一株纤维素酶产生菌的筛选鉴定

用CMC平板筛选方法,从草丛土样中筛选到一株产纤维素酶的菌株,该菌在20℃～50℃、pH4.0～10.0、NaCl1.0%～10.0%的条件下均能生长,在26℃～39℃、pH6.0～8.0、NaCl4.0%～6.0%条件下生长快速。经采用Biolog进行碳源利用鉴定判定为黄曲霉,命名为AF-1。AF-1的CMCase为19.26U/mL,FPA为6.22U/mL,纤维分解率为20.6%。采用单因素法对AF-1产酶条件进行了初步测定。

芽孢杆菌E2菌株纤维素酶形成条件的研究

芽孢杆菌 E_2菌株(Bacillus sp.strain E_2)能在55℃下良好生长并在培养液中大量积累胞外纤维素酶(190 mu/ml 培养液),所产生的纤维素酶为单一的 CMCase。对芽孢杆菌 E_2菌株产酶条件进行了研究.该菌产酶的最适培养基装量为200ml/500ml 三角瓶,最适起始pH 为6.5,最适产酶温度为45℃,产酶高峰在培养时间8—12小时。E_2菌株不能利用单一的无机氮源形成纤维素酶。酩蛋白是试验过的供 E_2菌株形成纤维素酶的最好氮源,其用量为3g/L。CMC-Na,纤维二糖,能作为碳源供芽孢杆菌 E_2菌株形成纤维素酶。高浓度的葡萄糖(8g/L)对芽孢杆菌 E_2菌株纤维素酶的形成有抑制作用。天然纤维素不能作为芽孢杆菌 E_2菌株形成纤维素酶的碳源。

Screening for a Novel Trichoderma vride Strain Highly Producing Cellulase via Ultraviolet Mutagenesis

[Objective] The aim of this study was to find out a new Trichoderma vride K strain highly producing cellulase.[Method] Ultraviolet（UV） was used to induce mutagenesis on T.vride K and to select out a new Trichoderma vride strain highly producing cellulase from the first round and further selection.[Result] A new T.vride strain K6 with high yield of cellulase was obtained with the enzyme production amount of 1.39 times over that of starting strain K.This strain showed highest cellulase yield under the culture condition of 28 ℃ for 96 h.[Conclusion] The strain K6 selected out from induced mutation is endowed with better capacity of producing cellulase,which provides a new method for the utilization of straw.

Comparison of hydroxyl radical-scavenging abilities between white rot fungus Lentinula edodes and five mildews on lignocellulose substance

The hydroxyl radical（-OH）-scavenging ability of culture filtrates from submerged culture of Lentinula edodes AX3 and five mildews on lignocellulose substance was analysed. Only L. edodes AX3 showed significant -OH-scavenging ability which reached 52.2% at about the 48th hour. All mildews could hardly scavenge -OH under the experimental conditions. -OH-scavenging ability is considered related to the mode and ability of lignocellulose degradation of a strain. The degradation or bioconversion products might be the substance base for.this effect.

Improvement of Cellulase Producing Capacity of Aspergillus niger by Ultraviolet Mutation

[Objective] The research aimed to breed the high-yield production strain of cellulase.[Method] Aspergillus niger was used as the starting strain,and a high-yield production strain of cellulase was selected after UV mutation treatment.[Result] Under the suitable condition,the strain 2（15） with the highest CMC production capacity was selected,which nearly increased 50% than that of the starting strain.[Conclusion] The research provided the foundation for its appliation in the feed production in the future.

Optimization of Fermentation Conditions for Cellulase Production from Camellia Seed Shell Using Response Surface Methodology

