Poly(L-lactide) degradation by Saccharothrix waywayandensis

Poly(l-lactide) (PLA) was degraded by more than 95 mg from 100 mg PLA film by an actinomycete, Saccharothrix waywayandensis, growing in 100 ml liquid culture containing 0.1% (w/v) gelatin. In addition to degrading PLA, this strain assimilated the major degradation product of PLA, l-lactic acid.     

Development of fermentation process for PLA-degrading enzyme production by a new thermophilic Actinomadura sp. T16-1

The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-degrading Actinomadura sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at 50°C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2 % KH₂PO₄, and 0.02% MgSO₄ · 7H₂O. The optimal concentration of PLA film and gelatin obtained by response surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238% (w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter.     

Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma

A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family.     

Polylactide Degradation by an Amycolatopsis sp

By applying the plate count and clear-zone methods, it was confirmed that polylactide (PLA)-degrading microorganisms are sparsely distributed in soil environments. An Amycolatopsis isolate was successfully isolated. Microbial degradation of PLA film was demonstrated; i.e., about 60% of the 100-mg film initially added was degraded by the strain after 14 days of liquid culture.     

转化人胚肌腱细胞力学特性研究

探讨转化人胚腱细胞９Ｔｒａｎｓｆｏｒｍｅｄ ｈｕｍａｎ ｅｍｂｒｙｏｎｉｃ ｔｅｎｄｏｎ ｅｅｌｌｓ,ＴＨＥＴＣｓ）力学特性,以阐明在应变条件下进行细胞－支架材料复合培养时腱铁力学相容性。取人腱细胞（Ｈｕｍａｎ ｅｍｂｒｙｏｎｉｃ ｔｅｎｄｏｎ ｃｅｌｌｓ,ＨＥＴＣｓ）和ＴＨＥＴＣｓ,采用微吸管吸吮实验技术,测量单个细胞的粘生及其对聚乳酸（Ｐｏｌｙｌａｃｔｉｃ ａｃｉｄ,ＰＬＡ）和聚乳酸羟基乙酸共     

Isolation of a microorganism capable of degrading poly-(L-lactide)

The isolation of poly-(L-lactide) (PLA)-degrading microorganisms was investigated. A PLA-degrading actinomycete, strain No. 3118, was isolated and tentatively identified as a member of the genus Amycolatopsis. The optimum conditions for degradation of PLA were 43 degrees C at about pH 7 in a mineral salt medium with a low concentration of organic nutrients (0.002% yeast extract). The original shape of PLA film (Mw=2.3x10(5) after sterilization, 20 &mgr;m thick) disappeared within 2 weeks. Lactic acid was detected after the film was incubated with culture supernatant.     

应用原位双膜法快速筛选人Flt3配体甲醇酵母高表达转化子

原位双膜法是一种基于免疫原理的快速筛选高表达甲醇酵母转化子的方法,即首先将固体培养基上的菌落转印至醋酸纤维素薄膜上,再利用硝酸纤维素薄膜原位捕获穿过醋酸纤维素薄膜的菌落外泌蛋白,然后用免疫方法检测与硝酸纤维素薄膜结合的蛋白.利用此法筛选到人Flt3配体（hFL）的甲醇酵母高表达转化子,液体诱导表达量约20 mg/L.ELISA结果证明,原位双膜法所得的菌落染色强度与该菌落液体诱导表达水平正相关.蛋白质印迹结果显示,培养上清在25 ku处有明显杂交条带,而对照组杂交呈阴性,且表达量随诱导天数增加.原位双膜法是一种良好的筛选方法,可以快捷、准确地筛选高表达酵母转化子.     

Purification and Complete Primary Structure of the First PLA<Subscript>2</Subscript> from <Emphasis Type="Italic">Lachesis stenophrys</Emphasis> (the Central American Bushmaster) Snake Venom

The first PLA(2) (LsPA-1) from L. stenophrys snake venom was purified to homogeneity using three chromatographic steps and had its complete primary structure determined. An average molecular mass of 13,870.3 kDa was determined by mass spectrometry and a 3.3-fold increase in the PLA(2) activity was observed for LsPA-1 as compared to the whole venom. Multiple alignment of PLA(2) from Lachesis spp. snakes suggested the existence of two geographical clades for this genus in the New World, which is in accordance with morphological, behavioral and mtDNA data obtained by others. Phospholipases A(2) from Crotalus spp. snake venoms were similarly distributed into two groups. Intergroup analysis indicated that most amino acid substitutions were observed in the amino- and carboxy-terminal regions of the molecules in each clade. Both regions have been suggested to play important roles in determining the biological properties of PLA(2) from snake venoms. The dendogram derived for PLA(2) from Lachesis and Crotalus snakes highlighted the phylogenetic relationships between these two genera in the New World.     

