里氏木霉β-葡萄糖苷酶基因的克隆及序列分析

文章以里氏木霉 (Trichoderma reesei)的基因组 DNA为模板 ,根据 Gen Bank上检索的 β-葡萄糖苷酶基因DNA序列 ,设计特异性引物 ,用高保真酶 probest polymerase进行 PCR扩增 ,获得了 2 .5 0 kb的 DNA片段。将其克隆在 p U C18的 Sma I位点上。测序结果表明 ,所获得的 DNA序列与 Gen Bank上检索的 β-葡萄糖苷酶基因的核苷酸序列同源性达 99.90 % ,氨基酸序列同源性达 10 0 %。

Expression Activity of CLCuV Bidirectional Promoter in Agrobacterium tumefaciens

Cotton leaf curl virus (CLCuV) belongs to the subgroup III of geminiviruses with single strand DNA genome. Study demonstrated that the bidirectional promoter of CLCuV had activity in Agrobacterium tumefaciens (Smith et Townsend) Conn. This is the first report for the activity of the bidirectional promoter of geminivirus in A. tumefaciens. Results showed that the activity of the complementary sense promoter was stronger than that of virion sense promoter, and was detected 2-fold higher than that of CaMV 35S promoter in A. tumefaciens. Moreover, the promoter 5' deletion analysis indicated that the mean GUS activity driven by a 287 nucleotides complementary sense promoter fragment (from-287 to the translation initiation site) is 4 times higher than that driven by the whole complementary sense promoter in A. tumefaciens. This result suggested that there might exist negative regulatory elements in this deleted fragment. The function of other cis-elements included in CLCuV complementary sense promoter was also discussed in this paper.

Inactivation Kinetics of β-N-Acetyl-D-Glucosaminidase from Prawn (Penaeus vannamei) by Formaldehyde

β-N-Acetyl-D-glucosaminidase （NAGase, EC.3.2.1.52） is chitinolytic enzymes and disintegrate dimmer and trimer a composition of oligomers of N-acetyl-β-D-glucosamine （NAG） into monomer. Prawn （P. vannamei） NAGase is involved in digestion and molting processes. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating shell and threatens the survival of the animal. The effect of formaldehyde on prawn （P. vannamei） β-N-acetyl-D-glucosaminidase activity for the hydrolysis of pNP-NAG has been studied. The results show that formaldehyde, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC50 is estimated to be 1.05mol· L^-1. The inactivation mechanism obtained from Lineweaver-Burk plots shows that the inactivation of the enzyme by formaldehyde belongs to the competitive type. The inactivation kinetics of the enzyme by formaldehyde has been studied using the progress-of-substrate-reaction method described by Tsou, and the rate constants have been determined. The results show that k＋0 is much larger than k-0, indicating the free enzyme molecule is fragile in the formaldehyde solution.

Optimization of Quantitative Real-time PCR System on Amplification of Beta-glucosidase Gene Os1bglu4

[Objective] This study aimed to establish a quantitative real-time PCR （qRT-PCR） system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,using the primers of reference gene actin or ubiquitin.[Result] Actin was more suitable to be the reference gene than ubiquitin.More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results,so,0.4 μmol/L was selected as the optimal primer concentration in this study.The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃,therefore,annealing temperature was set at 60 ℃.Compared with the reaction system of 25 μl,the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So,the 10 μl reaction system was selected,which significantly reduces the research costs for the detection of a large amount of samples in future study.

Ethanol Production by Enzymatic Hydrolysis of Elephant Grass

The aim of this work is to evaluate the efficiency of ethanol production by fermentation of a hydrolysate obtained by the enzymatic hydrolysis of purple elephant grass （Pennisetum purpureum Schum.） using a blend of cellulases. Three hundred grams of 20-mesh granulated purple elephant grass was subjected to hot water pretreatment at 100 ℃ for a period of 25 min in a batch autoclave reactor. The pulp obtained from the pretreatment process was washed with water at 60 ℃ and submitted to enzymatic hydrolysis using a combination of exoglucanases, endoglucanases and beta-glucosidases or β-glucosidases from Novozymes. The pH of the system was kept constant at 4.8 by adjusting the levels of acetic acid or sodium acetate in the buffer solution. Enzymatic hydrolysis occurred at 50 ℃ upon agitation at 200 rpm on a shaker for 72 hours. The hydrolysate obtained after agitation was fermented using dried Saccharomyces cerevisiae yeast （manufactured by Dr. Oetker） at 30 ℃ for 10 hours. The liquids obtained after fermentation were analysed using HPLC to determine the quantity of ethanol produced. After 4 hours of fermentation, the maximum quantity of ethanol was 1.8 g/L. The stoichiometric yield of ethanol was approximately 95%. However, the step ofpretreatment was deemed unsatisfactory due to the loss of glucose during the pretreatment process.

