一株具有ACC脱氨酶活性固氮菌的筛选与鉴定

ACC（1-aminocyclopropane-1-carboxylate, 1-氨基环丙烷-1-羧酸）脱氨酶是近年来发现的许多植物促生细菌（Plant growth promoting bacteria, PGPB）共有的一个特征性酶,很多具有ACC脱氨酶活性的细菌能够增强植物抗逆性,缓解干旱、淹水、盐碱、高温、病虫害等对植物的危害。因此,ACC脱氨酶阳性细菌的筛选和研究对促进农业生产具有重要意义。本文从大量样品中分离、筛选到1株ACC脱氨酶阳性固氮菌,编号为7037,该菌株ACC脱氨酶活性为-丁酮酸2.530 mol /(hmg),protein,固氮酶活性为C2H410.068 nmol /(hmg), protein;具有较为广泛的碳源利用能力和很强的环境适应能力,被鉴定为节杆菌属（Arthrobacter sp.）的一个种。盆栽试验显示,小白菜接种7037菌株比对照组鲜重增加了139%,差异极显著。该菌株可望进一步研究开发成为微生物肥料的生产菌种。     

Molecular cloning and characterization of an alpha-amylase occurring in the pulp of ripening bananas and its expression in Pichia pastoris

Alpha-amylases (EC 3.2.1.1) are glycosyl hydrolases with endoglycolytic activity on the alpha-1,4-d-glucosidic linkages in starch. In bananas, the mobilization of starch accounts for sugar accumulation during ripening, and among several hydrolytic enzymes, alpha-amylase is the only enzyme argued to be able to attack the intact granules, indicating a pivotal role for this enzyme. A 1953 bp full-length banana alpha-amylase cDNA (MAmy), encoded for a sequence of 416 amino acids, was cloned and used for heterologous expression in Pichia pastoris. The cloned MAmy presented the highly conserved motifs common to alpha-amylases, and the amylolytic activity of the extracts from yeast transformed with MAmy demonstrated that it encodes for a functional alpha-amylase, suggesting a putative role for this gene in starch degradation during fruit ripening.     

Partial characterization of peroxidase and polyphenol oxidase activities in blackberry fruits

A partial characterization of peroxidase (POD) and polyphenol oxidase (PPO) activities in blackberry fruits is described. Two cultivars of blackberry (Wild and Thornless) were analyzed for POD and PPO activities. Stable and highly active POD and PPO extracts were obtained using insoluble poly(vinylpyrrolidone) and Triton X-100 in 0.05 M sodium phosphate, pH 7.5, buffer. Blackberry POD and PPO activities have a pH optimum of 6.5, in a reaction mixture of 0.2 M sodium phosphate. Optimal POD activity was found with 3% o-dianisidine. Maximum PPO activity was found with catechol (catecholase activity) followed by 4-methylcatechol. Polyacrylamide gel electrophoresis of blackberry extracts under non-denaturing conditions resolved in various bands. In the POD extracts of Wild fruits, there was only one band with a mobility of 0.12. In the Thornless POD extracts there were three well-resolved bands, with R(f) values of 0.63, 0.36, and 0.09. Both the Wild and Thornless blackberry cultivars produced a single band of PPO, with R(f) values of 0.1 for Wild and 0.06 for Thornless.     

Molecular cloning, recombinant expression, and immunological characterization of a novel allergen from tartary buckwheat

Buckwheat is generally regarded as a nutritionally rich food source. However, earlier studies prove that it also causes allergies to subjects. Allergenic proteins with a strong IgE-binding activity have been identified in common buckwheat (CB) and a 24 kDa allergen (rTBa) in tartary buckwheat (TB). The objective of this research was to clone and express a novel allergen in tartary buckwheat and to evaluate its structure and immunological activity. The 1773 bp full-length cDNA was amplified and cloned from the total RNA of TB by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Its nucleotide sequence had high similarity with legume-like 13S storage protein mRNA in CB. The deduced amino acid sequence included a putative signal peptide and 18 fragments as its epitope sites. The predicted full-length TB allergen sequence was found to have two domains, and the recombinant protein reacted with sera from patients with positive IgE binding to buckwheat and had a lower binding ability than the recombinant TBa and recombinant TBb (C- and N-terminal amino acid sequence of TBt codes for protein). This fact suggests that full-length TB allergen may hydrolyze to two domains in vivo, decreasing the IgE-binding ability.     