Morphogenesis and tissue cultures of three citrus species

Studies were performed to define tissue culture techniques and culture conditions for morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis (L.) Osb.), citron (C. medica L.) and lime (C. aurantifolia) (Christm. Swing). The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l^-1 for sweet orange and lime, and 3 mg l^-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l^-1 for sweet orange and citron, and 1 mg l^-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mg l^-1 NAA and 0.25 mg l^-1 BA. Callus was maintained by periodical subculture into the same medium supplemented with 10% (v:v) organge juice. In vitro plantlets of the three species were obtained by rooting of shoots developed from bud cultures, and of citron and lime by development of shoots from root cultures. The plants were successfully established on soil.     

γ-Irradiation of PEGd,lPLA and PEG-PLGA Multiblock Copolymers: I. Effect of Irradiation Doses

To evaluate the effects of different gamma irradiation doses on PEGd,lPLA and PEG-PLGA multiblock copolymers. The behaviour of the multiblock copolymers to irradiation was compared to that of PLA, PLGA polymers. PEGd,lPLA, PEG-PLGA, PLA and PLGA polymers were irradiated by using a ⁶⁰Co irradiation source at 5, 15, 25 and 50 kGy total dose. Characterization was performed on all samples before and after irradiation, by nuclear magnetic resonance (NMR), infrared absorption spectrophotometry (FTIR) and gel permeation chromatography (GPC). The effect of gamma irradiation on polymer stability was also evaluated. Results of NMR and FTIR suggest an increase in -OH and -COOH groups, attributed to scission reactions induced by irradiation treatment. Data of GPC analysis showed that the weight average molecular weight (Mw) of polymer samples decreased with increasing irradiation dose. The extent of Mw degradation expressed as percentage of Mw reduction was more prominent for polymers with high molecular weight as PEGd,lPLA and PLA. The dominant effect of gamma-irradiation on both polymer samples was chain scission. The multiblock copolymer PEGd,lPLA presented higher sensitivity to irradiation treatment with respect to PLA, likely due to the presence of PEG in the matrix. The effect of gamma irradiation continues over a much longer period of time after gamma irradiation has been performed. It is suggested that the material reacts with oxygen to form peroxyl free radicals, which may further undergo degradation reactions during storage after irradiation.     

Application of a novel disposable film culture system to photoautotrophic micropropagation of <Emphasis Type="Italic">Eucalyptus uro-grandis (Urophylia x grandis)</Emphasis>

Summary To overcome various disadvantages of conventional culture vessls for plant micropropagation, we previously developed the photoautotrophic micropropagation technique, with special mention for the first practical film culture system, the ‘Miracle Pack’ (MP), which was made of fluorocarbon polymer film (Neoflo^? PFA film) and supported by a polycarbonate frame. While the PFA film has superior thermal stability, high light transmittance and high gas permeability, making the MP system (MP-PFA) superior to conventional culture vessels for the micropropagation of various plant species, its high cost is a disadvantage. In this study, a possible alternative of lower-cost OTP^? film made of TPX (4-methyl-1-pentane polymer) and CPP (a polypropylene), which possesses similar characteristics to PFA film, is evaluated to develop a novel disposable film culture vesel, termed ‘Vitron’, for culturing Eucalyptus (urophylla x grandis), plantlets. The three film culture systems, MP-PFA, MP-OTP (MP with OTP film), and Vitron, were placed under CO₂ enrichment, low photosynthetic photon flux density (PPFD; 45 μmol m⁻² s⁻¹), and sugar-free medium, using phenol resin foam (Oasis^?) as a substrate. In vitro and ex vitro growth and development of Eucalyptus shoots from the four-leaf stage to the rooting stage were compared for all three culture systems. The effects of the duration and concentration of CO₂ enrichments on in vitro growth of Eucalyptus cultured in the Vitron film system were also examined. The best growth and quality of Eucalyptus plantlets was obtained for the Vitron vessel placed in 3000 ppm CO₂ enrichment for 24 hours per day at low PPFD with sugar-free liquid medium and Oasis as substate. Results of this study suggest that the novel Vitron culture system is suitable for the photoautotrophic micropropagation of Eucalyptus. These authors contributed equally to the research results.     

