产气肠杆菌几丁质酶的分离纯化及性质研究

从自然罹病死亡的草原毛虫(Gynephorap ruoergnesis)体内分离到一株产气肠杆菌(Enterobacter aerogenes),它在几丁质的诱导下能产生较高活性的几丁质酶。发酵液经硫酸铵盐析、DEAE纤维素柱层析和Sephadex G-100柱层析分离出几丁质酶。用SDSPAGE测得该酶的分子量为425kD。水解几丁质的Km值为2.88mg/mL-1。酶反应的最适温度为55℃,最适pH值为60,金属离子对几丁质酶活性影响较大,其中Zn2+、Ba2+、Ca2+和Mn2+对酶有较强的激活作用,而Hg2+、Co2+和Mg2+则有较强的抑制作用。     

球孢白僵菌两种胞外几丁质酶的诱导和纯化

球孢白僵菌突变株CH-1316在完全培养基中培养至对数前期后转入以胶体几丁质为唯一碳、氮源的液体诱导培养基中继续培养20～25h,几丁质酶被诱导产生;在对数生长期胞外几丁质酶活力最高。发酵液经(NH_4)_2SO_4沉淀、DEAE-纤维素层析和凝胶过滤分离出二种几丁质酶组分,在聚丙烯酰胺凝胶电泳图上显示出两条均一的带,并且每条带都具有几丁质酶活力。几丁质酶1既是外切酶又是内切酶,而几丁质酶2只表现内切酶活力。分子排阻法测得这两种酶的分子量分别为52000和39000。     

Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation

Fourteen Penicillium strains have been screened on wheat bran–crude chitin mixture medium for extracellular chitinase production in solid-state fermentation. Under the experimental conditions tested, Penicillium aculeatum NRRL 2129 (=ATCC 10409) was selected as the best enzyme producer. The optimum incubation period for chitinase production by the potent organism was found to be 72 h. Chromatofocusing was performed as the first step in the purification scheme, but high amount of contaminating proteins interfered with the method. Hence, ion-exchange chromatography experiments were carried out followed by gel filtration to separate and isolate chitinase isoenzymes. Four major chitinase peaks of molecular weight 82.7, 44.6, 28.2 and 26.9 kDa were observed after gel filtration chromatography while, on SDS-PAGE, three protein bands of molecular weights 82.6, 33.9 and 29.1 kDa were identified. The purified enzyme showed optimal temperature and pH at 50 and 5.5 °C, respectively.     

Purification and Characteristics of Membrane-Bound Chitinase of Anaerobic Ruminal Fungus Piromyces communis OTS1

A membrane-bound chitinase from cell wall fractions of the anaerobic ruminal fungus, Piromyces communis OTS1, was purified by affinity chromatography, gel filtration, and chromatofocusing. The molecular size of the chitinase was estimated by gel filtration to be 42.4 kDa and by SDS-PAGE to be 44.8 kDa, and its pI was 4.4. Activity was inhibited by Hg²⁺ and allosamidin. The activity at 39°C was greatest at pH 6.0. It had an ‘endo’ type action. Solubilization tests indicated that plasmalemma-bound chitinase was held in place by an electrostatic type interaction. Characterization of the membrane-bound chitinase was more similar to that of extracellular chitinase than cytosolic chitinase. This suggested that membrane-bound chitinase was the origin of extracellular chitinase. Received: 15 October 1997 / Accepted: 13 January 1998     

Isolation and partial characterization of immunologically reactive fractions from chitinase digested cell wall of Trichophyton mentagrophytes

Summary Cell wall preparations ofTrichophyton mentagrophytes were digested with chitinase following which various fractions were isolated by ultrafiltration and Sephadex gel filtration. All fractions isolated contained both polysaccharide and peptide material. A correlation was seen between those fractions capable of eliciting immediate and delayed skin reactions in sensitized guinea pigs and those capable of stimulating thein vitro proliferation of lymphocytes taken from sensitized guinea pigs. These immunologically active fractions also developed precipitin lines with antiserum taken from sensitized animals. A low molecular weight fraction was found to be completely reactive immunologically (UM_2(a)), and appeared to have a molecular weight in the range of 2,000–4,000 as assessed by ultrafiltration and gel filtration studies.This work was supported by grant number 3411 from the Medical Research Council of Canada.A. Kh. Al-Rammahy was supported by a scholarship from the Ministry of High Education, Iraq.     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。     

