餐厨垃圾厌氧消化产沼气过程中酶学表征

厌氧消化产沼气被认为是餐厨垃圾资源化利用的有效方式之一,其实质是在多种微生物综合作用下的生物化学过程.本文研究了在促进和抑制性因子作用下,餐厨垃圾厌氧发酵的酶学过程,对其中的脱氢酶和水解酶(β-葡萄糖苷酶,BAA-蛋白水解酶,碱性磷酸酶)活性变化进行了分析.研究表明,与空白对照组相比添加酵母粉后脱氢酶的最高活性提高了8...     

Definition and characterization of enzymes for maximal biocatalytic solubilization of prebiotic polysaccharides from potato pulp

Potato pulp is a high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp is particularly rich in pectin, notably galactan branched rhamnogalacturonan I polysaccharides, which are highly bifidogenic when solubilized. The objective of the present study was to characterize and compare four homogalacturonan degrading enzymes capable of catalyzing the required solubilization of these pectinaceous polysaccharides from potato pulp in a 1 min reaction. An additional purpose was to assess the influence of the pH and the potential buffer chelating effects on the release of these polysaccharides from the potato pulp. The pH and temperature optima of two selected pectin lyases from Emericella nidulans (formerly known as Aspergillus nidulans) and Aspergillus niger were determined to 8.6 and 4.0, respectively, at ≥100 °C within 1 min of reaction. The optima for the two selected polygalacturonases from E. nidulans and Aspergillus aculeatus were determined to pH 4.4 and 46 °C, and pH 3.7 and ≥80 °C, respectively. The polygalacturonase from A. aculeatus was 4-42 times more heat-resistant at 50 °C than the other enzymes. The difference in pH optima of the pectin lyases and the exceptional thermal stabilities of some of the enzymes are proposed to be related to specific amino acid substitutions, stabilizing hydrogen bonding and structural traits of the enzymes. The K_M and V_max values ranged from 0.3-0.6 g/L and 0.5-250.5 U/mg protein, respectively. Phosphate buffer induced release of a higher amount of dry matter than Tris-acetate buffer at pH 6, indicating a chelating effect of the phosphate. Moreover, the phosphate had a higher chelating effect at pH 6 than at pH 4. The optimal conditions for a high yield of polysaccharides from potato pulp were therefore: 1% (w/w) potato pulp treated with 1% (w/w) enzyme/substrate (E/S) pectin lyase from E. nidulans and 1% (w/w) E/S polygalacturonase from A. aculeatus at pH 6.0 and 60 °C for 1 min.     

Isoenzymes of lipolytic acyl hydrolase and esterase in potato tuber

Five varieties of potato (Solanum tuberosum) were shown by gel- and free-flow-electrophoresis to exhibit multiple forms of lipolytic acyl hydrolase (LAH) and esterase enzymes. The electrophoretic patterns of LAH and esterase activities and protein differed with the variety and were characteristic for a given variety. In the variety (Golden Wonder) with the highest LAH activity (p-nitrophenylpalmitate as substrate), this was 200-fold greater than the esterase activity (p-nitrophenylacetate as substrate) and isoenzyme patterns for both enzymes were the most complex. In the variety with a very low LAH activity (Désirée), the LAH and esterase activities were similar and more simple isoenzyme patterns for these enzymes were observed.     

Enzymatic browning reaction of apple juices prepared using a blender and a low-speed masticating household juicer

ABSTRACT

The aim of this study was to investigate the effect of juicer type (blender or LSM household juicer) on the browning reaction of apple juice and evaluate the remaining antioxidant activity in the juice. The blender apple juice showed a darker brown color and 4.5 times higher PPO activity than LSM apple juice. This result suggested that the blender caused severer damage to plastids in cells leading to leakage of PPO into the juice than the LSM juicer. The total polyphenol and flavonoid content of LSM apple juice was approximately 2 times higher than that of blender apple juice because polyphenols and flavonoids can be used as substrates by PPO. The antioxidant activity of LSM juice was higher than that of blender juice. Together, these results suggested that the LSM juicer is superior to the blender for preparation of fresh apple juices due to the minimization of enzymatic oxidation reactions.