Multiple organ dysfunction syndrome due to ingestion of fish gall bladder

OBJECTIVE: To investigate changes in renal function, urine N-acetyl-beta-D-glucosaminidase enzyme (N-AG),liver function, myocardial enzymes, the pathology of renal damage and the mechanism of acute renal failure (ARF) associated with fish gall bladder poisoning. METHODS: Eleven patients with acute fish gall bladder poisoning were consecutively admitted to our hospital from September 1997 to October 1999. Renal function, urine N-AG enzyme, liver function, and myocardial enzymes were assayed before and after treatment. One patient consented to a kidney biopsy and the pathology of renal damage was observed under light and electron microscopes. RESULTS: All patients had multiple organ dysfunction syndrome (MODS) and 11 patients suffered from ARF. Ten patients had liver dysfunction, ten patients had poisonous myocarditis, and 8 patients had gastrointestinal dysfunction. Renal function, urine N-AG enzyme, liver function, and myocardial enzymes were significantly improved after treatment compared with those of before treatment (P

Effects of Maize Residue Quality and Soil Water Content on Soil Labile Organic Carbon Fractions and Microbial Properties

Investigating the effects of residue chemical composition on soil labile organic carbon （LOC） will improve our understanding of soil carbon sequestration. The effects of maize residue chemical composition and soil water content on soil LOC fractions and microbial properties were investigated in a laboratory incubation experiment. Maize shoot and root residues were incorporated into soil at 40% and 70% field capacity. The soils were incubated at 20 ℃ for 150 d and destructive sampling was conducted after 15, 75, and 150 d. Respiration, dissolved organic carbon （DOC）, hot-water extractable organic carbon （HEOC）, and microbial biomass carbon （MBC） were recorded, along with cellulase and β-glucosidase activities and community-level physiological profiles. The results showed that the cumulative respiration was lower in root-amended soils than in shoot-amended soils, indicating that root amendment may be beneficial to C retention in soil. No significant differences in the contents of DOG, HEOC and MBC, enzyme activities, and microbial functional diversity were observed between shoot- and root-amended soils. The high soil water content treatment significantly increased the cumulative respiration, DOC and HEOC contents, and enzyme activities compared to the low soil water content treatment. However, the soil water content treatments had little influence on the MBC content and microbial functional diversity. There were significantly positive correlations between LOC fractions and soil microbial properties. These results indicated that the chemical composition of maize residues had little influence on the DOC, HEOC, and MBC contents, enzyme activities, and microbial functional diversity, while soil water content could significantly influence DOC and HEOC contents and enzyme activities.

Cover Crop and Irrigation Effects on Soil Microbial Communities and Enzymes in Semiarid Agroecosystems of the Central Great Plains of North America

Cover crops can have beneficial effects on soil microbiology by increasing carbon （C） supply, but these beneficial effects can be modulated by precipitation conditions. The objective of this study was to compare a fallow-winter wheat （Triticum aestivum L.） rotation to several cover crop-winter wheat rotations under rainfed and irrigated conditions in the semiarid US High Plains. Experiments were carried out at two sites, Sidney in Nebraska, and Akron in Colorado, USA, with three times of soil sampling in 2012--2013 at cover crop termination, wheat planting, and wheat maturity. The experiments included four single-species cover crops, a 10-species mixture, and a fallow treatment. The variables measured were soil C and nitrogen （N）, soil community structure by fatty acid methyl ester （FAME） profiles, and soil β-glucosidase,β-glucosaminidase, and phosphodiesterase activities. The fallow treatment, devoid of living plants, reduced the concentrations of most FAMEs at cover crop termination. The total FAME concentration was correlated with cover crop biomass （R = 0.62 at Sidney and 0.44 at Akron）. By the time of wheat planting, there was a beneficial effect of irrigation, which caused an increase in myeorrhizal and protozoan markers. At wheat maturity, the cover crop and irrigation effects on soil FAMEs had subsided, but irrigation had a positive effect on the β-glucosidase and phosphodiesterase activities at Akron, which was the drier of the two sites. Cover crops and irrigation were slow to impact soil C concentration. Our results show that cover crops had a short-lived effect on soil microbial communities in semiarid wheat-based rotations and irrigation could enhance soil enzyme activity. In the semiarid environment, longer time spans may have been needed to see beneficial effects of cover crops on soil microbial community structure, soil enzyme activities, and soil C sequestration.