HaSNPV几丁质酶的分离纯化与毒理学研究

利用重组工程菌GS115-pPIC 9k-ChiA 4.0发酵几丁质酶粗液,经(NH₄)₂SO₄盐析、透析、DEAE Sepharose Fast Flow离子交换层析以及Sephadex G-100凝胶过滤层析分离纯化,获得电泳纯的棉铃虫单粒包埋型核型多角体病毒(HaSNPV)几丁质酶,回收率为19.64%,纯化17.74倍;经HaSNPV几丁质酶毒理学研究,通过触杀作用和胃毒作用,几丁质酶使小菜蛾幼虫细胞液化,导致死亡,其作用效果与几丁质酶浓度和作用时间成正比,且胃毒作用大于触杀作用;经小麦根腐、烟草赤星、番茄灰霉和苹果炭疽等植物病原真菌的抑菌实验,表明几丁质酶可以通过酶解真菌细胞壁中的几丁质达到抑菌作用。     

Purification and characterization of chitinase from Streptomyces sp. M-20

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Streptomyces sp. M-20 and purified by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel filtration. No exochitinase activity was found in the culture filtrate. The molecular mass of the purified chitinase was 20 kDa, estimated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed by activity staining with Calcofluor White M2R. Chitinase was optimally active at pH of 5.0 and at 30 degrees C. The enzyme was stable from pH 4 to 8, and up to 40 degrees C. Among the metals and inhibitors that were tested, the Hg(+), Hg(2+), and p-chloromercuribenzoic acid completely inhibited the enzyme activity. The chitinase activity was high on colloidal chitin, chitotriose, and chitooligosaccharide. The purified chitinase showed antifungal activity against Botrytis cinerea, and lysozyme activity against the cell wall of Botrytis cinerea.     

One-step purification of chitinase from Serratia marcescens NK1, a soil isolate

AIMS: A simple single step technique of gel filtration was developed for the purification of chitinase from Serratia marcescens NK1. METHODS AND RESULTS: Chitinase from Ser. marcescens NK1 was purified to homogeneity by gel filtration chromatography with 9.2% recovery. The enzyme had a pH optimum of 6.2 and a temperature optimum of 47 degrees C. It was stable in a wide pH range of 3.0 to 10.0, retaining 60% activity at pH 3.0 and 65% activity at pH 10.5. It retained 70% activity at 28 degrees C after 72 h and nearly 50% activity at 50 degrees C up to 24 h. CONCLUSION: The chitinase from Ser. marcescens NK1 can be efficiently purified in a single step by gel filtration chromatography. The chitinase of Ser. marcescens NK1, a soil isolate, is highly stable and as active as that of other reported isolates of Ser. marcescens. SIGNIFICANCE AND IMPACT OF THE STUDY: This purification scheme is advantageous because of its simplicity and can therefore be applied for the purification of other enzymes. The yield is sufficient for initial characterization studies of the enzyme, and an improved resolution can be obtained if the chromatography is done under fast flow systems.     

Purification and characterization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes

A method was developed to purify a 30-kDa protein from jelly fig (Ficus awkeotsang) pericarp, including preparation of jelly curd from achenes, extraction of proteins from the curd, and isolation of the 30-kDa protein by anion-exchanger and gel filtration. Chitinase activity was detected in the purified 30-kDa protein by activity staining in both non-denaturing gel electrophoresis and SDS-PAGE. Isoelectrofocusing showed that the isoelectric point of the 30-kDa protein was lower than pH 3.5. The K(m), k(cat), optimal pH and temperature of this putative chitinase were determined to be 0.076 mM, 0.089 s(-1), pH 4, and 60 degrees C, respectively. The purified 30-kDa protein was thermostable (retaining activity up to 65 degrees C for several hours) and could be stored at 4 degrees C for a year without apparent loss of chitinase activity. Antifungal activity of this putative chitinase was measured in terms of inhibition of Colletotrichum gloeosporioides spore germination.     

荔枝壳多糖特性研究

多糖是荔枝壳的重要活性成分,本研究采用阴离子交换柱和凝胶过滤柱对荔枝壳多糖进行分离纯化。凝胶渗透色谱测定其分子量为14000 Dal。通过气相色谱测定其单糖组成为甘露糖、半乳糖和少量的阿拉伯糖,分子链由1,2键、1,3键和1,6键组成,不含1,4键。红外光谱分析表明甘露糖以β-D-甘露糖形式存在,不含羧基基团。     

Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae

A novel chitinase was detected in extracellular culture fluids of the entomopathogenic fungus Metarhizium anisopliae (ATCC 20500) grown in liquid medium containing chitin as a sole carbon source. A chitinase was purified to near homogeneity from culture broth of M. anisopliae by DEAE-Sephacel, CM-Sepharose CL-6B ion-exchange chromatography, and gel filtration with Superose 12HR. The molecular mass of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 60 kDa and the optimum pH of the enzyme was 5.0. This molecular mass is different from values of 33, 43.5, and 45 kDa for endochitinases and 110 kDa for an exochitinase (N-acetylglucosaminidase) from M. anisopliae ME-1 published previously. In addition, N-terminal sequences of 60-kDa chitinase are different from those of 43.4- and 45-kDa endochitinases. The purified enzyme showed high chitinolytic activity against colloidal, crystalline chitin of crab shells as well as against p-nitrophenyl-beta-d-N-acetylglucosamide, p-nitrophenyl-beta-d-N, N'-diacetylchitobiose, and p-nitrophenyl-N, N'-N"-triacetylchitotriose, indicating that this enzyme has both endo- and exochitinase activity.     