Abbreviations: LSM: low-speed masticating; PPO: polyphenol oxidase; ABTS: 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl     

Improvements in regeneration from protoplasts of potato and studies on chromosome stability

Summary Regeneration of plants from protoplasts of potato (Solanum tuberosum) cv. Maris Bard has been achieved from four different initial culture media (ET2, ET3, CLG, VkCLG). These media differed in their hormone, salt and sugar content. Plating efficiencies were highest in the VkCLG medium, but no correlation was found between plating efficiency and regeneration frequency (i.e. the percentage of calli producing shoots). Regeneration frequencies were high on all four media; up to 95% on ET3. Chromosome counts of up to 50 regenerants selected at random from the four treatments showed no significant differences between any of the treatments, in the proportions of plants which were euploid (48), aneuploid at the tetraploid level (48±), and aneuploid with high chromosome numbers (48+ +). Highly significant differences were present, however, between shoots which rooted quickly (predominantly euploid) and those which rooted only after transfer to a rooting medium (predominantly 48+ +). Overall more than 60% of the regenerants were normal (2n=4x=48) and this is a considerable improvement on our earlier work in this cultivar (4% normal). These findings are discussed in relation to factors affecting chromosome stability. Chromosome structural rearrangements are also described.     

Characterization, amyloid formation, and immobilization of a novel SGNH hydrolase from Listeria innocua 11262

A novel oligomeric hydrolase (LI22) from Listeria innocua CLIP 11262 was identified, characterized, and immobilized for industrial application. Sequence analysis of LI22 revealed a putative catalytic triad (Ser¹⁰-Asp¹⁷⁶-His¹⁷⁹), and a conserved sequence motif Ser(S)¹⁰-Gly(G)⁷⁷-Asn(N)⁷⁹-His(H)¹⁷⁹ with moderate identities (<30%) with other members of the SGNH-hydrolase superfamily. LI22 was able to hydrolyze p-nitrophenyl acetate, α- and β-naphthyl acetate, while the S10A mutant completely lost its activity. Structural properties of LI22 were investigated using gel filtration, circular dichroism (CD), fluorescence, molecular modeling, and gel filtration. We have shown that upon incubation in 30% TFE or 50% ethanol solution, LI22 was transformed into curly amyloid fibrils. Cross-linked enzyme aggregates of LI22 were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Higher thermal and chemical stability, as well as good durability after repeated use of the LI22-CLEA, highlight its potential applicability as a biocatalyst in the pharmaceutical and chemical industries.     

Structural studies of the Nudix hydrolase DR1025 from Deinococcus radiodurans and its ligand complexes

We have determined the crystal structure, at 1.4A, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked beta-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap(4)A (both at 1.6A resolution). In the Ap(4)A co-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Enzymatic characteristics of an ApaH-like phosphatase,PrpA, and a diadenosine tetraphosphate hydrolase,ApaH, from Myxococcus xanthus

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap₄A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards Ap_nA and ATP. In the presence of Mn²⁺, PrpA hydrolyzed Ap₄A into AMP and ATP, whereas in the presence of Co²⁺ PrpA hydrolyzed Ap₄A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards Ap_nA and ATP. Mn²⁺ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co²⁺ was required for Ap_nA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an Ap_nA hydrolase, respectively.     

