Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Biokinetic parameters and behavior of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> during anaerobic growth

The biokinetics of glucose metabolism were evaluated in Aeromonas hydrophila during growth in an anaerobic biosystem. After approx 34 h growth, A. hydrophila metabolized 5,000 mg glucose l⁻¹ into the end-products ethanol, acetate, succinate and formate. The maximum growth rate, μ _m, half saturation coefficients, K _s, microbial yield coefficient, Y, cell mass decay rate coefficient, k _d, and substrate inhibition coefficient, K _si were 0.25 ± 0.03 h⁻¹, 118 ± 31 mg glucose l⁻¹, 0.12 μg DNA mg glucose⁻¹, 0.01 h⁻¹, and 3,108 ± 1,152 mg glucose l⁻¹, respectively. These data were used to predict the performance of a continuous growth system with an influent glucose concentration of 5,000 mg l⁻¹. Results of the analysis suggest that A. hydrophila will metabolize glucose at greater than 95% efficiency when hydraulic retention times (HRTs) exceed 7 h, whereas the culture is at risk of washing out at an HRT of 6.7 h.     

Root cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> subsp. <Emphasis Type="Italic">angustifolium</Emphasis> elicited with chitosan and production of xanthone-rich extracts with antifungal activity

Hypericum perforatum is a well-known medicinal plant which contains a wide variety of metabolites, including xanthones, which have a wide range of biological properties, including antifungal activity. In the present study, we evaluated the capability of roots regenerated from calli of H. perforatum subsp. angustifolium to produce xanthones. Root biomass was positively correlated with the indole-3-butyric acid concentration, whereas a concentration of 1 mg l⁻¹ was the most suitable for the development of roots. High auxin concentrations also inhibited xanthone accumulation. Xanthones were produced in large amounts, with a very stable trend throughout the culture period. When the roots were treated with chitosan, the xanthone content dramatically increased, peaking after 7 days. Chitosan also induced a release of these metabolites into the culture. The maximum accumulation (14.26 ± 0.62 mg g⁻¹ dry weight [DW]) and release (2.64 ± 0.13 mg g⁻¹ DW) of xanthones were recorded 7 days after treatment. The most represented xanthones were isolated, purified, and spectroscopically characterized. Antifungal activity of the total root extracts was tested against a broad panel of human fungal pathogen strains (30 Candida species, 12 Cryptococcus neoformans, and 16 dermatophytes); this activity significantly increased when using chitosan. Extracts obtained after 7 days of chitosan treatment showed high antifungal activity (mean minimum inhibitory concentration of 83.4, 39.1, and 114 μg ml⁻¹ against Candida spp., C. neoformans, and dermatophytes, respectively). Our results suggest that root cultures can be considered as a potential tool for large-scale production of extracts with stable quantities of xanthones.     

Establishment of an <Emphasis Type="Italic">Agrobacteriuim</Emphasis>-mediated cotyledon disc transformation method for <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA), 0.05 mg l⁻¹ 3–indolebutyric acid (IBA), 1 mg l⁻¹ phosphinothricin and 500 mg l⁻¹ cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, 0.5 mg l⁻¹ gibberellic acid (GA3), 1 mg l⁻¹ phosphinothricin and 250 mg l⁻¹ cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l⁻¹ IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.     

Antioxidant Properties of Selected <Emphasis Type="Italic">Boletus</Emphasis> Mushrooms

Considering the growing interest for mushrooms and the demand search of natural antioxidants sources, the aim of this study was to investigate the antioxidant properties of two edible widely used Boletus species, Boletus edulis, and Boletus auranticus, collected from Istra region in Croatia in late summer 2007. To evaluate the antioxidant properties and content of antioxidant compounds, scavenging capacity on DPPH˙, OH˙, and O₂˙⁻ radicals, reducing power and capacity to inhibit lipid peroxidation has been investigated. It is determined that content of total phenols (41.82 ± 0.08 mg gallic acid equivalent per gram of dry extract) was higher for B. edulis. Using high performance liquid chromatography/diode array detector analysis, the main antioxidant compound, variegatic acid, has been detected and quantified. 1,1-Diphenyl-2-picryl-hydrazyl-hydrate assay was used as a preliminary free radical–scavenging evaluation. By this assay, it has been found that B. edulis dry mushroom extract exhibits 50% of inhibition value at the extract concentration of 0.016 ± 0.0003 mg/ml. The extracts were capable of reducing iron(III) and, thus, are capable of donating electrons. Using electron paramagnetic resonance spin-trapping and spin-probing techniques, activity against relevant reactive species, ˙OH and O₂˙⁻ radical, was analyzed for both mushroom extracts. Both investigated extracts are determined as good inhibitors for ˙OH radical reduction, and both exhibited significant capacity for scavenging O₂˙⁻ radical and for that could help to prevent or meliorate oxidative damage. Only B. edulis extract prevents lipid peroxidation. Investigated mushroom extracts could represent easily accessible natural antioxidant resource.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Genetic transformation of the medicinal plant <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated method

A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l⁻¹ BAP and 0.5 μmol l⁻¹ NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection medium (co-culture medium supplemented with 60 mg l⁻¹ kanamycin and 200 mg l⁻¹ cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l⁻¹ kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil. The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced in our lab in the past two years.     