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.     

A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1

A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose. The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase. The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36. The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.     

Purification and properties of a chitinase from Penicillium oxalicum autolysates

A chitinase (EC. 3.2.1.14) from autolysed culture filtrate of Penicillium oxalicum was purified by precipitation with ammonium sulphate, gel filtration and ion exchange chromatographies. The purified enzyme showed a single protein band in SDS gel electrophoresis. The enzyme is an acidic protein with a pI of 4.5 and has a molecular weight of 54 900 as estimated from SDS gel electrophoresis and 21 500 from gel filtration. The optimum pH and temperature were 5.0 and 35°C, respectively. The enzyme was stable at temperatures up to 45°C and in a pH range between 4.0 and 6.0. The K_m was 2.5 mg ml^-1 for colloidal chitin, Hg²⁺ and Ag⁺ were effective inhibitors. The viscosimetric study carried out using carboxymethyl chitin as substrate revealed the endotype action of this enzyme.     

Immunochemistry of a Formamide-extracted Antigen from Clostridium perfringens Cell Walls

The type-specific antigen of a strain of Clostridium perfringens involved in food poisoning was isolated from the cell wall by the use of hot formamide. The antigen appears to consist of polysaccharide or mucopeptide. The formamide extract was shown to be heterogeneous by gel filtration on Sephadex G-200. The serologically active fraction contained about 25% of the amount of protein present in the original formamide extract. Hexosamine, acetyl groups, and carbohydrate also were detected. The formamide extract showed a high degree of serological activity. The serological activity was increased twofold on Sephadex gel filtration.     

Purification and characteristics of cytosolic chitinase from Piromyces communis OTS1

Abstract A chitinase was purified from the cytosolic fraction of the anaerobic rumen fungus Piromyces communis OTS1 by affinity chromatography using regenerated chitin, gel filtration and chromatofocusing. The chitinase was most active at pH 6.2 and at 60 °C in a 20-min assay. The molecular mass of the purified protein was estimated by SDS-PAGE to be 42 kDa and its pI was 4.9. The enzyme activity, which was of the 'endo' type, was inhibited by A⁺, Hg²⁺ and allosamidin. N -Acetyl- β -glucosaminidase and 'exo' type chitinase activity were absent from the purified preparation.     

聚丙烯酰胺凝胶电泳配合荧光增白剂显色检测木霉几丁质酶同工酶谱

为提高木霉几丁质酶检测方法的准确性和灵敏度,建立一种快速检测几丁质酶同工酶的方法。采用活性凝胶电泳、变性凝胶电泳、原位显色凝胶电泳结合荧光增白剂（Calcofluor white M2R）显色从绿色木霉LTR-2发酵产物中检测几丁质酶同工酶。活性凝胶电泳在粗酶液浓缩5倍时显示两条活性谱带,变性凝胶电泳在浓缩10倍时显示一条活性谱带,原位显色凝胶电泳在浓缩20倍时显示两条不清晰的活性谱带,SDS-PAGE显示这两条活性谱带的分子量分别为65kDa和42kDa。结果表明活性聚丙烯酰胺凝胶电泳和Calcofluor white M2R显色相结合的方法在几丁质酶上样量为0.47U时具有较好的分辨能力,是检测木霉几丁质酶同工酶的有效的方法。     

凝胶过滤法纯化裂褶多糖检测方法的改进

凝胶过滤洗脱液中多糖含量一般采用硫酸-苯酚等化学显色法测定,然后根据洗脱曲线得到纯化的组分,但化学方法费时且消耗试剂.本研究采用350 nm非特征性吸收波长对2种不同纯度裂褶多糖样品PSG1和PSG2的洗脱液直接测定吸光度,与硫酸-苯酚法490 nm测定得到的洗脱曲线基本一致,即分别可得到4个和2个组分,同时采用HPLC对分离效果进行了检测.研究表明,采用350 nm波长直接检测可实现不同纯度裂褶多糖的分离.利用本法检测其他多糖的效果有待进一步研究.