Objective] This study was conducted to optimize cameI ia seed sheI fer-mentation conditions for ceI uIase production by Trichoderma koningi using response surface methodoIogy. [Method] Fermentation conditions for ceI uIase production from Trichoderma koningi were optimized with response surface method （RSM） by taking carboxymethyI ceI uIase （CMCase） activity as a response indicator. Three factors that affecting CMCase activity were screened out using singIe factor test among pretreatment methods of raw material, nitrogen sources, initial pH values, inocuIum voIume, fermentation time and voIume of Iiquid medium, they were fermentation time, initial pH value, and voIume of Iiquid medium. The optimum conditions of fer-mentation and interaction of the three factors were determined through Box-Behnken design and regression analysis using Design-Expert software. [Result] Pretreatment of cameI ia seed sheI with alkaline was most conducive to CMCase production. The use of 0.2% （NH4）2SO4 as nitrogen source, inocuIum size of 5%, initial pH value of 5.8 and voIume of Iiquid medium at 22 mI were the best fermentation conditions for maximizing CMCase production by T. koningi from cameI ia seed sheI . Under these conditions, 179.15 U/mI of CMCase was obtalned after 5 d of fermentation, which was improved by 24.52% compared with the maximum CMCase activity of singIe factor test. [Conclusion] The resuIts wiI provide some references for use of cameI ia seed sheI and ceI uIase production.

LOW DOSE PIRFENIDONE SUPPRESSES TRANSFORMING GROWTH FACTOR BETA-1 AND TISSUE INHIBITOR OF METALLOPROTEINASE-1, AND PROTECTS RATS FROM LUNG FIBROSIS INDUCED BY BLEOMYCIN

Objective To investigate the optimal dosage of pirfenidone for the treatment of pulmonary fibrosis induced by bleomycin in Wistar rats, and the alteration of expressions of transforming growth factor beta-1 （ TGF-β1 ）, tissue inhibitor of metalloproteinase-1 （ TIMP-1 ）, and matrix metalloproteinase-13 （ MMP-13 ） in lung tissue. Methods Male Wistar rats were endotracheally instilled with bleomycin or normal saline. Pirfenidone （25-800 mg · kg^-l · d^-1 ）, dexamethasone （3 mg/kg）, or 1% carboxymethylcellulose sodium were given daily by feed 2 days before instillation of bleomycin. Groups T7 and T14 were fed pirfenidone 50 mg · kg^-1 · d^-1 at 7 days or 14 daYs after bleomycin instillation. Lungs were harvested at 28 days after bleomycin instillation. Patholological changes in luffg tissues were evaluated with HE staining. Lung collagen was stained by sirius red and measured by content of hydroxypro- line. Expression of proteins of TGF-β1 TIMP-1, and MMP-13 were detected by Western blotting. Results At doses of 25, 50, and 100 mg· kg^- 1 · d ^- 1, pirfenidone had significant anti-fibrotic effects for bleomy- cin-induced rat pulmonary fibrosis, and these effects were most significantly attenuated at the dosage of 50 mg · kg^-1 ·d^ -1（ HE： P 〈 0. 01, P 〈 0.01, and P = 0.064; sirius red： P 〈0.05, P 〈 0.01, and P 〈 0.05 ; hydroxyproline： P = 0.595, P 〈 0.01, and P = 0.976）. Pirfenidone at a dosage of 50 mg · kg^- l · d^-1 inhibited protein expression of TGF-131 and TIMP-1 in lung tissue in the early phase （0.79 and 0.75 times of control group）, but had no effect on ex- nr^eelnn nf MMP-13. Conclusion Low dose pirfenidone, especially at dosage of 50 mg · kg^-1 · d^-1, has significant anti-fibrotic effects on bleomycin-induced rat pulmonary fibrosis. Pirfenidone partially inhibits the enhancement of the expression of TGF-131 and TIMP-β1 in lung tissue.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

Effect of expression of TGF-beta and TNF-alpha with Qidan granule treatment in rats by bleomycin-induced pulmonary fibrosis

Objective: To evaluate the therapeutic effects and mechanisms of Qidan granule in blemycinA5-induced pulmonary interstitial fibrosis (PIF)in rats. Methods: PIF models were established by blemycinA5-induced in rats. They were treated by Qidan granule and Hydrocortisone respectively. The pathological changes and collagen protein disposition were observed, and the expression of TGF-β, TNF-α proteins were measured by immunohistochemical technique. Results: The pulmonary alveolitis and fibrosis were alleviated remarkably in Qidan granule group compared with those in the model control group and hydrocortisone group (P<0.01). The expression of TGF-β and TNF-α protein were higher in Qidan granule group than those in normal group ,and were significantly less than those in the model control group and in hydrocortisone group (P<0.01). Conclusion: Qidan granule would ameliorate the pulmonary alveolitis and fibrosis. TGF-β and TNF-α might play an important role in the development of alveolitis and fibrosis in rats.