Combined effects of zinc and algal food on the competition between planktonic rotifers, <Emphasis Type="Italic">Anuraeopsis fissa</Emphasis> and <Emphasis Type="Italic">Brachionus rubens</Emphasis> (Rotifera)

We evaluated the combined effects of algal (Chlorella vulgaris) food levels (low, 0.5 × 10⁶ (or 2.9 μg C ml⁻¹); and high, 1 × 10⁶ cells ml⁻¹ (or 5.8 μg C ml⁻¹)) and zinc concentrations (0, 0.125, and 0.250 mg l⁻¹ of ZnCl₂) on the competition between two common planktonic rotifers Anuraeopsis fissa and Brachionus rubens using their population growth. Median lethal concentration data (LC₅₀) (mean ± 95% confidence intervals) showed that B. rubens was more resistant to zinc (0.554 ± 0.08 mg l⁻¹) than A. fissa (0.315 ± 0.07 mg l⁻¹). A. fissa when grown alone or with Zn was always numerically more abundant than B. rubens. When grown in the absence of zinc, under low- and high-food levels, the peak abundances of A. fissa varied from 251 ± 24 to 661 ± 77 ind. ml⁻¹, respectively, and the corresponding maxima for B. rubens were 52 ± 3 and 102 ± 18 ind. ml⁻¹. At a given food level, competition for food reduced the peak abundances of both rotifers considerably. Increase in Zn concentration also lowered the rotifer abundances. The impact of zinc on competition between the two-rotifer species was evident at low-food level, mainly for A. fissa. At zinc concentrations of 0 and 0.125 mg l⁻¹, the populations of both rotifers continued to grow for about 10 days, but thereafter B. rubens began to decline. Role of zinc on the competitive outcome of the two species is discussed in relation to the changing algal densities in natural water bodies.     

Stimulation of saponin production in <Emphasis Type="Italic">Panax ginseng</Emphasis> hairy roots by two oligosaccharides from <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l⁻¹. HS and OS at 30 mg l⁻¹, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to ∼20 g l⁻¹ from 13 g l⁻¹ and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

Biosynthesis of Dibenzyl Trisulfide (DTS) from somatic embryos and rhizogenous/embryogenic callus derived from Guinea hen weed (<Emphasis Type="Italic">Petiveria alliacea</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) leaf explants

A procedure for producing somatic embryos enriched with dibenzyl trisulfide (DTS) using a hormone-dependent culture system is reported for Petiveria alliacea L. (Guinea hen weed). Leaf explants were cultured on a Murashige and Skoog medium supplemented with a range of naphthaleneacetic acid (NAA) concentrations and a fixed concentration of benzyladenine (BAP) at 11.0 μM and sucrose or glucose at 30 g l⁻¹. Leaf explants cultured on all media types started to form callus at the cut surfaces of the discs 10–14 d after initiation. The type of sugar used influenced average fresh weight, the propensity to form roots, as well as the embryogenic response. The highest mean fresh weight (337.7 ± 26.18 mg) and mean root number (23.7 ± 1.69) was produced on media enriched with sucrose and supplemented with 53.7 μM NAA and 11.0 μM BAP. An ethanol extract of rhizogenic/embryogenic callus or somatic embryos was subjected to high-performance liquid chromatography analysis, which revealed the presence of DTS in both extracts. UV spectral analysis and the use of standard quantitation procedures showed that the quantity of DTS in the somatic embryo extract, at 0.16% (w/v), was approximately 30-fold higher than in rhizogenic/embryogenic callus (0.0055% w/v) of similar fresh weight. These results indicate that it is possible to biosynthesize approximately 6 mg of natural DTS from 3,808 mg of fresh somatic embryos within 10 wk from less than three leaf explants.     

Highly efficient plant regeneration and <Emphasis Type="Italic">in vitro</Emphasis> polyploid induction using hypocotyl explants from diploid mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis> Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO₃. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l⁻¹ 6-benzylamino purine, 0.3 mg l⁻¹ indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO₃) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l⁻¹ indole-3-butyric acid and were successfully established in the soil.     

