Arginine Deiminase(ADI) was purified to homogeneity using ammonium sulfate precipitation,Q-Sepharose Fast Flow anion exchange chromatography and SephadexG-75 gel filtration chromatography. This purification protocol resulted in a 34.5-fold purification of ADI with 31.4% final yield. A molecular weight of about 190 kD determined by native gradient polyacrylamide gel electrophoresis. The enzyme has only one kind of 46 kD subunit determined by SDS-PAGE. Combining the results from the two kinds of electrophoresis,the authors deduce that the enzyme may be a tetramer. The optimum pH and temperature for lipolytic activity of ADI was pH 6.5 and 50℃,respectively. It was extremely stable at 45℃ and retained 97.9% of its original activity for 30 min. The stability declined rapidly as soon as the temperature rose over 50℃. ADI was highly stable in the pH range from pH 5-8. ADI acted on L-arginine but not on D-arginine. ADI catabolism was dependent on metal ions. At their adequate concentration,Mn2+,Mg2+ and Co2+ were the effective promoter,while superfluous Zn2+and Co2+ inhibited ADI activity. L-citrulline did not act on ADI,but L-ornithine inhibited ADI activity. The degradation of L-arginine with ADI catalysis was according to simple Michaelis-Menten equation. The Michaelis constant was 3.2686 mmol/L and the maxi-mum velocity was 2.44 μmol/min.

Extraction of arginine deiminase(ADI) from NJ402 using aqueous two-phase system(ATPS) provides a method for purification of arginine deiminase.The aqueous two-phase system(ATPS) is composed of polyethylene glycol(PEG) and(NH4)2SO4.Approximately 91.1% of arginine deiminase with the purification factor of 2.35 could be extracted from NJ402 under the optimal conditions that the average relative molecular mass of PEG is 1000,w(PEG1000)=15%,w[(NH4)2SO4]=20%,and pH=6.5 at room temperature.

The result of extraction of arginine deiminase(ADI) using reversed micellar system is reported,which provides a method for the purification of arginine deiminase.In the system,cetyltrimethylammonium bromide(CTAB) was used as surfactant,octane was used as solvent and butanol was used as co-solvent.The optimum conditions were established that the concentration of CTAB is controlled at 0.01 mol/L,pH at 7,shaking time at 15 minutes,the concentration of NaCl at 0.75 mol/L,and the initial enzyme mass concentration at 30 g/L.The yield of ADI can reach 85%,while the specific activity can reach 1.107 U/mg which is 4.52 times that of the original deiminase.

D-arginine was produced by means of biotransformation of DL-arginine in the presence of arginine deiminase which was found in the Streptococcus faecalis.The yield of D-arginine and L-citrulline was 87% and 88% respectively.Factors which effected on the enzyme activity were studied.The result shows that the optima conditions are as below: 37(°C),pH 6.0 in the biocatalytic system.70.0g of DL-arginine can be converted by 1.0 g of bacteria cells used for 7 times with the conversion rate against L-arginine being 100% with all D-arginine remained.

L-theanine (γ-glutamylethylamide) is the main free amino acid component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for optically pure L-theanine production with a new L-aminoacylases-production fungi Cunnighamella echinulata 9980 was developed. The optimum conditions of reaction were: 0.05 M N-Acyl-DL-theanine, 0.04 g/ml pellets, 5×10-4CoCl2, pH7. This was the first report that Cunnighamella echinulata was the source of aminoacylases, and aminoacylase, regardless of its source, was applied in DL-theanine resolution.

Optically pure D-glutamine was prepared by biotransformations of DL-glutamine manufactured by chemical synthesis. Firstly, DL-glutamine was synthesized from DL-glutamic acid on larger scale. Protected DL-glutamic acid was prepared by using inexpensive phthaloyl as protecting group. Then N-phthaloyl-DL-glutamic acid anhydride was synthesized with protected DL-glutamic acid and acetic anhydride under reflux for 15 min. Under ambient temperature and normal pressure, N-phthaloyl-DL-glutamine was produced by reacting N-phthaloyl-DL-glutamic acid anhydride with 2 mol/L ammonia solution. After the phthaloyl was removed from the intermediate with 0.5 mol/L hydrazine hydrate under ambient temperature for 48 h DL-glutamine mixture was obtained in 57% yields. L-Glutamine in the racemic mixture was selectively decarboxylated to 4-aminobutanamide by the decarboxylase of E. coli AS1.505. Under the following conditions: temperature of 37 degrees C, reaction time of 8 h, pH of 4.8, substrate concentration of 30 g/L, D-glutamine was efficiently produced.

