土壤分离转谷氨酰胺酶生产菌株

根据转谷氨酰胺酶催化反应结果的特性 ,设计了一个利用廉价的酶作用底物实施的蛋白质交联凝絮 -沉淀性能测定方法 ,并将其作为初筛手段 ,用于从土壤中分离生产转谷氨酰胺酶微生物菌种。从 13 9株放线菌中经初筛和摇瓶发酵测定酶活的复筛试验筛选得到 10株产酶菌株 ,其中一株酶活达 0 2 4U/mL ,并对其进行分类鉴定实验 ,确定为链霉菌属 ,编号为Streptomycessp .WZFF .W 12。

Effect of Chitinase-Producing Strain V-8 on Controlling Cotton Fusarium Wilt

[Objective] This study aimed to screen endophytic bacteria which is antag- onistic to cotton Fusarium wilt. [Method] Fresh cotton plants collected from cotton- growing areas in Jingzhou City, Hubei Province were used as experimental materials to isolate endophytic bacteria. Through chitinase test and co-culturing both micro-or- ganisms side by side on the same PDA culture plate, antagonistic strains to cotton Fusarium wilt were screened. [Result] A total of 83 bacterial isolates were obtained from cotton plants grown in the fields, six of which were chitinase-productive bacte- ria. Through chitinase test and co-culturing both micro-organisms side by side on the same PDA culture plate, strain V-8 which had the strongest antagonistic effect on Fusarium oxysporum f. sp. vasinfectum was screened. Strain V-8 had a wider anti- fungal spectrum with certain inhibitory effect on all the six important pathogenic fungi including Fusarium oxysporum f. sp niveum; it colonized stably in the rhizospheric soil of cotton, with a colonization density of up to 6.2x10s cfu/g fifty days after inoc- ulation; the relative effect on controlling cotton Fusarium wilt in pot test was 73.2%. The Findings of this study suggested that strain V-8 had great potential for biological control of cotton Fusarium wilt and could be taken as a substantial material for the cloning of chitinase genes. [Conclusion] The results from this study provides bases for the control of cotton fusarium wilt, as well as the exploitation of endophytic bac- teria resources in cotton and the development of novel biological pesticides.

Enhanced Production of Hybrid Extracellular β-Glucanase by Re-combinant Escherichia coli Using Experimental Design Method

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid ex-tracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L-1. After optimization of the medium, 297.71U·ml-1 of β-glucanase activity in the medium and 352350U·g-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those ob-tained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.

Production of Alkaline Protease by Bacillus Licheniformis

In this research the results of studies on optimization of alkaline protease production by Bacillus licheniformis are reported. The parameters, which were taken into consideration, are pH, temperature, time course of enzyme production, stirring rate and kinetics parameters. The effect of various carbon and nitrogen sources in culture medium compound on enzyme production was also considered The result of optimization revealed that maximum protease production was obtained at 37 ℃, pH equivalent tol 0.0 and with 150 rpm will occur after 72 hours. By comparing the effect of 5 carbon sources （maltose, glucose, starch, casein and lactose） in enzyme production, it has been known that using lactose will increase about 1.5 times enzyme production, compared to condition in which maltose is used. The result of studies on the effect of five nitrogen sources （i.e., peptone, tryptone, ammonium sulfate, urea and corn steep liquor） shows that corn steep liqour increases enzyme production more than others, while peptone can also be considered as a good nitrogen source; but, ammonium sulfate and urea reduce enzyme production considerably. It was concluded that protease production occurs in the stationary phase of growth. Studying the kinetics parameters resulted that the best model for the enzyme above is Lineweaver-Burk model according to which Km is 0.64 mmol and Vmax is 88 lamol/min.

Optimum Production and Characterization of an Acid Protease from Marine Yeast Metschnikowia reukaufii W6b

The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease.The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 ℃.The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts.The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 ℃ and a shaking speed of 140 rmin-1.Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level.The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecu-lar-weight nitrogen sources.Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability.The acid protease produced by M.reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

Selection of Strains Producing Lipase with Transesterification Activity and Its Characterization

The interest for lipase production is due to the ability of this enzyme to catalyze some reactions, such as the transesterification. Although industrial biodiesel is produced chemically, there are several problems associated with this technology that can be prevented through the use of lipases. The present work aimed to select microorganisms with potential for production of lipase with transesterification activity. The lipase from Burkholderia cepacia was the one with the most promising results for this type of reaction, showing results of hydrolytic activity at 37 ℃and pH 8.0. The pH and volume of crude enzyme extract that showed favorable for synthesis ofbiodiesel is at about pH 6.0 and 3.75 mL, respectively, which represents approximately 42% of water in the system, ensuring the conversion of nearly 60% to biodiesel.

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumberApostichopus japonicus

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopusjaponicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes （protease, amylase, and cellulase） were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16S rDNA amplified with Mbo L Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A.japonicus gut was Bacillus, which can produce protease, amylase, and cellulase. Virgibacillus, however, can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.

Screening and Preservation of Douchi Fibrinolytic Enzyme Producing Strain

[Objective] To isolate and preserve a high-activity Douchi fibrinolytic enzyme producing strain from Douchi products. [Method] The Douchi flbrinolytic enzyme producing strain was screened on the selected medium prepared with self-made pork blood powder, the strain with the highest activity was screened out according to the size of hydrolysis cycle, and then preserved in LB medium. [ Rebait] A Douchi fibrinolytic enzyme producing strain with high thrombolytic activity was successfully screened out from the Douchi produced in Hunan, Guangdong and Jiangxi Prov- inces. [ Ceadusioe] The study lays foundation for the development of new-type thrombolytic drugs.

Isolation of a novel alginate-degrading bacterium, Ochrobactrum sp. and production of alginate-degrading enzyme

An alginate-degrading bacterium, identified as Ochrobactrum sp. on the basis of 16S rDNA gene sequencing, was isolated from brown algal samples collected from the waters in close vicinity to the Dongtou isles of the East China Sea. The strain, designated WZUH09-1, is able to depolymerize alginates with higher enzyme activity than that of others reported so far.

Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q_(10)

The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E.coli strain for coenzyme Q10 production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution(multiple copy integration) and replicon-free and markerless chromosomal integration(single copy integration), respectively. A coenzyme Q10 hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution,replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway.The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass(DCM) of coenzyme Q10 when supplemented with 0.075 g·L-1of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q10 production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.