Discovery and functional verification of endogenous glucanases for scleroglucan hydrolysis in Sclerotium rolfsii / 生物工程学报

Scleroglucan is a high-molecular water-soluble microbial exopolysaccharide and mainly applied in the fields of petroleum, food, medicine and cosmetics. The high molecular weight of scleroglucan produced by microbial fermentation leads to low solubility, high viscosity and poor dispersibility, thus bringing a series of difficulties to extraction, preservation and application. It is important to explore suitable degradation method to adjust the molecular weight of scleroglucan for expanding its industrial application. Taking Sclerotium rolfsii WSH-G01 as a model strain, in which functional annotations of the glucanase genes were conducted by whole genome sequencing. Based on design of culture system for culture system for differential expression of β-glucanase, endogenous β-glucanase genes in S. rolfsii WSH-G01 were excavated by transcriptomics analysis. Functions of these potential hydrolases were further verified. Finally, 14 potential endogenous hydrolase genes were obtained from S. rolfsii. After heterologous overexpression in Pichia pastoris, 10 soluble enzymes were obtained and 5 of them had the activity of laminarin hydrolysis by SDS-PAGE and enzyme activity analysis. Further investigation of the 5 endogenous hydrolases on scleroglucan degradation showed that enzyme GME9860 has positive hydrolysis effect. The obtained results provide references not only for obtaining low and medium molecular weight of scleroglucan with enzymatic hydrolysis, but also for producing different molecular weight of scleroglucan during S. rolfsii fermentation process with metabolic engineering.

Conversion of gypenosides by enzymatic hydrolysis with β-glucanase / 国际药学研究杂志

Objective: To investigate the enzymatic hydrolysis of gypenosides by β-glucanase 26130 CN to prepare some hydrolyzed secondary saponins, so as to provide material basis for further biological studies. Methods: Using β-glucanase 26130 CN, the total saponins from Herba Gynostemmatis were hydrolyzed with the enzyme catalysis, and the hydrolytic products were analyzed by ultra high- performance liquid chromatography coupled with quadrupole time- of- flight mass spectrometry(UHPLC- Q- TOF/MSE)to identify the converted products. Then, the main components of Herba Gynostemmatis, gypenosides XLIX and A, were used as substrates of the β-glucanase 26130 CN for convertsion to secondary saponin products. The products were separated by preparative highperformance liquid chromatography(HPLC)and identified by NMR and MS. Results: Twenty eight triterpenoid saponins were identified in the total saponin hydrolysate on the basis of their high-resolution MS data, by comparison with the data in the literature, and seven of them were validated to be the converted products. It was found that the β-glucanase 26130 CN could hydrolyze the glycosidic bond of terminal glucose or xylose in the molecule of gypenosides. By the enzymatic hydrolysis of gypenoside XLIX and gypenoside A, gypenoside I(the one glucosyl-lost gypenoside XLIX)and gypenoside UL1(the one xylosyl-lost gypenoside A)were obtained via the preparative HPLC separation of the gypenoside XLIX and gypenoside A hydrolysates, respectively. Conclusion: β-glucanase 26130 CN could effectively catalyze the hydrolysis of terminal glucosyl and xylosyl groups in gypenosides, with a relatively high hydrolytic conversion rate, which could be used to prepare some secondary saponins or aglycones.

Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Research progresses in microbial 1,3-1,4-β-glucanase: protein engineering and industrial applications / 生物工程学报

1,3-1,4-β-glucanase (E.C.3.2.1.73) is an important industrial enzyme which cleave β-glucans into oligosaccharides through strictly cutting the β-1,4 glycosidic bonds in 3-O-substituted glucopyranose units. Microbial 1,3-1,4-β-glucanase belongs to retaining glycosyl hydrolases of family 16 with a jellyroll β-sandwich fold structure. The present paper reviews the industrial application and protein engineering of microbial β-glucanases in the last decades and forecasts the research prospects of microbial β-glucanases.