A novel synthesis method of dipeptides containing glutamine is reported. Protected L-amino acids were prepared by using inexpensive phthaloyl as the protecting group. Then the phthaloyl-L-amino acids were condensed with glutamine salts by the mixed anhydride method to afford phthaloyl dipeptides. Subsequently, the phthaloyl was removed from the dipeptides with hydrazine hydrate. As a result, optically pure glutamine-containing dipeptides were obtained in good yields.

L-citrulline is an important marker to many diseases. A simple, accurate and rapid enzymatic method is proposed for the determination of L-citrulline for its bioconversion by the ornithine transcarbamoylase (EC2. 1.3.3) and the carbamate kinase (EC 2.7. 2.2). The optimized conditions for the bioconversion were 30 degrees C similar to 35 degrees C, pH 6 similar to 7. The reserved special activity of immobilized enzymes was up to 95% after they were kept for 60 days in 4 degrees C. The linear range was 2 similar to 10 mg/L (R =0. 9987), the recovery for the detrmination was more than 99%. The enzymatic method is simple and accurate. It has been applied successfally to the determination of urinary L-citrulline from 3 sufferers of rheumatoide arthritis.

Process of fermentation of arginine deiminase, which was a new tumor-growth inhibitor, was studied. Effects of various carbon sources, nitrogen sources and other factors on arginine deiminase production in Enterococcus faecalis NJ402 were investigated in single factor test. The optimal medium was selected as follows (g/L) : sucrose 15, peptone 5, extract of yeast 5, extract of beef 2.5, NaCl 3, KH2PO4 2, MgSO4 0.01, MnSO4 0.0025, L-arginine 15, pH 7.5 with 4% inoculum in 100L fermentor. E. faecalis could reach high yield of arginine deiminase after 10 h at 37°C, the specific activity could reach 2.57 U/mL with yield of 40 mg/mL biomass.

Optically pure D-glutamine is initially prepared by biotransformations of D,L-glutamine in the presence of L-glutamine decarboxylase in Escherichia coli AS1.505.Factors that affected the enzymatic activity such as converting temperature,pH value,etc.,were studied.The results show that the specific enzyme's activity can come up to 4200U,and L-glutamine can be completely degraded by the decarboxylase for 8 h under the optical conditions as follows:37℃,4.8 of pH value,5 g/L of cell concentration,0.15 g/L of tween-80,14 h of strain age,and 40 g/L of the substrate concentration.

A novel Enterococcus faecalis strain designated NJ402 was found with high activity of arginine deiminase (ADI). The optimum condition for catalytic activity was determined in terms of temperature (about 40℃),thermostability (available 37℃) and pH (6-7). The effects of substrate and product concentration were studied.The effects of various metal ions added in reaction mixtures on the biocatalyst were investigated and ADI of NJ402was found to exhibit Co2+ dependence, different from previous reports. Surfactant, cetyl trimethyl ammonium bromide, was one of the most important keys for producing L-citrulline. The enzyme in resting cells possessed the quality of high stability for reuse.

考察了大肠杆菌AS1.505的L-谷氨酸脱羧酶对L-谷氨酸的专一脱羧作用,分析了转化体系温度、pH值等因素对L-谷氨酸脱羧酶活力的影响.实验结果表明最佳工艺为:温度37℃,转化体系pH值4.8,菌体浓度6 g/L,吐温-80 0.15 g/L,菌龄14 h,底物浓度50 g/L.L-谷氨酸脱羧酶在最适转化条件下比酶活可以达到15036 U.1 g湿菌体可重复使用3次共转化DL-谷氨酸25 g,其中L-谷氨酸可以完全转化为γ-氨基丁酸.D谷氨酸及γ-氨基丁酸的总收率可以分别达到理论收率的87%和85%.