Kinetic Characterization of Trichoderma reesei CL847 TR3002： An Engineered Strain Producing Highly Improved Cellulolytic Cocktail

Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck of the production process of bioethanol from lignocellulose. It is generally admitted that the most efficient organism for the production ofcellulolytic enzymes is the fungus Trichoderma reesei, mostly due to its high secretion capacity. Unfortunately, this fungus secretes very low concentrations of β-glucosidase, thereby often requiring β-glucosidase supplementation for complete cellulose hydrolysis. It is especially important to have sufficient quantities of β-glucosidase in order to prevent inhibition of cellobiohydrolases by cellobiose. In order to optimize the produced cocktail, a more efficient β-glucosidase was cloned into T. reesei CL847 strain. The new strain, called CL847 TR3002, secretes the evolved β-glucosidase and was tested for cellulase production in laboratory-scale reactors. Its growth kinetics and cellulase production were characterized using fed-batch and chemostat modes under various culture conditions. The cellulase activities of the evolved strain were compared with activities of the parent strain. In addition, hydrolysis of a steam exploded wheat straw was performed at three different enzyme-loading levels （5 mg/g, 10 mg/g and 20 mg/g of dry matter） and a new kinetic model was developed.

Expression of PEPT2 mRNA in the lung of rat with bleomycin-induced pulmonary fibrosis

Objective：Pulmonary fibrosis is a common pathological phenomena in lung cancer patients after chemotherapy or radiotherapy, which is a key factor hindering to transport ion of high concentrated drug to the lung tissue, peptide trans-porter has become targets of the rational design of peptides and peptide drug. The purpose of the study is to investigate the expression of PEPT2 mRNA in the lung of rats with bleomycin （BLM）-induced pulmonary fibrosis. Methods：Fifty healthy adult Spragne-Dawley rats were randomized into five groups, the rats in BLM 7d, 14d and 28d groups were treated with a single instil ation of 5 mg/kg of BLM, to induced pulmonary fibrosis models. On days 7, 14 and 28, the animals were kil ed by exsan-guination respectively. Normal saline （NS） group were treated by NS, on days 14, the animals were kil ed by exsanguinations. Control group were untreated. The lung samples were processed for light microscopy and determined the hydroxyproline （HYP） concentration. The expression of PEPT2 mRNA were measured by RT-PCR. PEPT2 cDNA fragments were tested by dideoxy chain termination. Results：Compared with control and NS group, HYP levels increased on day 7 of BLM group, but there was no statistical significant dif erence （P〉0.05）. HYP levels markedly increased on days 14 and 28 of BLM group, there was statistical significant dif erence （P〈0.01）. The morphological study showed that col agenous fiber proliferated on days 14 and 28 of BLM group, especial y on day 28, formed pulmonary fibrosis. There were no significant changes of pulmo-nary PEPT2 mRNA expression at dif erent groups （P〉0.05）. Conclusion：The pulmonary fibrosis models of SD rats can be induced by a single instil ation of 5 mg/kg of bleomycin on 28d. There were no significant changes of PEPT2 mRNA expression in the lung of rats with bleomycin-induced pulmonary fibrosis.

Cooperation effects of GA3, IAA and uniconazole-P on tension wood formation and gravitropism in Fraxinus mandshurica seedlings

The cooperation effects of GA3, IAA and uniconazole-P were studied on the gravitropism and wood formation in Fraxinus mandshurica Rupr. var.japonica Maxim. Seedlings using traditional paraffin section technology. Our results are： （1） Gravitropism of stems was strongly inhibited only in B, whereas promoted significantly in D, E, F, H and I treatments; （2） Xylem formation was increased on both sides in H, I and J treatments and on the lower side in E and F which also show the synergistic effect; （3） On the radial direction, cell wall thickness was enhanced on the upper side, whereas decreased on the lower side in C, and was also significantly promoted on the upper in E, G, H, I and J treatments. On the tangential direction, cell wall thickness was increased on the lower side in E, H, I and J treatments respectively; （4） Gelatinous layer of wood fibers was observed on the upper side in all treatments. These results suggest that both GA3 and IAA affected negative gravitropism and wood formation significantly. However, appliedor unapplited-uniconazole-P does not affect G-layer formation, indicating GA does not play the key role on G-layer formation, and ratio of GA3/IAA or IAA may be more important in regulating G-layer formation.