Anti-fungal substances of Bacillus subtilis against Ganoderma boninense

Aims@#To determine the optimum culture incubation time for β-glucanase and chitinase production by Bacillus subtilis as well as optimum pH and temperature condition for enzymatic activity against Ganoderma boninense. The suitable solvent (methanol, ethyl acetate or hexane) for the extraction of bacterial metabolites from B. subtilis were also determined. @*Methodology and results@#In vitro antagonistic activity of antifungal metabolites derived from B. subtilis to inhibit the growth of G. boninense was evaluated based on time of culture incubation, extraction solvent of the metabolites, and enzymatic treatments conditions including pH and temperature. The results showed that β-glucanase could be optimally produced (with a specific activity 4.222 U/mg-protein) after 28 h of incubation. The optimum pH and temperature for the activity of β-glucanase were 7.5 and 45 °C respectively when 1% laminarin used as the substrate. B. subtilis showed optimum chitinase activity (0.0514 U/mL) after 8 h of incubation. Optimum pH and temperature of chitinase were at pH 6.0 and 40 °C, respectively using 1% colloidal chitin as the substrate. β-glucanase crude enzyme showed strongest antifungal activities against the mycelial growth of G. boninense better than crude enzyme of chitinase with an inhibition rate of 47.75% at 5 days of incubation. Furthermore, cultivation of B. subtilis over 48 h produced antifungal metabolites which could inhibit the growth of G. boninense the most. The best solvent to extract metabolite from B. subtilis was identified as ethyl acetate that rendered an inhibition value of 38.91%. @*Conclusion, significance and impact of study@#Bacillus subtilis could be a potential biological control agent against G. boninense.

Cellulolytic ability of a promising Irpex lacteus (Basidiomycota: Polyporales) strain from the subtropical rainforest of Misiones province, Argentina / Capacidad celulolítica de una cepa prometedora de Irpex lacteus (Basidiomycota: Polyporales) de la selva subtropical de la provincia de Misiones, Argentina

Abstract The cellulolytic activity of fungi growing in the subtropical rainforest of Misiones (Argentina) represents a challenge in the technological development of the production of cellulosic bioethanol in the region using native sources. These fungi are promising to obtain sustainable enzyme cocktails using their enzymes. Cellulolytic ability of 22 white-rot fungi isolated from the subtropical rainforest of Misiones-Argentina in agar medium with two types of cellulosic substrates, carboxy-methylcellulose or crystalline cellulose, were comparatively analyzed, and the activity of two cellulolytic enzymes was evaluated in liquid medium. Although all isolates were able to grow and degrade both substrates in agar medium, and to produce total cellulase Filter paper (FPase) and endo-β-1,4-glucanase (EG) activities in broth, the isolate Irpex sp. LBM 034 showed the greatest enzymatic levels (FPase, 65.45 U L-1; EG, 221.21 U L-1). Therefore, the ITS sequence of this fungus was sequenced and analyzed through a phylogenetic analysis. These results indicate that the isolate LBM 034, corresponding to Irpex lacteus, has a promising cellulolytic ability and enzymes such as EG useful in sustainable saccharification of cellulosic materials in the region. Rev. Biol. Trop. 66(3): 1034-1045. Epub 2018 September 01.

Resumen La actividad celulolítica de hongos autóctonos asociados a la selva subtropical de Misiones (Argentina) representa un desafío en el desarrollo tecnológico de la producción de bioetanol celulósico en la región, mediante el uso de recursos nativos. Los sistemas enzimáticos de estos hongos tienen potencial aplicación en la obtención de cocteles enzimáticos rentables. La habilidad celulolítica de 22 hongos causantes de pudrición blanca se analizó comparativamente, que fueron aislados de la selva subtropical de Misiones-Argentina, en cultivos agarizados con dos tipos de sustratos celulósicos, carboxi-metilcelulosa o celulosa cristalina. También se evaluó la actividad de dos enzimas celulolíticas en cultivos líquidos. Aunque todos los aislamientos fueron capaces de crecer y degradar ambos sustratos en medio agarizado y revelar actividad celulolítica total y endo-β-1,4-glucanasa en cultivo líquido, el aislamiento Irpex sp. LBM 034 mostró las mayores actividades en papel de filtro con 65.45 U L-1 y endo-β-1,4-glucanasa con 221.21 U L-1, respectivamente. Por tanto, se secuenció y analizó la secuencia ITS de este hongo a través de un análisis filogenético. Estos resultados indicaron que el aislamiento LBM 034, correspondiente a Irpex lacteus, tiene una habilidad celulolítica prometedora en la producción de enzimas con actividad endo-β-1,4-glucanasa, útil en la sacarificación sustentable de materiales celulósicos de la región.

