Impairment of Peroxisome Degradation in Pichia methanolica Mutants Defective in Acetyl-CoA Synthetase or Isocitrate Lyase

Single recessive mutations of the methylotrophic yeast Pichia methanolica acs1, acs2, acs3 and icl1 affecting acetyl-CoA synthetase and isocitrate lyase, and growth on ethanol as sole carbon and energy source, caused a defect in autophagic peroxisome degradation during exposure of methanol-grown cells to ethanol. As a control, a mutation in mdd1, which resulted in a defect of the ‘malic’ enzyme and also prevented ethanol utilization, did not prevent peroxisome degradation. Peroxisome degradation in glucose medium was unimpaired in all strains tested. Addition of ethanol to methanol-grown cells of acs1, acs2, acs3 and icl1 mutants led to an increase in average vacuole size. Thickening of peroxisomal membranes and tight contacts between groups of peroxisomes and vacuoles were rarely observed. These processes proceeded much more slowly than in wild-type or mdd1 mutant cells incubated under similar conditions. No peroxisomal remnants were observed inside vacuoles in the cells of acs1, acs2, acs3 and icl1 mutants after prolonged cultivation in ethanol medium. We hypothesize that the acs and icl mutants are defective in synthesis of the true effector—presumably glyoxylate—of peroxisome degradation in ethanol medium. Lack of the effector suspends peroxisome degradation at an early stage, namely signal transduction or peroxisome/vacuole recognition. Finally, these defects in peroxisome degradation resulted in mutant cells retaining high levels of alcohol oxidase which further led to increased levels of acetaldehyde accumulation upon incubation of mutant cells with ethanol. © 1997 by John Wiley & Sons, Ltd.     

Multiplicity of mechanisms of carbon catabolite repression involved in the synthesis of alcohol oxidase in the methylotrophic yeast Pichia pinus

The effect of various carbon compounds on the synthesis of alcohol oxidase in a medium with methanol was studied in the wild type strain of Pichia pinus as well as in gcr1 and ecr1 mutants defective in glucose and ethanol repression of methanol metabolic enzymes, respectively. Compounds repressing the synthesis of alcohol oxidase in the wild type strain were divided into four groups. Repression of alcohol oxidase by compounds of the first group (glucose, fructose, mannose, galactose, L -sorbose and xylose) was impaired only in the gcr1 mutant and that by compounds of the second group (ethanol, acetate, 2-oxoglutarate and erythritol) only in the ecr1 mutant. Repression by compounds of the third group (malate, dihydroxyacetone) was not impaired in both these regulatory mutants and that by compounds of the fourth group (succinate, fumarate, L -arabinose, sorbitol, salicin, xylitol and cellobiose) was partially reduced in both gcr1 and ecr1 strains. Mutation gcr1 causes a significant decrease in phosphofructokinase activity. It also led to a six- to seven-fold increase in intracellular pools of glucose-6-phosphate and fructose-6-phosphate and to a two-fold decrase in the intracellular pool of fructose-1,6-bisphosphate. In ecr1 strains, a decrese in 2-oxoglutarate dehydrogenase activity accompanied by an increae in activities of NAD- and NADP-dependent isocitrate dehydrogenases and NAD- and NADP-dependent glutamate dehydrogenases was demonstrated. The intracellular pool of 2-oxoglutarate was increased 2·5-fold in ecr1 strains. Genes GCR1 and ECR1 are not linked. The mechanisms of catabolite repression of alcohol oxidase in methylotrophic yeasts are discussed.     

Peroxisome-deficient mutants of Hansenula polymorpha

As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol-utilization-defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase. Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol- and ethanol utilizing-deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome-deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol. The mutations complemented and therefore were recessive mutations in different genes.     

Deviant Pex3p Levels affect Normal Peroxisome Formation in Hansenula polymorpha: A Sharp Increase of the Protein Level Induces the Proliferation of Numerous,Small Protein-import Competent Peroxisomes

Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::P_AOX.PEX3^7x), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density (1·18 g cm⁻³) after sucrose gradient centrifugation of WT::P_AOX.PEX3^7x homogenates, compared to normal peroxisomes (1·23 g cm⁻³). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the P_AOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. © 1997 John Wiley & Sons, Ltd.     