Interactions between organic and inorganic carbon sources during mixotrophic cultivation of Synechococcus sp.

Glucose at 3 g l(-1) markedly accelerated growth of Synechococcus sp. PCC 7002. The net photosynthesis rate was 263 micromol O2 (mg Chl a h)(-1) for mixotrophic culture and 146 micromol O2 (mg Chl a h)(-1) for photoautotrophic culture. Additing 1 g NaHCO3 l(-1) to the glucose-supplemented culture enhanced the photosynthetic rate by 18%, and the total carbon consumption rate was raised to 2.5 mg l(-1) (mg chl a h)(-1) from a previously negative value. An interaction between organic and inorganic carbon metabolism was established.     

In vitro rooting of sentang shoots (Azadirachta excelsa L.) and acclimatization of the plantlets

Summary In vitro proliferated sentang shoots were cultured onto half-strength Murashige and Skoog (MS) medium containing combinations of 1-naphthyleneacetic acid (NAA) and indole-3-butyric acid (IBA). Sentang shoots were unable to root in the absence of both auxins. A combination of 0.5 mg NAA per and 1 mg IBA per 1 induced the most shoots to form roots. With the addition of 2.5 g activated charcoal per 1 into half-strength MS medium containing 0.5 mg NAA per 1 and 1 mg IBA per 1, roots were more numerous and longer. Substances like gelrite and phloroglucinol and sugar content which would commonly influence in vitro rooting were inhibitory to adventitious root formation of sentang. Maximal rooting of 100% was achieved in “Culture Pack,” made of fluorocarbon polymer film containing charcoal-free medium with 0.5 mg NAA per 1 and 1 mg IBA per 1. Rooted shoots were acclimatized for 4 wk. Overall survival was 80%. These findings suggest the use of Culture Pack as the culture vessels, with 0.5 mg NAA per 1 and 1 mg IBA per 1 in half-stength MS media to effectively induce roots in sentang shoots.     

Degradation of poly(tetramethylene succinate) by thermophilic actinomycetes

Various thermophilic actinomycetes were screened for their ability to degrade a high melting point, aliphatic polyester, poly(tetramethylene succinate) (PTMS), at 50 °C. By using the clear zone method, Microbispora rosea, Excellospora japonica and E. viridilutea were found to have PTMS-degrading activity. In a liquid culture with 100 mg PTMS film, M. rosea subsp. aerata IFO 14046 degraded about 50 mg film sample after 8 days. Degradation at the amorphous regions of the PTMS film was observed by scanning electron microscopy. This strain was also able to completely degrade poly(-caprolactone).     

Morphogenetic pathway in petiole derived callus of Amorphophallus albus in vitro

Two different morphogenetic pathways, adventitious bud and corm-like structure (CLS), were observed on organogenic calli derived from the petioles of Amorphophallus albus in vitro. The organogenic calli was established via culture of petiole segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.0 mg l⁻¹ 6-benzyladenine (BA) and subculture of the petiole-derived calli on MS medium with 0.5 mg l⁻¹ NAA and 0.5 mg l⁻¹ BA. These organogenic calli were used to induce morphogenesis via culture on MS medium with various concentrations of NAA and BA. BA alone favoured adventitious bud differentiation (57.0 ± 8.3% at maximum) from the organogenic calli but inhibited CLS formation. In the presence of NAA and BA, both adventitious bud and CLS were observed in a same culture system. The maximum CLS formation (71.2 ± 9.3%) were found on MS medium with 0.5 mg l⁻¹ NAA and 2.0 mg l⁻¹ BA, associated with 26.7 ± 8.6% adventitious bud differentiation. A small part of the adventitious buds developed into normal shoots which needed rooting culture phase to form complete plants. About 80% survival rate was obtained with these plants after transplantation to soil. More than 90% of the CLSs produced complete plants with shoots and root systems, regardless of the rooting media tested. Transplantation of the CLS-derived plants to soil gave 100% survival rate. Histological observations revealed both the two morphogenetic events originated from the meristematic cells located in superficial layers of callus tissue.     