Establishment of fingerprint of Astragali Radix polysaccharides based on endo-1,3-β-glucanasehydrolysis and identification of Astragali Radix of different germplasm resources / 中草药

Objective Enzymatic hydrolysis of Astragali Radix polysaccharides from different germplasm resources Astragalusmembranaceus var. mongholicus (MG) (cultured and natural) or Astragalusmembranaceus (MJ) (cultured and natural) was carried out by the best enzymolysis conditions of endo-1,3-β-glucanase. Saccharide fingerprints were obtained for the identification and evaluation of the germplasm resources of Astragali Radix by Fluorophore-assisted Carbohydrate Electrophoresis (FACE). Methods The data were analyzed by principal component analysis and t test using SMICA software to distinguishdifferential sugar segments among different germplasm resources of Astragali Radix. Results Pentasaccharide and hexasaccharide of endo-1,3-β-glucanasehydrolyzate could be used as differentiated saccharide fragments between natural MG and MJ.Trisaccharide, tetrasaccharide, and pentasaccharide could be used as differentiated saccharide fragments to distinguish the cultured MG and MJ.The pentasaccharide and hexasaccharide can be used as differential fragments to distinguish MJ (culturedandnatural). Conclusion Thepolysaccharide products degraded by endo-1,3-β-glucanase can well distinguish Astragali Radix species (MG and growth mode (cultured and natural Astragali Radix). This study laid the foundation for the quality evaluation of Astragali Radix and screening of active oligosaccharides.

Isolation and characterization of a novel endo-beta-1, 4-glucanase from a metagenomic library of the black-goat rumen

ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.

Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere

Abstract Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30 °C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.

Potential application of β-1, 3 glucanase from an environmental isolate of Pseudomonas aeruginosa MCCB 123 in fungal DNA extraction.

Pseudomonas aeruginosa MCCB 123 was grown in a synthetic medium for β-1,3 glucanase production. From the culture filtrate, β-1,3 glucanase was purified with a molecular mass of 45 kDa. The enzyme was a metallozyme as its β-1,3 glucanase activity got inhibited by the metal chelator EDTA. Optimum pH and temperature for β-1,3 glucanase activity on laminarin was found to be 7 and 50 °C respectively. The MCCB 123 β-1,3 glucanase was found to have good lytic action on a wide range of fungal isolates, and hence its application in fungal DNA extraction was evaluated. β-1,3 glucanase purified from the culture supernatant of P. aeruginosa MCCB 123 could be used for the extraction of fungal DNA without the addition of any other reagents generally used. Optimum pH and temperature of enzyme for fungal DNA extraction was found to be 7 and 65 °C respectively. This is the first report on β-1,3 glucanase employed in fungal DNA extraction.

Endo-and exoglucanase activities in bacteria from mangrove sediment

The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.

Production, purification and characterization of a thermostable β-1,3-glucanase (laminarinase) produced by Moniliophthora perniciosa

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70 percent) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20 percent of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

A enzima glucanase de Moniliophthora perniciosa foi produzida em meio líquido e purificada a partir do sobrenadante da cultura. A metodologia de superfície de resposta (MSR) foi usada para avaliar os efeitos das variáveis, incluindo indutor (extrato de levedura) e tempo de fermentação, na atividade da glucanase de M. perniciosa detectada no meio de cultura. A enzima presente no sobrenadante foi purificada em duas etapas: precipitação com sulfato de amônio (70 por cento) e cromatografia de filtração em gel em Sephacryl S-200. A produção da enzima glucanase foi maior na concentração de 5,9 g L-1 de extrato de levedura e 13 dias de fermentação. Os resultados mostraram três diferentes isoformas (GLUI, GLUII e GLUIII) com fatores de purificação de 4,33, 1,86 e 3,03, respectivamente. O extrato enzimático parcialmente purificado mostrou um pH ótimo de 5,0 e uma temperatura ótima de 40°C. A atividade enzimática aumentou na presença de KCl em todas as concentrações estudadas. A atividade da glucanase foi maior na presença de NaCl 0,2 M. A enzima apresentou alta estabilidade térmica, perdendo apenas 10,20 por cento de sua atividade específica após 40 minutos de incubação a 90°C. Os resultados de termoestabilidade e a atividade em baixo pH mostraram que a enzima glucanase de M. perniciosa tem características promissoras para futuras aplicações industriais.

Molecular cloning, purification and characterization of thermostable -1,3-1,4 glucanase from Bacillus subtilis A8-8.

A gene encoding a -1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant -1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60oC, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60oC and retained 30% of its original activity at 70oC for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.