Isolation and characterization of Kluyveromyces marxianus mutants deficient in malate transport

In malic acid-grown cells of the strains ATCC 10022 and KMS3 of Kluyveromyces marxianus the transport of malic acid occurred by a malate-proton symport, which accepted l-malic, d-malic, succinic and fumaric acids, but not tartaric, malonic or maleic acids. The system was inducible and subjected to glucose repression. Mutants of the strain KMS3, unable to grow in a medium with malic acid, were isolated and checked for their capacity to utilize several carbon sources and to transport dicarboxylic acids by the malate-proton symport. Two distinct clones affected on malate transport were obtained. Both were able to grow on a medium with glycerol or ethanol but not with dl-malic, succinic, oxoglutaric and oxaloacetic acids as the sole carbon and energy sources. However, while one of the mutants (Mal7) displayed activity levels for the enzymes malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase similar to those of the wild strain, in the other mutant type (Mal6) the activities for the same enzymes were significantly reduced. Plasma membranes from derepressed cells of the wild strain and of the mutants Mal6 and Mal7 were isolated and the protein analysed by SDS–PAGE. The electrophoretic patterns of these preparations differed in a polypeptide with an apparent molecular mass of about 28 kDa, which was absent only in the mutant Mal7. The results indicated that Mal7 can be affected in a gene that encodes a malate carrier in K. marxianus. © 1998 John Wiley & Sons, Ltd.     

Isolation and preliminary characterization of Pichia pinus mutants insensitive to glucose repression

A new method for the isolation of glucose repression-insensitive mutants in the methylotrophic yeast Pichia pinus was developed. The method is based on screening of small suspension samples derived from 2-deoxyglucose-resistant colonies for alcohol oxidase activity. Alcohol oxidase activity was evaluated by determination of formaldehyde excreted by cells. Mutants with glucose non-repressible alcohol oxidase and catalase synthesis were obtained. All mutants grew poorly on D -xylose compared to the wild type, whereas growth on L -arabinose was similar to the wild type. Changes in the glucose transport system were suggested to be responsible for altered growth characteristics and defective glucose repression.     

Glucose metabolism,enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum

The physiology of Hanseniaspora uvarum K₅ was studied in glucose-limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0·28 h^?1, glucose was completely metabolized in biomass and CO₂. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO₂ were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.     

Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl-coenzyme A synthetase.

The expression of the glyoxylate cycle enzymes is required for growth of the yeast Yarrowia lipolytica on acetate or fatty acids as sole carbon source. Acetyl-coenzyme A, which is produced by acetyl-coenzyme A synthetase (ACS) from acetate, is needed for induction of this expression. Acetate-non-utilizing mutants of this yeast were investigated in order to identify mutants which express no or strongly reduced activity of this enzyme. Mutations in gene ICL2 exhibited the strongest effects on the activity. In icl2 mutants, lack of ACS activity resulted in a non-induced glyoxylate cycle on acetate; however, induction on fatty acids was not affected. Gene ICL2 was identified as the structural gene encoding the monomer of ACS. It is shown that a high level of ACS activity is necessary for full expression of the glyoxylate cycle enzymes. Mutations in gene ICL1, which encodes isocitrate lyase, resulted in overproduction of ACS without any growth on acetate. A new gene (GPR1 = glyoxylate pathway regulation) was detected in which trans-dominant mutations inhibit expression of ACS and the glyoxylate cycle on acetate as carbon source.     

Pectinase Activity of Vegetable Spoilage Bacteria in Modified Atmosphere

A modified atmosphere of 10% CO₂, 5% O₂ and 85% N₂ was used to determine effects on pectinase activities of vegetable spoilage bacteria. Cultures of Erwinia carotovora, Pseudomonas fluorescens, and Xanthomonas campestris, grown at 25°C in liquid medium held under the modified atmosphere were assayed for polygalacturonase and pectate lyase activities. Erwinia and Pseudomonas exhibited higher polygalacturonase and pectate lyase activities than Xanthomonas whether grown under modified atmosphere or air. In some cases, bacteria grown under air exhibited higher pectinase activity. In general modified atmosphere did not significantly reduce pectinase activities in any of the bacteria tested.     