Protected L-glutamic acid was prepared using inexpensive phthaloyl as protecting group.Then N-phthaloyl-L-glutamic acid anhydride was synthesized with protected L-glutamic acid and acetic anhydride under reflux for 10 minutes.Under ambient temperature and pressure,N-phthaloyl-L-theanine and N-phthaloyl-L-glutamine were produced by reaction of N-phthaloyl-L-glutamic acid anhydride with 2 mol/L ethylamine solution and 2 mol/L ammonia solution respectively.Subsequently,the phthaloyl group was removed from the intermediates with 0.5 mol/L hydrazine hydrate under ambient temperature for 48 h.As a result,L-theanine and L-glutamine were obtained in 61% and 57% yields respectively.

DL-茶氨酸经乙酰化可生成N-乙酰-DL-茶氨酸,然后利用米曲霉氨基酰化酶拆分N-乙酰-DL-茶氨酸可以获得L-茶氨酸.本文对氨基酰化酶酶促反应也进行了初步研究,考察了反应温度、反应时间、pH值和底物浓度等因素对酶促反应的影响.实验表明,转化温度40℃、转化时间30h、pH为7.0和底物浓度0.2mol/L时,目的产物L-茶氨酸的产率最高.

介绍了微波促L-苯丙氨酸消旋反应是一种新的可适合于工业化的生产方法,具有对环境友好的优点.使用微波作为热源不仅可加速反应和节约能源,也可提高反应的收率和产品的纯度.本方法确定了在微波辐射下,以1.0 mol/L氢氧化钠水溶液替代低级脂肪酸作为反应溶剂,用物质的量比为0.10的水杨醛为催化剂,L-苯丙氨酸可以快速消旋;在此条件下,消旋反应随微波辐射功率的提高而加快,在66%输出功率下反应已接近最大反应速率.从实验中得出微波促L-苯丙氨酸消旋的反应是一级不可逆反应.考察了此消旋反应的影响因素,同时讨论了微波作用下L-苯丙氨酸的消旋反应机理.

L-Arginine can be converted to L-citrulline with arginine deiminase which was found in the Streptococcus faecalis.Many factors affecting on the enzymatic activity were studied.The result shows that the optimum conditions are as follows:the strain age 20 h,37 ℃,pH 6.0,ρ(cetyltrimethylammonium bromide(CTAB))=0.30 g/L.Some metal ions such as Cu~(2+),Zn~(2+)and Co~(2+) also have effect on the enzymatic activity.

A simple,accurate and rapid spectrophotometric method is proposed for the determination of L-citrulline in enzynatic conversion solution.The method is based on the absorbance of complex at 490 nm being proportional to L-citrulline concentrations.Effect of L-arginine,L-ornithine,buffer solution,the time of reaction and dosage of diacetylmonoxime on the determination of L-citrulline is also studied.The linear range is 0～0.04(mg/mL)(r=0.999),and the mean recovery is 99.58%.

How to get text information from this huge information space becomes a more important problem with the rapid growth of the Internet. A semantic indexing system GHUNT based on concept space is proposed to solve the problem. Some new technologies are integrated in GHUNT to obtain good performance. GHUNT is an all-sided solution for information retrieval on the Internet.

OBJECTIVE To investigate into the antibiotic activity of extract of fermented bamboo parasitic fungus cultivated artificially so as to develop new natural antibiotics. METHOD Limited cultivation matrix was adopted to cultivate fermented bamboo parasitic fungus and then the fermentation fluid was extracted with acetic ether. Experiments were carried out on the antibiotic activity of the extracts. RESULT Three strains of anamorph bamboo parasitic fungus were isolated and fermented. The in vitro experiment on the antibiotic activity of the extract showed that its lowest bacteria-inhibiting concentration for colibacillus and staphylococcus aureus was 0.4 mg/mL and the lowest bacteria-destroying concentration was 3.19 mg/mL. These two figures for geotrichumcandidum and aspergillus niger were both 0.024 mg/mL. CONCLUSION The extract of the fermented bamboo parasitic fungus contains natural substances with antibiotic activity, with wide antibacterial spectra.