Production of fructose from inulin using mixed inulinases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

Two new effective microbial producers of inulinases were isolated from Jerusalem artichoke tubers grown in Thailand and identified as Aspergillus niger TISTR 3570 and Candida guilliermondii TISTR 5844. The inulinases produced by both these microorganisms were appropriate for hydrolysing inulin to fructose as the principal product. An initial inulin concentration of ∼100 g l⁻¹ and the enzyme concentration of 0.2 U g⁻¹ of substrate, yielded 37.5 g l⁻¹ of fructose in 20 h at 40°C when A. niger TISTR 3570 inulinase was the biocatalyst. The yield of fructose on inulin was 0.39 g g⁻¹. Under identical conditions, the yeast inulinase afforded 35.3 g l⁻¹ of fructose in 25 h. The fructose yield was 0.35 g g⁻¹ of substrate. The fructose productivities were 1.9 g l⁻¹ h⁻¹ and 1.4 g l⁻¹ h⁻¹ for the mold and yeast enzymes, respectively. After 20 h of reaction, the mold enzyme hydrolysate contained 53% fructose and more than 41% of initial inulin had been hydrolysed. Using the yeast enzymes, the hydrolysate contained nearly 38% fructose at 25 h and nearly 36% of initial inulin had been hydrolysed. The A. niger TISTR 3570 inulinases exhibited both endo-inulinase and exo-inulinase activities. In contrast, the yeast inulinases displayed mainly exo-inulinase activity. The mold and yeast crude inulinases mixed in the activity ratio of 5:1 proved superior to individual crude inulinases in hydrolysing inulin to fructose. The enzyme mixture provided a better combination of endo- and exo-inulinase activities than did the crude extracts of either the mold or the yeast individually.     

Optimization and mechanism of diacetyl accumulation by <Emphasis Type="Italic">Enterobacter</Emphasis><Emphasis Type="Italic">aerogenes</Emphasis> mutant UV-3

A mutant designated as UV-3 was obtained from wild-type Enterobacter aerogenes 10293 through u.v. radiation. The activities of α-acetolactate decarboxylase (Ald), lactate dehydrogenase (Ldh) and diacetyl reductase (Dr) in UV-3 were strongly attenuated, with the lowest activities at pH 7.0–7.5, and temperature between 36 and 39°C. Compared to the wild-type, the yield of diacetyl by UV-3 was increased 18.7-fold, up to 1.05 ± 0.01 g l⁻¹. Acetoin and ethanol productions were decreased by 48.4 and 71.4%, respectively, but acetate yield was increased by 34.6%. Optimum medium for diacetyl production by UV-3 contained 10% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.01% (NH₄)₂SO₄, 0.1% citric acid, 0.2% MnSO₄ and 0.2% MgSO_4, and this was determined by one-factor-at-a-time approach. Data from the five level central composite designs demonstrated that initial pH of 7.0, temperature of 37°C and rotational speed of 180 rev/min were optimum processing parameters for diacetyl production. The maximum yield of diacetyl could reach 1.35 g l⁻¹ in a 5-l bioreactor. These results showed an enhancement of the non-enzymatic oxidative decarboxylation of α-acetolactate and a decrease in the activities of Ald, Ldh and Dr as a consequence of diacetyl accumulation in UV-3.     

Utilization of <Emphasis Type="Italic">fadA</Emphasis> knockout mutant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> for overproduction of medium chain-length-polyhydroxyalkanoate

The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l⁻¹ stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l⁻¹ with a cell dry weight of 10.3 g l⁻¹.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Growth and laccase production kinetics of <Emphasis Type="Italic">Trametes versicolor</Emphasis> in a stirred tank reactor

White rot fungi are a promising option to treat recalcitrant organic molecules, such as lignin, polycyclic aromatic hydrocarbons, and textile dyes, because of the lignin-modifying enzymes (LMEs) they secrete. Because knowledge of the kinetic parameters is important to better design and operate bioreactors to cultivate these fungi for degradation and/or to produce LME(s), these parameters were determined using Trametes versicolor ATCC 20869 (ATCC, American Type Culture Collection) in a magnetic stir bar reactor. A complete set of kinetic data has not been previously published for this culture. Higher than previously reported growth rates with high laccase production of up to 1,385 U l⁻¹ occurred during growth without or glucose limitation. The maximum specific growth rate averaged 0.94 ± 0.23 day⁻¹, whereas the maximum specific substrate consumption rates for glucose and ammonium were 3.37 ± 1.16 and 0.15 ± 0.04 day⁻¹, respectively. The maximum specific oxygen consumption rate was 1.63 ± 0.36 day⁻¹.