Two glutamic acids in chitosanase A from Matsuebacter chitosanotabidus 3001 are the catalytically important residues.

Chitosanase is the glycolytic enzyme that hydrolyzes the glucosamine GlcN-GlcN bonds of chitosan. To determine the catalytically important residues of chitosanase A (ChoA) from Matsuebacter chitosanotabidus 3001, we performed both site-directed and random mutagenesis of choA, obtaining 31 mutants. These mutations indicated that Glu-121 and Glu-141 were catalytically important residues, as mutation at these sites to Ala or Asp drastically decreased the enzymatic activity to 0.1-0.3% of that of the wild type enzyme. Glu-141 mutations remarkably decreased kinetic constant k(cat) for hydrolysis of chitosan, meanwhile Glu-121 mutations decreased the activities to undeterminable levels, precluding parameter analysis. No hydrolysis of (GlcN)(6) was observed with the purified Glu-121 mutant and extremely slow hydrolysis with the Glu-141 mutant. We also found that Asp-139, Asp-148, Arg-150, Gly-151, Asp-164, and Gly-280 were important residues for enzymatic activities, although they are not directly involved in catalysis. In addition, mutation of any of the six cysteine residues of ChoA abrogated the enzymatic activity, and Cys-136 and Cys-231 were found to form a disulfide bond. In support of the significance of the disulfide bond of ChoA, chitosanase activity was impaired on incubation with a reducing agent. Thus, ChoA from M. chitosanotabidus 3001 uses two glutamic acid residues as putative catalytic residues and has at least one disulfide bond.     

无花果沙雷氏菌CH0203壳聚糖酶的纯化及生化性质研究

目的：得到纯化的无花果沙雷氏菌CH02503的壳聚糖酶,并研究其生化性质。方法：将发酵粗酶液先后通过硫酸铵分级沉淀,superdex75凝胶柱和羧甲基纤维素离子交换柱层析,壳聚糖酶得到纯化。结果：经测定,该酶为内切酶,其相对分子质量为29kDa,等电点9．4,在45℃和pH4．0—7．5之间稳定,最适温度是45％,最适pH3．6,Mn^2＋、Co^2＋能够激活,Pb^2＋、Cu^2＋、Ni^2＋、Cr^3＋能够抑制该酶的活性,该酶最适底物是脱乙酰度85％的壳聚糖,对脱乙酰度低于45％的壳聚糖不能作用,对羧甲基甲壳素和羧甲基纤维素不能作用,以完全脱乙酰的壳聚糖为底物时,最终水解产物是单糖、二糖、三糖,反应的米氏常数为0．44mg／ml。     

Purification and Characterization of Two Types of Chitosanase from a Microbacterium sp.

Two extracellular chitosanases (ChiX and ChiN) were extracted from Microbacterium sp. OU01 with Mr values of 81 kDa (ChiX) and 30 kDa (ChiN). ChiN was optimally active at pH 6.2 and 50°C and ChiX at pH 6.6 and 60°C (assayed over 15 min). Both the activities increased with the degree of deacetylation (DDA) of chitosan. ChiN hydrolyzed oligomers of glucosamine (GlcN) larger than chitopentaose, and chitosan with 62–100% DDA; but ChiX acted on chitosan and released GlcN. Hydrolysis of chitosan with 99% DDA by ChiN released chitobiose, chitotriose and chitotetraose as the major products.     

Purification and Characterization of Three Chitosanase Activities from Bacillus megaterium P1

Bacillus megaterium P1, a bacterial strain capable of hydrolyzing chitosan, was isolated from soil samples. Chitosan-degrading activity was induced by chitosan but not by its constituent d-glucosamine. Extracellular secretion of chitosanase reached levels corresponding to 1 U/ml under optimal conditions. Three chitosan-degrading proteins (chitosanases A, B, and C) were purified to homogeneity. Chitosanase A (43 kilodaltons) was highly specific for chitosan and represented the major chitosan-hydrolyzing species. Chitosanases B (39.5 kilodaltons) and C (22 kilodaltons) corresponded to minor activities and possessed comparable specific activities toward chitosan, chitin, and cellulose. Chitosanase A was active from pH 4.5 to 6.5 and was stable on the basis of activity up to 45 degrees C. The optimum temperature for enzymatic chitosan hydrolysis was 50 degrees C. Kinetic studies on chitosanase A suggest that the enzyme is substrate inhibited. The apparent K(m) and V(max) determined at 22 degrees C and pH 5.6 were 0.8 mg/ml and 280 U/mg, respectively. End products of chitosan hydrolysis by each of the three chitosanases were identified as glucosamine oligomers, similar to those obtained for previously reported chitosanase digestions.     