Production of poly(l-lactide)-degrading enzyme by Amycolatopsis orientalis for biological recycling of poly(l-lactide)

Efficient production of poly(l-lactide)(PLA)-degrading enzyme was achieved by addition of 0.1% (w/v) silk fibroin powder into a liquid culture medium of an actinomycete, Amycolatopsis orientalis, without other complex nitrogen sources, such as yeast extract and peptone. Scaled-up production of the enzyme in a 5-l jar fermenter showed the possibility of producing this enzyme on an industrial scale at low production cost. The extracellular PLA-degrading enzyme showed potent degrading activity, which is effective for biological recycling of PLA, i.e., 2,000 mg/l of PLA powder was completely degraded within 8 h at 40°C using 20 mg/l purified enzyme. An optically active l-lactic acid with 600 mg/l was obtained as degradation product of PLA without undesirable racemization.     

Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation

Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5mg/L benzylamino purine (BAP), 100mg/L kanamycin and 250mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.     

Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan

The mature peptide of Aspergillus niger xylanase A (AnxA) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant AnxA (reAnxA) was secreted into culture medium. After 96-h 0.25% methanol induction, the activity of reAnxA in the culture supernatant reached the peak, 175 U/mg, which was 1.9 times as high as that of the native AnxA (92 U/mg). Studies on enzymatic properties showed that the optimum temperature and optimum pH of reAnxA were 50 degrees C and 5.0, respectively. The reAnxA was very stable in a wide pH range of 3.0-8.0. After incubation at the pH 3.0-8.0, 25 degrees C for 1h, all the residual activities of reAnxA were over 80%. The K(m) and k(cat) values for reAnxA were 4.8 mg/ml and 123.2s(-1), respectively. HPLC analysis showed that xylotriose was the main hydrolysis product of birchwood xylan and bran insoluble xylan by reAnxA.     

Comparison of cytocompatibility of zein film with other biomaterials and its degradability in vitro

Cytocompatibility of particle zein (Pzein) and film zein (Fzein) was evaluated and compared with polyhydroxybutyrate (PHB), its copolymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), polylactic acid (PLA), and collagen, using HL-7702 cells, in terms of cell attachment rate within 3 h, and cell viabilities at 3 and 6 days determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The zein degradation test was carried out using collagenase and trypsin, and the degradation product was added to the culture medium at different concentrations in order to examine the concentration-dependent cytotoxic effect. RESULT: The adhesion rate of the HL-7702 cells on both Pzein and Fzein was higher than that on collagen film. Cell viabilities were higher on both Pzein and Fzein than on films of PLA, PHB, PHBV, and collagen from fish skin. Zein can be degraded by both trypsin and collagenase, and the degradation product can enhance cell viability within a certain range of concentrations.     

培养基组分及培养条件对蜡梅花粉萌发及花粉管生长的影响

采用液体培养法研究不同培养基组分和培养条件对蜡梅花粉萌发和花粉管生长的影响。结果表明:(1)PEG-4000是蜡梅花粉离体培养所必需的培养基成分,当培养基中无PEG-4000时,花粉不能正常萌发。(2)培养基内低浓度蔗糖对花粉萌发和花粉管的生长无显著影响,但随着蔗糖浓度的升高,则对花粉萌发和花粉管生长表现出强烈的抑制作用,且浓度越高,抑制效应越强。(3)培养基内其它组分分别在一定浓度范围(0～250g/L PEG-4000、0～50mg/L硼酸、0～30mg/L硝酸钙)内对花粉萌发及花粉管生长有促进作用,但超过上述高限值时则起抑制作用。(4)培养基内镁和钾的浓度对花粉萌发及花粉管生长影响不显著。研究表明,蜡梅最适花粉液体培养基组分为250g/L PEG-4000+50mg/L H3BO3+30mg/L Ca(NO3)2.4H2O,且在pH 5.5、温度15℃和600lx的光照培养条件下蜡梅花粉萌发和花粉管生长最佳。