Inactivation Kinetics of Polyphenol Oxidase in an Aqueous Model System under Stand-Alone and Combined Ultrasound and Ultraviolet Treatments

The objectives of this work were to study the ultrasound- and ultraviolet light-induced inactivation kinetics of polyphenol oxidase extracted from different sources in a model system. The polyphenol oxidase crude extract was obtained from bananas, apples, quince, eggplants, plums, dill, and cultured mushrooms, which exhibited high enzyme activity. The polyphenol oxidase crude extract was treated with ultrasound and ultraviolet light at 40°C temperature for 40 min. The study showed that the polyphenol oxidase enzyme was inactivated between 12 and 100% during ultrasound only treatment; between 4 and 29% during ultraviolet light only treatment; and between 80 and 100% during simultaneous ultrasound and ultraviolet light treatment. Based on the measurements, an exponential decay model for determining polyphenol oxidase inactivation kinetics was developed. The model provides high determination coefficients (R²): 0.968–0.999 with ultrasound only treatment, 0.881–0.990 with ultraviolet only treatment, and 0.975–1.000 with simultaneous ultrasound and ultraviolet treatment. The polyphenol oxidase kinetics evaluation showed that different treatments provided different inactivation times, or D-values. The D-values were 7.0–656.1 min for ultrasound only treatment (D_US), 251–1887 min for ultraviolet only treatment (D_UV), and 3.3–59.4 min for combined ultrasound and ultraviolet treatment (D_US+UV).     

Control of Phytophthora nicotianae disease,induction of defense responses and genes expression of papaya fruits treated with Pseudomonas putida MGP1

BACKGROUND: Biological control is a potential strategy to reduce post‐harvest decay in several fruits. Little research has been carried out on the effects of endophytic bacterium on post‐harvest blight caused by Phytophthora nicotianae in papaya. In this work, the biocontrol activity of Pseudomonas putida MGP1 on this disease and its possible mechanisms, including changes of defensive enzyme activities, total phenolic content and mRNA levels of two important genes, were investigated. RESULTS: Fruits treated with MGP1 showed a significant lower disease index and demonstrated increases in chitinase, β‐1,3‐glucanase, phenylalanine ammonia‐lyase, peroxidase, polyphenol oxidase and catalase activities and total phenolic content. In addition, the expression levels of pathogenesis related protein 1 gene (PR1) and non‐expressor of PR1 gene (NPR1) in papaya fruits were elevated by MGP1 treatment. CONCLUSION The results indicated that papaya fruits were responsive to the endophytic bacterium Ps. putida, which could activate defensive enzymes and genes and thereby induce host disease resistance. © 2012 Society of Chemical Industry     

利用CRISPRi技术构建乙醛酸生物合成Corynebacterium glutamicum工程菌

以谷氨酸棒状杆菌(Corynebacterium glutamicumATCC 13032)为出发菌株,敲除其支流代谢关键酶乳酸脱氢酶合成基因lldh,建立规律间隔成簇短回文重复序列干扰(clustered regularly interspaced short palindromic repeats interfer...     

Purification and some properties of a novel raw starch‐digesting amylase from Aspergillus carbonarius

A medium was developed to obtain the maximum yield of raw starch‐digesting amylase from Aspergillus carbonarius (Bainier) Thom IMI 366159 in submerged culture with raw starch as the sole carbon source. The amylase was purified to apparent homogeneity by sucrose concentration and ion exchange chromatography on S‐ and Q‐Sepharose (fast flow) columns. SDS‐PAGE revealed two migrating protein bands corresponding to relative molecular masses of 31.6 and 32 KDa. The enzyme was optimally active at pH 6.0–7.0 and 40 °C, was uninfluenced across a relatively broad pH range of 3.0–9.0 and retained over 85% activity between 30 and 80 °C after 20 min incubation. The enzyme was strongly activated by Co²⁺ and only slightly by Fe²⁺, while Ca²⁺, Hg²⁺, EDTA and N‐bromosuccinamide elicited significant repression of the enzyme activity. The enzyme hydrolysed amylopectin (K_m 0.194 mg ml ⁻¹), glycogen (K_m 0.215 mg ml ⁻¹), pullulan (K_m 0.238 mg ml ⁻¹), amylose (K_m 0.256 mg ml ⁻¹) and raw potato starch (K_m 0.260 mg ml ⁻¹), forming predominantly maltose and relatively smaller amounts of glucose. © 2000 Society of Chemical Industry     

Sanitizer treatments alleviate lignification of sliced few-flower wildrice (Zizania latifolia Turcz.)