Influence of pectinase treatment on fruit spirits from apple mash, juice and pomace

The current investigation was conducted to determine the influence of pectinase treatment on fruit spirits produced from apple mash, juice, and pomace. Crispin apples were processed into apple mash, juice, and pomace in our pilot-plant, and fermented with a commercial Red Star wine yeast (Sachharomyces cerevisiae Davis 904). After fermentation, the samples of fermented apple mash, juice, and pomace were distilled, and the distillates were analyzed by HPLC with a Bio-Rad Aminex HPX 87H column and a refractive index detector. Methanol, ethanol, n-propanol, iso-butanol, and iso-amyl alcohol were identified as the major alcohols in all the apple spirits. Student's t-test results indicate that there are significant differences between the methanol concentrations of pectinase treated and non-pectinase treated apple spirits. Duncan's multiple range tests show significant differences in the concentrations of methanol of the fruit spirits made from apple mash, juice, and pomace. Apple pomace yielded significantly higher methanol concentrations than apple mash and juice. Pectinase treatment had little effect on the concentrations of n-propanol, iso-butanol, and iso-amyl alcohol. It is concluded that fruit spirits made from the pectinase treated mash, juice, and pomace of Crispin apples had methanol concentrations significantly above the United States FDA guidance of 0.35% by volume or 280 mg/100 mL of fruit brandy containing 40% ethanol.     

Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure–activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z′ value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.     

Expression, purification and crystal structure of a truncated acylpeptide hydrolase from Aeropyrum pernix K1

Acylpeptide hydrolase （APH） catalyzes the N-terminal hydrolysis of N^α-acylpeptides to release N^α-acylated amino acids. The crystal structure of recombinant APH from the thermophilic archaeon Aeropyrum pernix K1 （apAPH） was reported recently to be at a resolution of 2.1 A using X-ray diffraction. A truncated mutant of apAPH that lacks the first short α-helix at the N-terminal, apAPH-△（1-21）, was cloned, expressed, characterized and crystallized. Data from biochemical experiments indicate that the optimum temperature of apAPH is decreased by 15℃ with the deletion of the N-terminal α-helix. However, the enzyme activity at the optimal temperature does not change. It suggests that this N-terminal α-helix is essential for thermostability. Here, the crystal structure of apAPH-△（1-21） has been determined by molecular replacement to 2.5 A. A comparison between the two structures suggests a difference in thermostability, and it can be concluded that by adding or deleting a linking structure （located over different domains）, the stability or even the activity of an enzyme can be modified.     

Purification and properties of the allophanate hydrolase from Chlamydomonas reinhardii

Allophanate hydrolase was purified to homogeneity from extracts of Chlamydomonas reinhardii grown phototrophically using urea as sole source of nitrogen. The following sequence of steps comprised the purification procedure: (1) protamine sulfate precipitation; (2) ammonium sulfate fractionation; (3) poly(ethylene glycol) fractionation; (4) batch-wise DEAE-cellulose adsorption; (5) Sepharose 6-B gel filtration; (6) hydroxyapatite chromatography. This procedure yielded an allophanate hydrolase preparation which was homogenous as judged by polyacrylamide gel electrophoresis. The molecular weight, as determined by gradient polyacrylamide electrophoresis and gel filtration, was 110 000 and 100 000, respectively. The pH optimum of this enzyme was approximately 9.0, while the K_m for allophanate was 0.55 mM. Allophanate hydrolase was sensitive to N-ethylmaleimide but was protected from this inhibition by allophanate. Malonic acid, oxaloacetic acid, and acetoacetic acid were inhibitory to allophanate hydrolysis.     

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h, glycerol (20 g L⁻¹) and corn steep liquor (CSL) (30 g L⁻¹) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L⁻¹ h⁻¹) and continuous feeding of CSL (1 g L⁻¹ h⁻¹) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L⁻¹; maximum EH production was 351.2±13.1 U L⁻¹ with a specific activity of 14.3±0.5 U g⁻¹. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.     