Transformation of Compound K from Saponins in Leaves of Panax notoginseng by Immobilized β-Glucanase / 中草药·英文版

Objective To prepare an active anti-tumor component, compound K (C-K), from saponins in leaves of Panax notoginseng (SLPN) using immobilized β-glucanase. Methods Two entrapments, alginate gel-1 (Alg 1) and alginate gel-2 (Alg 2), were evaluated for their ability to immobilize β-glucanase. The amount and purity of C-K obtained from the transformation process were analyzed by HPLC, and the immobilizing parameters were optimized. Results β-Glucanase can be immobilized and reused with either of the entrapment. However, using AIg 1 resulted in higher enzyme activity than Alg 2. The optimal concentration of the immobilized enzyme was 10%; The optimal crosslinking time was 4-6 h; and the optimal concentration of the crosslinking agent was 6%-7%. Conclusion Immobilized β-glucanase shows sustained enzyme activity, good ethanol tolerance, and was reusable for the preparation of C-K from SLPN.

Stem Blight Control of Schizonepeta tenuifolia Caused by Phytophthora nicotianae Using Trichoderma spp / 中草药·英文版

Objective To control stem blight disease of Schizonepeta tenuifolia caused by Phytophthora nicotianae.Methods The antagonist effect of 13 Trichoderma strains(including T.viride and T.hamianum)was evaluated upon mycelia growth of P.nicotianae.Trichoderma strains with high antagonistic activities against the pathogen were used to control stem blight of S.tenuifolia in the field.Results Of 13 Trichoderma strains tested,T.viride strain M3 showed maximum mycelia growth inhibition(83.2%)to the pathogen,followed by T.viride strain Tv04-2(78.2%)and then T.harziamum strain ThB(65.0%),in vitro.Fungal cell wall degrading enzymes,protease,and β-1,3-glueanase were analyzed qualitatively and quantitatively in further study.T.viride strains M3,Tv04-2,and T.harzianum strain ThB efficiently against P.nicotianae were used to control stem blight of S.tenuifolia in the field,and T.viride strain M3 showed the best biocontrol potential.Conclusion Trichoderma spp.can be used as alternatives of pesticides to control stem blight,one of the serious soilhome diseases of S.tenuifolia caused by P.nicotianae.However,though T.viride strains Tv04-2 aad T.harzianum strain ThB are also highly against P.nicotianae in vitro,the controlling efficacy of them on stem blight disease is not as excellent as T.viride strains M3 in the field.

Utilização do triticale e de enzimas em dietas para suínos: digestibilidade e metabolismo / Utilization of triticale and of enzymes in pig diets: digestibility and metabolism

Avaliou-se o efeito da inclusão de níveis de triticale sobre o valor nutritivo de dietas para suínos com ou sem enzimas. Foram utilizados 24 suínos machos, castrados, com peso inicial de 59kg, alojados em gaiolas metabólicas. O delineamento foi de blocos ao acaso em arranjo fatorial 3 x 2 (níveis de triticale, 0, 30 e 60 por cento, com ou sem enzimas), com quatro repetições cada. A inclusão de triticale na dieta em até 60 por cento e a adição de enzimas não influenciou (P>0,05) a digestibilidade da matéria seca, fósforo, energia bruta, metabolização da energia, energia digestível e metabolizável e o balanço do N. Os valores médios de energia digestível e metabolizável foram de 3.537 e 3.435kcal. Houve interação triticale x enzimas na proteína digestível aparente (PDa) (P<0,01). Na dieta com 60 por cento de triticale sem adição de enzimas a PDa foi 6 por cento inferior à controle. Na dieta com 30 e 60 por cento de triticale com enzimas, a PDa foi similar à controle e 5 por cento superior à dieta com 60 por cento de triticale sem enzimas. A inclusão de 60 por cento de triticale em dietas para suínos reduz a PDa. A adição de enzimas melhora a PDa em dietas com 30 e 60 por cento de triticale.

The effect of triticale levels with or without enzyme supplementation on nutritive value of pig's diet was evaluated using twenty four barrows weighting 59kg, housed in metabolic crates. A complete randomized experimental block design in a 3 x 2 factorial arrangement (triticale levels -0, 30 e 60 percent with or without enzymes) with four replicates each was utilized. No effects of triticale levels and enzymes supplementation (P>0.05) were observed on dry matter digestibility, phosphorus, crude energy, metabolization of energy, digestible and metabolizable energy and nitrogen balance. The average values of digestible and metabolizable energy were 3,537 e 3,435kcal. An interaction triticale x enzymes effect on apparent digestible protein (DPa) (P<0.01) was observed. Apparent digestible protein for 60 percent triticale diets without enzymes supplementation was 6 percent lower than control diet while DPa of 30 and 60 percent triticale diets with enzymes supplementation were similar to DPa of the control diet and 5 percent higher than 60 percent triticale diets without enzyme supplementation. The inclusion of 60 percent triticale in pig diets reduces the DPa. Enzymes supplementation improves the DPa for diets containing 30 and 60 percent of triticale.