Four natural epothilone derivatives isolated from Sorangium cellulosum strain So0157-2

Four natural epothilone analogues, including two 14-membered epothilones L （1）, L1 （2） and two epoxide opened epothilones A （3） and B （4）, were isolated from the extract of fermentation adsorber resin of Sorangium cellulosum strain So0157-2. Their structures were elucidated on the basis of comprehensive NMR and MS data. They are all isolated from the wild type S. cellulosum strain So0157-2 as natural products for the first time.

Effects of rapamycin against paraquat-induced pulmonary fibrosis in mice

Background and aims： Ingestion of paraquat （PQ）, a widely used herbicide, can cause severe toxicity in humans, leading to a poor survival rate and prognosis. One of the main causes of death by PQ is PQ-induced pul- monary fibrosis, for which there are no effective therapies. The aim of this study was to evaluate the effects of ra- pamycin （PAPA） on inhibiting PQ-induced pulmonary fibrosis in mice and to explore its possible mechanisms. Methods： Male C57BL/6J mice were exposed to either saline （control group） or PQ （10 mg/kg body weight, intraper- itoneally; test group）. The test group was divided into four subgroups： a PQ group （PQ-exposed, non-treated）, a PQ＋RAPA group （PQ-exposed, treated with RAPA at I mg/kg intragastrically）, a PQ＋MP group （PQ-exposed, treated with methylprednisolone （MP） at 30 mg/kg intraperitoneally）, and a PQ＋MP＋RAPA group （PQ-exposed, treated with MP at 30 mg/kg intraperitoneally and with PAPA at 1 mg/kg intragastrically）. The survival rate and body weight of all the mice were recorded every day. Three mice in each group were sacrificed at 14 d and the rest at 28 d after intox- ication. Lung tissues were excised and stained with hematoxylin-eosin （H＆E） and Masson＇s trichrome stain for his- topathological analysis. The hydroxyproline （HYP） content in lung tissues was detected using an enzyme-linked im- munosorbent assay （ELISA） kit. The expression of transforming growth factor-β1 （TGF-β1） and α-smooth muscle actin （α-SMA） in lung tissues was detected by immunohistochemical staining and Western blotting. Results： A mice model of PQ-induced pulmonary fibrosis was established. Histological examination of lung tissues showed that PAPA treatment moderated the pathological changes of pulmonary fibrosis, including alveolar collapse and interstitial collagen depo- sition. HYP content in lung tissues increased soon after PQ intoxication but had decreased significantly by the 28th day after PAPA treatment. Immunohistochemical staining and Western blotting showed that PAPA treatment significantly down-regulated the enhanced levels of TGF-β1 and e-SMA in lung tissues caused by PQ exposure. However, PAPA treatment alone could not significantly ameliorate the lower survival rate and weight loss of treated mice. MP treatment enhanced the survival rate, but had no significant effects on attenuating PQ-induced pulmonary fibrosis or reducing the expression of TGF-β1 and a-SMA. Conclusions： This study demonstrates that PAPA treatment effectively suppresses PQ-induced alveolar collapse and collagen deposition in lung tissues through reducing the expression of TGF-β1 and a-SMA. Thus, RAPA has potential value in the treatment of PQ-induced pulmonary fibrosis.

Lysyl oxidase promotes bleomycin-induced lung fibrosis through modulating inflammation

Enzymes involved in collagen biosynthesis, including lysyl oxidase （LOX）, have been proposed as potential therapeutic targets for idio- pathic pulmonary fibrosis. LOX expression is significantly upregulated in bleomycin （BLM）-induced lung fibrosis, and knockdown of LOX expression or inhibition of LOX activity alleviates the lung fibrosis. Unexpectedly, treatment of the mice with LOX inhibitor at the inflammatory stage, but not the fibrogenic stage, efficiently reduces collagen deposition and normalizes lung architecture. Inhibition of LOX impairs inflammatory ceU infiltration, TGF-β signaling, and myofibroblast accumulation. Furthermore, ectopic expres- sion of LOX sensitizes the fibrosis-resistant Balb/c mice to BLM-induced inflammation and lung fibrosis. These results suggest that LOX is indispensable for the progression of BLM-induced experimental lung fibrosis by aggravating the inflammatory response and subse- quent fibrosis process after lung injury.