Use of a Bioaugmented Organic Soil Amendment in Combination with Gypsum for Withania somnifera Growth on Sodic Soil

Limited availability of organic matter is a problem to sustain crop growth on sodic soil. Organic soil amendments are a costeffective source of nutrients to enhance crop growth. A field study was conducted to evaluate the effect of an organic soil amendment bioaugmented with plant growth-promoting fungi(SF_(OA) ) in combination with gypsum on soil properties and growth and yield attributes of Withania somnifera, one of the most valuable crops of the traditional medicinal system in the world, on a sodic soil at the Aurawan Research Farm of CSIR-National Botanical Research Institute, Lucknow, India. The SF_(OA) used was prepared by pre-enriching farm waste vermicompost with plant growth-promoting fungi before mixing with pressmud and Azadirachta indica seed cake. The application of SF_(OA) at 10 Mg ha^(-1)after gypsum(25.0 Mg ha^(-1)) treatment significantly(P < 0.05) increased root length(by 96%) and biomass(by 125%) of Withania plants compared to the control without SF_(OA) and gypsum. Similarly, the highest withanolide contents were observed in leaves and roots of Withania plants under 10 Mg ha^(-1)SF_(OA) and gypsum. Combined application of SF_(OA) and gypsum also improved physical, chemical and enzymatic properties of the soil, with the soil bulk density decreasing by 25%, water-holding capacity increasing by 121%, total organic C increasing by 90%, p H decreasing by 17% and alkaline phosphatase, β-glucosidase, dehydrogenase and cellulase activities increasing by 54%, 128%, 81% and 96%, respectively, compared to those of the control. These showed that application of the SF_(OA) tested in this study might reclaim sodic soil and further support Withania cultivation and results were better when the SF_(OA) was applied after gypsum treatment.

Characterization of β-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones

An extracellular β-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isofiavone. Matrix-assisted laser desorption/ionization with tandem time-of- flight/time-of-flight mass spectrometry （MALDI-TOF/TOF MS） revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified 13-glucosidase showed optimal activity at pH 5.0 and 65℃ and was very stable at 50℃. Moreover, the enzyme exhibited good stability over pH 3.0-8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters Km （apparent Michaelis- Menten constant） and Vmax （maximal reaction velocity） for p-nitrephenyl-β-D-cjlucopyranoside （pNPG） were 1.73 mmol/L and 42.37 U/mg, respectively. The Krn and Vmax for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/（L.h） for daidzein, 0.72 mmol/（L.h） for genistein, and 0.19 mmol/（L.h） for glycitein. This is the first report on the application of A. terreus β-glucosidase for converting isoflavone glycosides to their aglycones in soybean products.

A simplified and miniaturized glucometer-based assay for the detection of β-glucosidase activity

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants.The drawbacks of the existing methods are high consumption of both time and reagents,complexity in operation,and requirement of expensive instruments and highly trained personnel.The present study provides a simplified,highly selective,and miniaturized glucometer-based strategy for the detection ofβ-glucosidase activity.Single-factor experiments showed that optimumβ-glucosidase activity was exhibited at 50°C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min.The procedure for detection was simplified without the need of a chromogenic reaction.Validation of the analytical method demonstrated that the accuracy,precision,repeatability,stability,and durability were good.The linear ranges ofβ-glucosidase in a buffer solution and rat serum were 0.0873–1.5498 U/mL and 0.4076–2.9019 U/mL,respectively.The proposed method was free from interference fromβ-dextranase,snailase,β-galactosidase,hemicellulase,and glucuronic acid released by baicalin.This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid(DNS)assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter.Miniaturization of the method resulted in a microassay forβ-glucosidase activity.The easy-to-operate method was successfully used to detect a series ofβ-glucosidases extracted from bitter almonds and cultured by Aspergillus niger.In addition,the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing ofβ-glucosidase in many fields,including medical diagnostics,food safety,and environmental monitoring.