To investigate the effects of sanitation processing on the lignification of few-flower wildrice (FFW), fresh-cut FFW was treated with water (control), 80 mg/L peroxyacetic acid (PAA), 100 mg/L ClO₂ or 50 mg/L H₂O₂, packed in polyethylene film bags and stored at 2 °C for 21 days. Firmness, lignin, cellulose, hemicellulose and lignin forming related enzymes activities were examined. Sanitizer treatments inhibited the increases of firmness, lignin and cellulose than the control. The changes of hemicelluloses did not show a difference between the control and PAA treatment. An increase in the activities of phenylalanine ammonia lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO), and a reduction in catalase (CAT) and superoxide dismutase (SOD) activities, were detected in the sliced FFW in the incompatible interaction. Compared with the control, sanitizers suppressed the activities of PAL, POD and PPO, but enhanced the activities of SOD and CAT during 21 days of storage. These enzymatic activities varied with different sanitizers. These results show that 80 mg/L peroxyacetic acid (PAA), 100 mg/L ClO₂ or 50 mg/L H₂O₂ sanitation processing can maintain tenderness, alleviate the lignification and fibrosis of fresh-cut FFW.     

Enhancing disease resistance in peach fruit with methyl jasmonate

BACKGROUND: Peaches are susceptible to microbial decay during postharvest distribution at ambient temperature. To search for effective alternatives to currently used fungicides for disease control, in this study the effect of methyl jasmonate (MeJA) on disease resistance and fruit decay of peaches after harvest in response to pathogen attack was investigated. RESULTS: Freshly harvested peaches were treated with 1 µmol L^?1 MeJA vapour at 20 °C for 24 h. At 0, 12, 24 and 36 h after this treatment, both treated and untreated fruits were artificially wounded and inoculated with Penicillium expansum, Botrytis cinerea or Rhizopus stolonifer spore suspension (1 × 10⁵ spores mL^?1) and then incubated at 20 °C for 6 days. MeJA treatment significantly reduced the postharvest diseases. Incubation for 12 h was the optimal length of time after MeJA treatment, resulting in the lowest disease incidence and lesion diameter for all pathogens. The activities of defence enzymes including chitinase, β‐1,3‐glucanase, phenylalanine ammonia‐lyase, polyphenol oxidase and peroxidase were enhanced by MeJA treatment, and the level of total phenolics in MeJA‐treated fruit was also higher than that in control fruit. In addition, MeJA affected hydrogen peroxide (H₂O₂)‐metabolising enzymes such as superoxide dismutase, catalase and ascorbate peroxidase and induced a higher level of H₂O₂ during incubation, which might serve as a signal to induce resistance against P. expansum. CONCLUSION: MeJA was effective in reducing decay and might enhance disease resistance in peach fruit by increasing levels of antipathogenic proteins and antimicrobial phenolic compounds. Copyright © 2009 Society of Chemical Industry     

Inactivation and reactivation of horseradish peroxidase treated with supercritical carbon dioxide

Effects of supercritical carbon dioxide (SCCO₂) on the activity of horseradish peroxidase (HRP) in pH 5.6 acetate buffer solution were investigated. SCCO₂ treatment could effectively inactivate HRP. Higher pressure, higher temperature, and longer treatment time caused more inactivation. The maximum reduction of HRP activity reached nearly 90% at 30 MPa and 55 °C for 60 min. Analysis of first-order reaction kinetic data (characterized by a rate constant k and by a decimal reduction time D) showed that D value was closely related to the pressure and temperature of SCCO₂ treatment. Higher pressures or higher temperatures resulted in lower D values (higher k), the D value of HRP was minimized to 64.52 min treated by the combination of 30 MPa and 55 °C. The Z _p, representing the range of applied pressure between which the D values change by a factor of 10, was 114.81 MPa. The activity of HRP treated by SCCO₂ was reactivated significantly after initial 7-day storage at 4 °C apart from the samples at 30 MPa for 60 min, indicating the HRP inactivation may be reversible and the reactivation of HRP is dependent on the pressure level and treatment time.     