Partial characterization of polyphenol oxidase activity in raspberry fruits.

A partial characterization of polyphenol oxidase (PPO) activity in raspberry fruits is described. Two early cultivars harvested in May/June (Heritage and Autumm Bliss) and two late cultivars harvested in October-November (Ceva and Rubi) were analyzed for PPO activity. Stable and highly active PPO extracts were obtained using insoluble poly(vinylpyrrolidone) (PVP) and Triton X-100 in sodium phosphate, pH 7.0 buffer. Polyacrylamide gel electrophoresis of raspberry extracts under nondenaturing conditions resolved in one band (R(f)()(1) = 0.25). Raspberry PPO activity has pH optima of 8.0 and 5.5, both with catechol (0.1 M). Maximum activity was with D-catechin (catecholase activity), followed by p-coumaric acid (cresolase activity). Heritage raspberry also showed PPO activity toward 4-methylcatechol. Ceva and Autumm Bliss raspberries showed the higher PPO activity using catechol as substrate.     

Functional characterization of FaCCD1: a carotenoid cleavage dioxygenase from strawberry involved in lutein degradation during fruit ripening

A gene encoding a carotenoid cleavage dioxygenase class 1 enzyme (FaCCD1) was identified among a strawberry fruit expressed sequence tag collection. The full-length cDNA was isolated, and the expression profiles along fruit receptacle development and ripening, determined by quantitative real time polymerase chain reaction, showed that FaCCD1 is a ripening-related gene that reaches its maximal level of expression in the red fully ripe stage. FaCCD1 was expressed in Escherichia coli, and the products formed by the recombinant protein through oxidative cleavage of carotenoids were identified by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses. The FaCCD1 protein cleaves zeaxanthin, lutein, and beta-apo-8'-carotenal in vitro. Although beta-carotene is not a good substrate for FaCCD1 in vitro, the expression of FaCCD1 in an engineered carotenoid-producing E. coli strain caused the degradation of beta-carotene in vivo. Additionally, the carotenoid profile in strawberry was analyzed by high-performance liquid chromatography-photodiode detection, and a correlation between the increase of the expression level of FaCCD1 during ripening and the decrease of the lutein content suggests that lutein could constitute the main natural substrate of FaCCD1 activity in vivo.     

Effects of ethylene and 1-methylcyclopropene (1-MCP) on gene expression and activity profile of alpha-1,4-glucan-phosphorylase during banana ripening

Starch phosphorylases are enzymes that can use starch as substrate, and they are supposed to act in both in starch synthesis and degradation. This paper reports the effects of ethylene and 1-methylcyclopropene (1-MCP) on the degradation of starch and phosphorylase activity and gene expression. The results indicate that phosphorylase activity is induced during ripening and that it is associated with the onset of starch degradation. The regulation of banana phosphorylase activity is mainly dependent on gene expression, and the absence of ethylene perception by 1-MCP had a positive effect. However, this effect can be precluded by increased levels of ethylene, both autocatalytic and exogenous.     

Molecular cloning and characterization of oryzacystatin-III, a novel member of phytocystatin in rice (Oryza sativa L. japonica)

On the basis of cDNA sequences, we found that the calli of rice encodes an amino acid sequence that shares 56% and 89% identity, respectively, with oryzacystatin-I and oryzacystatin-II. This sequence differs from that of oryzacystatin-II in the N-terminal region (Gln(7)-Ala(19) in the oryzacystatin-III numbering), and this region contained a glycine residue (Gly(14)), which is evolutionarily conserved in the cystatin superfamily. We named this novel protein oryzacystatin-III. Nucleotide sequencing of the 5'-flanking region of the oryzacystatin-III gene showed that it is highly homologous to the oryzacystatin-II gene but distinct from the oryzacystatin-II locus. Oryzacystatin-III inhibited papain, ficin, and human cathepsin B. The inhibition constants for papain and ficin differ from those of oryzacystatin-I and -II, and cathepsin B activity is affected only by oryzacystatin-III, showing differences in the interaction of these inhibitors with enzymes. These data suggest that the above three inhibitors may play unique physiological roles in the regulations of rice cysteine proteinases.     