Purification,Characterization, and Gene Analysis of Cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both β-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the β-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to β-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Gene Cloning, Purification, and Characterization of a Phosphodiesterase from Delftia acidovorans

A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans, the gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 65°C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(p-nitrophenyl)phosphate with a K_m of 2.9 ± 0.1 mM and a k_cat of 879 ± 73 min⁻¹. The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E. coli. The IS1071 transposon insertion sequence was found downstream of pdeA.     

壳聚糖酶的分离纯化及性质研究

以实验室分离的产壳聚糖酶的LS菌株出发,制备壳聚糖酶粗酶液,经(NH_4)_2SO_4盐析、透析、DEAE-Sephadex A25阴离子交换、Sephadex G-100凝胶过滤分离纯化后,SDS-PAGE显示为一条带,壳聚糖酶被纯化了22.4倍,回收率为34.2%。同时对其酶学性质进行了初步研究,发现该酶在低于35℃和pH 6.O～7.5范围内较稳定,最适反应温度为55℃,反应pH 5.0～5.5;Zn~(2 )、Ag~ 、Ca~(2 )、Co~(2 )、Mn~(2 )对该酶有明显的促进作用,而Fe~(3 )、Hg~(2 )对该酶有强烈的抑制作用;该酶的米氏常数(K_m)为2.50 mg/mL,最大反应速度(V_(max))为4.19μmol/(mL·min);SDS-PAGE测定的相对分子质量为30.9×10~3。     

Purification,Characterization and Gene Analysis of N-Acetylglucosaminidase from Enterobacter sp. G-1

Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45°C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetyl-glucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N′-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)₂ or PNP-(GlcNAc)₃.     

Characterization of a Chitosanase from Aspergillus fumigatus ATCC13073

Chitosanase II was purified from the culture filtrate of Aspergillus fumigatus ATCC13073. The purified enzyme had a molecular mass of 23.5 kDa. The N-terminal amino acid sequence of chitosanase II was identical to those of other Aspergillus chitosanases belonging to glycoside hydrolase family 75. The optimum pH and temperature were pH 6.0 and 40 °C. Chitosanase II hydrolyzed 70% deacetylated chitosan faster than fully deacetylated chitosan. Analysis of the degradation products generated from partially N-acetylated chitosan showed that chitosanase II split GlcN-GlcN and GlcNAc-GlcN bonds but not GlcNAc-GlcNAc or GlcN-GlcNAc, suggesting that it is a subclass I chitosanase. It degraded (GlcN)(6) to produce (GlcN)(3) as main product and small amounts of (GlcN)(2) and (GlcN)(4). Reaction rate analyses of mono-N-acetylated chitohexaose suggested that the (+3) site of chitosanase II recognizes the GlcNAc residue rather than the GlcN residue of its substrate.     

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K_m values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K_i and K_i′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn²⁺ or Co²⁺ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH₂-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.     

芽孢杆菌Bacillus sp. S-1壳聚糖酶基因的克隆与序列分析

从连云港海滩晒虾蟹壳的泥土里筛选出一株产壳聚糖酶能力较高的菌株S-1,根据其形态特征、生理生化以及16S rDNA鉴定,初步认定该菌为芽孢杆菌属（Bacillus）。利用NCBI数据库中已经报道的Bacillus壳聚糖酶序列设计兼并引物,以菌株Bacillus sp. S-1的基因组DNA为模板进行聚合酶链式反应（PCR）,克隆到壳聚糖酶基因的部分序列;利用Clontech公司Universal GenomeWalker试剂盒构建该菌株的基因组步移文库,根据已测定的序列信息设计特异性引物,结合两步法PCR技术分别克隆两端未知序列,拼接获得壳聚糖酶基因的全长序列（该基因全长1362 bp编码453个氨基酸,注册号：EU924147）,并对该序列进行了生物信息学方面的分析。     