The Identification of Antagonistic Bacteria B-04 against Botrytis cinerea and Improving Biocontrol Effect through Genetic Modification with ?-1,3-glucanase Gene / 中国生物工程杂志

Bacillus cereus B-04 antagonist to Botrytis cinerea were isolated from samples of tomato soil infected by Botrytis cinerea in Zibo, which are identified through a series of morphological and biochemical characteristics and the sequence of 16SrDNA. Aiming at enhancing the inhibitory effect of this strain, a 4.1kb DNA fragment containing ?-1,3-glucanases gene from pUC1940 was inserted into vector pBE2 and pHY300PLK to construct recombination plasmids, PBE2-glu and pHY300PLK-glu, which were transferred into Bacillus cereus B-04, resulting in a new strain named B-04-glu. Restriction enzyme digestion and ?-1,3-glucanases plate culture confirmed that B-04-glu contained a functional ?-1,3-glucanases gene. Compared to the wild strain B-04, B-04-glu had an increased inhibitory effect against Botrytis cinerea on tomato.

Transgenic Atractylodes macrocephala with double defense genes exhibiting resistanceto Rhizoctonia solani / 中草药

Objective The transgenic Atractylodes macrocephala resistance to Rhizoctonia solani was obtained by gene engineering. Methods On the base of the efficient regeneration system of Baizhu via shoot organogenesis, the rice chitinase gene (RCH10) and the alfalfa ?-1, 3-glucanase gene (AGLU) were tandem-inserted into the transformation vector pB101, which was transformed into A. macrocephala with gene gun. Transformants were confirmed by PCR, GUS assay, and disease resistant. Results Twenty-five independent transformants possessed desired genes were observed by PCR detection, and among them five transformants exhibiting resistance to Rhizoctonia solani. Conclusion The disease resistant variety has been obtained by transformation, which provides a shorten avenue for the direct introduction of novel traits into A. macrocephala through genetic engineering without the need for numerous back-crossing in breeding programs that slow down cultivar improvement, meanwhile it improves disease resistant gene pool.

Expression of the Trichoderma reesei Endoglucanse IV in Pichia pastoris / 微生物学通报

In this work,the Pichia pastoris expression system was applied to express the T.reesei EGⅣ.The eg4 gene was isolated from rice hull induced T.reesei culture through RT-PCR,and was ligated with the Pichia expression vector pPICZ?A,resulting in the recombinant plasmid pPICZ?A-eg4.The recombinant plasmid pPICZ?A-eg4 was then linearized and transformed into P.pastoris GS115,and the eg4 gene was in frame integrated into the Pichia genome through homologous recombination,resulting the recombinant strain P.pastoris-EGⅣ1.With methanol induction,the recombinant strain P.pastoris-EGⅣ1 expressed and secreated EGⅣ into the culture supernatant with CMC activity of 2.11U/mL.The SDS-PAGE analysis showed that the apparent molecular weight of expressed protein was about 50kD,slightly less than that produced by T.reesei.

PRODUCTION OF CELLULASE BY PENICILLIUM SP. NXP25 AND ITS PROPERTIES / 微生物学通报

Cellulase was produced by growing Penicillium sp. NXP25 in liquid medium consisted of 5% com cob powder, 3% wheat bran, 0.35% nitrogen source No 10 and 0.3% calcium chloride. The optimum culture conditions were initial pH 5.0, 10% mycelial inoculum, temperature 29℃, shaking speed 280r/min and cultivation time 72h. When determining enzyme activity at 50℃, endo-1, 4-?-glucanase activity, extro-1, 4-?-glucanase activity, ?-glucosidase activity and filter paper enzyme activity of the supernatant of the culture were 841u/mL, 13u/mL, 24u/mL and 46u/mL, respectively. The optimum pH and temperature for the action of the above enzymes were pH 4.8 and60℃, pH5.0 and 50℃, pH 4.5 and 70℃, pH 5.0 and 55℃, respectively. Stable pH range of the above enzymes were 3.0-7.0, 4.0~6.0, 4.0~7.0 and 4.0~6.0, respectively. After incubating the enzyme complex at 65C for 30min, 24% of endo-1, 4-?-glucanase activity, 7% of extro-1, 4-?-glucanase activity, 89%of ?-glucosidase activity and 8% of filter paper enzyme activity were remained, respctively.