1-MCP乙烯受体阻断剂对香蕉果实采后生理和品质的影响

1-甲基环丙烯(1-MCP)可以显著延迟香蕉果实软化，但1-MCP对综合食用品质变化的影响不甚清楚。该试验以国产品种广东高州矮香蕉为试材，用200 nL/L 1-MCP处理24 h后贮藏于20℃条件下，分析了1-MCP对香蕉综合品质的影响。结果表明：1-MCP处理显著地降低了香蕉果肉中可溶性糖和可溶性固形物含量的上升速率，延缓了果实硬度的下降。1-MCP能延缓香蕉果实的后熟进程，但并不会降低香蕉的综合食用品质。即使外源乙烯的加入也不能加快1-MCP处理后香蕉果实的后熟进程。     

Root-associated bacteria containing 1-aminocyclopropane-1-carboxylate deaminase improve growth and nutrient uptake by pea genotypes cultivated in cadmium supplemented soil

The effect of inoculation with Pseudomonas brassicacearum Am3, Pseudomonas marginalis Dp1 and Rhodococcus sp. Fp2 containing 1-aminocyclopropane-1-carboxylate deaminase (ACCD) on growth and uptake of N, P, K, Ca, S, Fe and Cd in shoots of pea (Pisum sativum) genotypes VIR188, VIR1658, VIR3429 and VIR4488 was studied in pot experiment with non-polluted and Cd-supplemented (10 mg Cd kg⁻¹) sod-podzolic soil. The growth-promoting effect of bacteria depended on plant genotype and bacterial strain. Only Rhodococcus sp. Fp2 had no ACCD activity in vitro in the presence of Cd and did not stimulate pea growth in Cd-supplemented soil. Inoculation with bacteria counteracted the Cd-induced inhibition of nutrient uptake by plants probably through stimulation of root growth and enhancement of nutrient uptake processes. Nutritional effects of the bacteria were specific with respect to the nutrient.     

Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp

Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.     

Molecular cloning and characterization of the gene encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4

The gene bgaP encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the BgaP gene revealed an open reading frame of 2031 bp encoding for a protein of 677 amino acid residues. The BgaP was heterologously expressed in E. coli and purified followed by Ni2+ affinity chromatography. The molecular mass of the native enzyme was approximately 156 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the BgaP indicated molecular masses of 78 and 77.311 kDa, respectively, suggesting that the BgaP is a dimer. The purified BgaP had an isoelectric point of 4.8 and exhibited maximal activity at 20 degrees C and pH 6.8 under the assay conditions used. The enzyme is particularly thermolabile, losing all activity in only 10 min at 45 degrees C. It was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG); the Km values with ONPG and lactose were calculated to be 5.4 and 20.4 mM at 5 degrees C, respectively. The catalytic efficiencies of BagP for lactose at 5 and 20 degrees C had 14 and 47 times more than that of E. coli beta-galactosidase at 20 degrees C, respectively. Therefore, cold-active beta-galactosidase from the psychrotrophic and halotolerant Planococcus sp. L4 could conceivably be developed to fulfill the practical requirements to enable its use for lactose removal in milk and dairy products at low temperature or a reporter enzyme for psychrophilic genetic systems.     

盐胁迫油菜根际中含假单胞菌（Pseudomonas spp.1）的氨基环丙烷-1-羧酸 (ACC) 脱氨基酶特征研究

When exposed to biotic or abiotic stress conditions,plants produce ethylene from its immediate precursor 1-aminocyclopropane-1-carboxylate(ACC),leading to retarded root growth and senescence.Many plant growth-promoting rhizobacteria contain the enzyme ACC deaminase and this enzyme can cleave ACC to form α-ketobutyrate and ammonium,thereby lowering levels of ethylene.The aim of this study was to isolate and characterize ACC deaminase-producing bacteria from the rhizosphere of salt-stressed canola(Brassica napus L.).Out of 105 random bacterial isolates,15 were able to utilize ACC as the sole source of nitrogen.These 15 isolates were also positive for indole acetic acid(IAA) production.Phylogenetic analysis based on partial 16 S rDNA sequences showed that all isolates belonged to fluorescent Pseudomonas spp.In the canola rhizosphere investigated in this study,Pseudomonas fluorescens was the dominant ACC deaminase-producing species.Cluster analysis based on BOX-A1R-based repetitive extragenic palindromic-polymerase chain reaction(BOX-PCR) patterns suggested a high degree of genetic variability in ACC deaminase-producing P.fluorescens strains.The presence of indigenous ACC-degrading bacteria in the rhizosphere of canola grown in saline soils indicates that these bacteria may contribute to salinity tolerance.     

