (9.5) g/mol for Ps. stutzeri on succinate and 32.3 (20.4) g/mol for Pa. denitrificans on succinate. The almost identical growth yields indicate that the ATP output of the anaerobic processes in the nitrate (nitrite) ammonifying organism and Ps. stutzeri are nearly the same. In mixed cultures with either Ps. stutzeri or Pa. denitrificans, C. freundii was the best competitor for nitrate. These results show that in anaerobic environments C. freundii may compete successfully with denitrifying organisms.     

Further characterization of phytase from Phaseolus aureus

Phytase purified to homogeneity from germinated mungbean cotyledons was inhibited by EDTA although it did not show any absolute requirement for divalent cations. Sodium fluoride, sodium citrate, mercaptoethanol and pCMB also inhibit the phytase activity but l-phenylalanine has no effect on activity. The phytase has a low affinity for inositol monophosphate. The relative rate of dephosphorylation of myo-inositol-1 -phosphate and myo-inositol-5 phosphate by phytase is 6 and 18% respectively of that of myo-inositol-hexaphosphate. Mungbean phytase cannot cleave myo-inositol-2-phosphate, 1,2-cyclic inositol phosphate, Na-β-glycerophosphate or p-nitrophenylphosphate. The relative rates of hydrolysis of different isomers of inositol hexaphosphate are in the following order: myo-IP₆, > neo-IP₆ > scyllo-IP₆ = d-chiro-IP₆, > l-chiro-IP₆. This enzyme seems to be most active with myo-inositol hexaphosphate.     

卡氏德巴利酵母植酸酶在毕赤酵母中的异源表达及优化

【背景】植酸是一种能螯合金属离子和蛋白质的有机磷类化合物,广泛存在于植物组织中,影响动物对营养元素的吸收。在饲料中加入植酸酶可有效降解植酸。【目的】构建毕赤酵母异源表达卡氏德巴利酵母(Debaryomyces castellii,D. castellii)植酸酶的菌株,促进卡氏德巴利酵母植酸酶的研究及工业应用。【方法】将卡氏德巴利酵母植酸酶基因进行密码子优化后转入毕赤酵母GS115中,通过筛选多拷贝、敲除蛋白酶、过表达分子伴侣及转运蛋白的方法获取优势菌株。【结果】所得重组菌株GS115/DCphy(ΔPep4)(BFR2)的产酶酶活是低拷贝菌株的7倍。【结论】研究结果为卡氏德巴利酵母植酸酶的异源表达及潜在工业应用提供了一定的指导。     

Transgenic maize plants expressing a fungal phytase gene

Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.     

Recombinant Pichia pastoris overexpressing bioactive phytase

Phytase genephyA2, whose signal peptide encoding sequence and intron sequence had been removed, was modified. The Arg-encoding codons CGG and CAG inphyA2 were mutated into synonymous codon AGA. The modifiedphyA2 was fused behind a-factor signal sequence under the control ofAOX1 promoter in plasmid pPIC9, then introduced into the hostPichia pastoris by electroporation. The results of Southern blotting analysis and Northem blotting analysis demonstrated that thephyA2 gene had integrated into the genome ofP. pastoris and transcribed. The result of SDS-PAGE of the phytase expressed by P.pastoris showed that the modifiedphyA2 had been overexpressed and secreted. The concentration of the phytase expressed by P.pastoris with modifiedphyA2 exceeded 15 000 U/mL, which had a 3 000-fold increase over that of originAspergillus niger 963 and was 37 times higher than that of recombinantP. pastoris with non-modifiedphyA2. Project supported by the “863” program, National Science and Technology Commission of China.