牡蛎中糖蛋白成分的分离纯化及其性质研究

本文以青岛产牡蛎为原料,通过低温水提取工艺得到牡蛎糖蛋白粗提物,然后用凝胶柱层析(Sephacryls-100 HR)进行纯化,最后用高效液相色谱制备得到纯度较高的物质F22.由SDS-聚丙烯酰胺凝胶电泳证实F22是纯度较高的单一组分;分子量为34.2 kDa;等电聚焦的结果显示等电点为5.5;红外色谱呈现出典型的糖的特征吸收峰;气相色谱分析结果显示F22的中性单糖是由葡萄糖这一种单糖组成的同多糖;β-消去反应表明F22的糖肽键应为N-型糖苷键.     

Purification,Characterization, and Antiproliferative Activity of a Single-Chain Lectin from Vicia palaestina (Fabaceae) Seeds

Vicia palaestina Boiss. is an annual herb that grows in dry areas of eastern Mediterranean countries. It belongs to section Cracca subgenus Vicilla, which is characterized by having a high content in the non-protein amino acid canavanine. The seeds from some of these vetches are also rich in lectins. The purification and characterization of a single-chain lectin from the seeds of V. palaestina is described here. This lectin was the most abundant protein in albumin extracts. It has affinity for the glycoconjugate N-acetylgalactosamine and inhibits proliferation of the cancerous Caco-2 and THP-1 cell lines. In addition to their high nutritional value, the seeds from V. palaestina represent a source of lectins with health promoting and pharmacological potential because of their antiproliferative activity.     

纤维素酶中具有壳聚糖水解酶活性成分的鉴定

在壳聚糖酶的研究过程中,目前已发现37种酶具有非专一性地降解壳聚糖的能力[1].对这些非专一性酶水解壳聚糖的机理有两种看法:一些人认为,由于这些酶大都来自商业酶制剂,未经过进一步的纯化,故有人认为其中所含的少量杂质可能是产生水解活力的原因;但也有人认为,在所有的酶制剂中都存在同一种杂质似乎是不可能的,因为这些酶来源于广泛的微生物、真菌、哺乳动物和植物等.众所周知,酶具有高度的专一性,即对所催化的反应和底物有严格的选择性,一种酶往往只能催化一种或一类反应;有如此多的不同种类的酶能非专一性地水解壳聚糖.因而探讨具有水解…     

Purification and Characterization of a Lectin from Chinese Quince (Chaenomeles sinensis) Fruit

Chaenomeles sinensis lectin (CSL) was isolated from Chinese quince fruit by affinity chromatography. The molecular weight of native CSL was estimated to be 16 kD by gel filtration chromatography. This lectin was found to contain approximately 57% carbohydrates. The molecular weight of deglycosylated CSL was estimated to be 3.6 kD by tricine-SIDS-PAGE under reduced conditions. Our results suggest that CSL may be a homodimer. The hemagglutinating activity of CSL was inhibited by N-acetyllactosamine and chicken ovalbumin, and it was drastically decreased at pH levels of 9.0 or greater. CSL may be a useful tool for the purification of glycoconjugates.     

热带假丝酵母酰基辅酶A氧化酶的纯化及性质研究

利用热带假丝酵母由烷烃生产二元酸时,二元酸面临被β氧化降解的代谢途径。酰基辅酶A氧化酶是二元酸β氧化的限速酶。以热带假丝酵母1230菌株为材料,经硫酸铵分级沉淀、阴离子交换柱层析、BlueSepharose亲和柱层析,得到电泳均一的酰基辅酶A氧化酶。该酶有两种亚基,分子量分别为74kD和78kD。酶作用最适pH和最适温度分别为80和50℃。金属离子Ag+、Pb2+完全抑制酶活性,Ba2+、Mg2+、Ca2+对酶活性有明显抑制作用。丙烯酸是酶的反竞争性抑制剂,Ki为0633mmol/L,维生素C是竞争性抑制剂,Ki为2.01×10-3mmol/L。