Crystal structure of beta-D-xylosidase from Thermoanaerobacterium saccharolyticum, a family 39 glycoside hydrolase

1,4-beta-D-Xylan is the major component of plant cell-wall hemicelluloses. beta-D-Xylosidases are involved in the breakdown of xylans into xylose and belong to families 3, 39, 43, 52, and 54 of glycoside hydrolases. Here, we report the first crystal structure of a member of family 39 glycoside hydrolase, i.e. beta-D-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. This study also represents the first structure of any beta-xylosidase of the above five glycoside hydrolase families. Each monomer of T. saccharolyticum beta-xylosidase comprises three distinct domains; a catalytic domain of the canonical (beta/alpha)(8)-barrel fold, a beta-sandwich domain, and a small alpha-helical domain. We have determined the structure in two forms: D-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-alpha-D-xylosyl-enzyme intermediate complex, thus providing two snapshots in the reaction pathway. This study provides structural evidence for the proposed double displacement mechanism that involves a covalent intermediate. Furthermore, it reveals possible functional roles for His228 as the auxiliary acid/base and Glu323 as a key residue in substrate recognition.     

Prokaryotic expression,purification, and sub-cellular localization of a novel alcohol acyltransferase from apple

The coding region of the alcohol acyltransferase gene (MdAAT2) from apple was sub-cloned into expression vectors, pET30a and pET32a, and introduced into E. coli for expression. The purified pET30a/MdAAT2 fusion proteins were used to immunize rabbits following standard protocols. The partially soluble fusion proteins had alcohol acyltransferase activity and were detected only in the pET32a/Origami B(DE3) expression system. Immunolocalization analysis indicated that MdAAT2 is mainly in the cytoplasm, in agreement with the prediction of sub-cellular localization obtained by the LOCSVMpsi program. Western blot analysis indicated that ester biosynthesis in different apple cultivars was related positively to the accumulation of MdAAT2.     

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.     

Extraction, purification and chemical characterisation of xylogalacturonans from pea hulls

Pea hulls contained 925 mg/g sugar including 659 mg/g cellulosic glucose and 90 mg/g uronic acid. They were de-esterified by NaOH (pH>13 at 4°C, 2 h) and treated with HCl (0.1 mol/l, 80°C, 24 h). The HCl-soluble fraction represented 95 mg/g initial pea hulls. It was rich in galacturonic acid (259 mg/g), xylose (93 mg/g) and rhamnose (91 mg/g), which co-eluted in anion-exchange chromatography. The HCl-soluble fraction was degraded by a rhamnogalacturonan-hydrolase and the reaction products were fractionated by size-exclusion chromatography. Two fractions, representing together 18 mg/g initial pea hulls, were composed almost exclusively of galacturonic acid and xylose and could be defined as xylogalacturonans. The first fraction exhibited a high molar mass, a molar ratio Xyl/GalA of 1 and contained almost 5% of rhamnose. The molar mass of the second fraction was much lower and the molar ratio Xyl/GalA was 0.6. Methylation analysis showed the presence in both fractions of a (1→4) galacturonan backbone highly substituted on O-3 either by terminal xylosyl residues or by short side-chains of (1→2) linked xylosyl residues.     

Field trials using single potato plants as whole plots, with and without nematicide treatment, to assess tolerance of attack by Globodera rostochiensis

It is important to assess the tolerance of new potato cultivars with partial resistance to potato cyst nematode attack. The experiments described here are intended to quantify tolerance adequately using a minimum of resources. When test plants were spaced widely in the field trials it was also possible to estimate their resistance levels but standard plant spacing allowed roots of neighbouring plants to encroach on a test plant's root space and to interfere with attempts to assay resistance. Standard plant spacing did, however, give more realistic yields per plant and competition from neighbouring plants was standardised by surrounding each test plant by four identical guard plants (cv. Désirée). Regressions of yield on severity of nematode attack were drawn when nematode infestation varied naturally across the trial site or when different levels of attack were created by applying nematicide at a range of dose rates. Cara was the most tolerant cultivar tested, with a complete spectrum of tolerance shown by other cultivars/clones. Some clones with a high degree of resistance were intolerant and it is precisely these that this type of test seeks to identify.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.