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Effect of a novel edible composite coating based on gum arabic and chitosan on biochemical and physiological responses of banana fruits during cold storage

The composite effects of gum arabic (GA) (5, 10, 15, and 20%) and chitosan (CH) (1.0%) on the biochemical and physiological characteristics of banana fruits stored at 13 ± 1 °C and 80 ± 3% relative humidity (RH) for 28 days and afterward for 5 days at simulated marketing conditions (25 °C, 60% RH) were investigated. Significant (P ≤ 0.05) differences were observed for the entire GA plus CH treatments as compared to the control. However, the results showed that after 33 days of storage, the weight loss and soluble solids concentration of fruits treated with 10% GA plus 1.0% CH composite coating were 24 and 54% lower, whereas fruit firmness, total carbohydrates, and reducing sugars were 31, 59, and 40% higher than the control, respectively. Furthermore, the composite edible coating of 10% GA plus 1.0% CH delayed color development and reduced the rate of respiration and ethylene evolution during storage as compared to the control. Similarly, sensory evaluation results also proved the effectiveness of 10% GA plus 1.0% CH composite coating by maintaining the overall quality of banana fruits. Consequently, the results of scanning electron microscopy also confirmed that the fruits coated with 10% GA plus 1.0% CH composite edible coating had very fewer cracks and showed a smooth surface. These findings suggest that 10% GA plus 1.0% CH as an edible composite coating can be used commercially for extending the storage life of banana fruits for up to 33 days.     

A novel method for the synthesis of cellulose nanofibril whiskers from banana fibers and characterization

Alkali treatment coupled with high pressure defibrillation and acid treatment have been tried on banana fibers obtained from the pseudo stem of the banana plant Musa sapientum. The structure and morphology of the fibers have been found to be affected on the basis of the concentration of the alkali and acid and also on the pressure applied. Steam explosion in alkaline medium followed by acidic medium is found to be effective in the depolymerization and defibrillation of the fiber to produce banana nanowhiskers. The chemical constituents of raw and steam exploded fibers were analyzed according to the ASTM standards. Structural analysis of steam exploded fibers was carried out by FTIR and XRD. The fiber diameter and percentage crystallinity of the modified fibers were investigated using X-ray diffraction studies. Characterization of the fibers by SFM and TEM supports the evidence for the development of nanofibrils of banana fibers.     

Degradation of cyhalofop-butyl (CyB) by Pseudomonas azotoformans strain QDZ-1 and cloning of a novel gene encoding CyB-hydrolyzing esterase

Cyhalofop-butyl (CyB) is a widely used aryloxyphenoxy propanoate (AOPP) herbicide for control of grasses in rice fields. Five CyB-degrading strains were isolated from rice field soil and identified as Agromyces sp., Stenotrophomonas sp., Aquamicrobium sp., Microbacterium sp., and Pseudomonas azotoformans; the results revealed high biodiversity of CyB-degrading bacteria in rice soil. One strain, P. azotoformans QDZ-1, degraded 84.5% of 100 mg L(-1) CyB in 5 days of incubation in a flask and utilized CyB as carbon source for growth. Strain QDZ-1 could also degrade a wide range of other AOPP herbicides. An esterase gene, chbH, which hydrolyzes CyB to cyhalofop acid (CyA), was cloned from strain QDZ-1 and functionally expressed. A chbH-disrupted mutant dchbH was constructed by insertion mutation. Mutant dchbH could not degrade and utilize CyB, suggesting that chbH was the only esterase gene responsible for CyB degradation in strain QDZ-1. ChbH hydrolyzed all AOPP herbicides tested as well as permethrin. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > CyB ≈ fluazifop-P-butyl > diclofop-methyl ≈ haloxyfop-P-methyl; the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, whereas the substitutions in the aromatic ring had only a slight influence.