Effects of Bacillus velezensis zk1 on the physiology and metabolism of peaches

Bacillus velezensis zk1 is the dominant bacterium that causing rot in peaches. However, the mechanisms through that this bacterium causes rot have not been elucidated. Here, we explored the mechanisms of peach decay caused by B. velezensis zk1. The invasion of B. velezensis zk1 in peaches resulted in an increase in glucose and arabinose contents in fruit tissues. Moreover, the relative conductivity of the fruit reached 84% after 4 days of culture with bacterial invasion. With the destruction of cells, the malondialdehyde content increased, whereas the vitamin C, dialdehyde, flavonoid and total phenol contents decreased. Polyphenol oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and ammonia lyase activities also decreased. Overall, these findings demonstrated that B. velezensis zk1 infection damaged peach chloroplasts, mitochondria, respiratory chain activity and related free radical scavenging enzyme systems, thereby disrupting the normal physiological metabolism of peaches and causing rot.     

LC-MS/MS multi-analyte method for mycotoxin determination in food supplements

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in food supplements is presented. The analytes included A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin), aflatoxins (aflatoxin-B₁, aflatoxin-B₂, aflatoxin-G₁ and aflatoxin-G₂), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B₁, fumonisin-B₂ and fumonisin-B₃), ochratoxin A, zearalenone, beauvericin and sterigmatocystin. Optimization of the simultaneous extraction of these toxins and the sample pretreatment procedure, as well as method validation were performed on maca (Lepidium meyenii) food supplements. The results indicated that the solvent mixture ethyl acetate/formic acid (95:5, v/v) was the best compromise for the extraction of the analytes from food supplements. Liquid–liquid partition with n-hexane was applied as partial clean-up step to remove excess of co-extracted non-polar components. Further clean-up was performed on Oasis HLB? cartridges. Samples were analysed using an Acquity UPLC system coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer equipped with an electrospray interface operated in the positive-ion mode. Limits of detection and quantification were in the range of 0.3–30 ng g^?1 and 1–100 ng g^?1, respectively. Recovery yields were above 60% for most of the analytes, except for nivalenol, sterigmatocystine and the fumonisins. The method showed good precision and trueness. Analysis of different food supplements such as soy (Glycine max) isoflavones, St John's wort ( Hypericum perforatum), garlic (Allium sativum), Ginkgo biloba, and black radish (Raphanus niger) demonstrated the general applicability of the method. Due to different matrix effects observed in different food supplement samples, the standard addition approach was applied to perform correct quantitative analysis. In 56 out of 62 samples analysed, none of the 23 mycotoxins investigated was detected. Positive samples contained at least one of the toxins fumonisin-B₁, fumonisin-B₂, fumonisin-B₃ and ochratoxin A.     

Thermal inactivation kinetics of quality-related enzymes in cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis><Emphasis Type="Bold">)</Emphasis>

Thermal inactivation of quality-related enzymes in both cauliflower crude enzyme extracts and fresh tissue samples was studied in temperature range 50–100 °C. For crude enzyme extracts, several parameters, reaction rate constants (k) and activation energy (E _a) as well as decimal reduction time (D) and (z) values, were used to characterize the thermal stability. The rates of inactivation were found to follow first-order inactivation kinetics. Activation energies varied between 101.18 and 208.42 kJ mol⁻¹ with z values of 10.59–24.09 °C. The examined kinetics indicated that lipoxygenase was the most heat resistant followed by peroxidase, polyphenol oxidase, pectin methyl esterase and ascorbic acid oxidase. Furthermore, the obtained results from the blanched fresh tissues indicated that inactivation of lipoxygenase secured disappearing of any other enzyme activities. Therefore, this study recommends using lipoxygenase as an indicator enzyme to optimize the thermal treatments of cauliflower products.