排序方式: 共有36条查询结果,搜索用时 15 毫秒
21.
烟曲霉菌壳聚糖酶基因的克隆及在大肠杆菌中的表达 总被引:3,自引:0,他引:3
根据GenBank中发布的烟曲霉菌壳聚糖酶(Aspergillus fumigatus chitosanase,EC3.2.1.132)基因序列人工合成8条DNA长链及4条引物链。DNA链的设计上在不改变壳聚糖酶氨基酸组成的前提下选择大肠杆菌使用频率高的密码子。PCR拼接法扩增壳聚糖酶基因并克隆入pGEM_T easy载体进行序列分析,进一步亚克隆入表达载体pGEX_3X。重组质粒pGEX_Csn转化E.coli DH5α,IPTG诱导表达,亲和层析及Factor Xa酶解纯化重组Csn。所得重组壳聚糖酶具有降解壳聚糖的生物活性,其活性受温度及pH值的影响。 相似文献
22.
Fuguet R Théraud M Vey A 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2004,138(2):149-161
The production of macromolecular insecticidal toxins in Adamek's medium by two selected strains of Beauveria bassiana was investigated. Filtrates and dialysates of the melanizing strain 618 were toxic when injected into the lepidopteran insect Galleria mellonella. Separation by DEAE chromatography revealed that peaks eluted respectively with 100 and 200 mM NaCl (P 100 and P 200) had an insecticidal activity and induced cuticular blackening. A hydrophilic protein, Bclp, which causes the formation of brownish spots of the integument, was purified from P 200 by means of chromatographic and electrophoretic methods. Bclp exhibited clear sequence homologies with fungal chitosanases of Fusarium solani. It has a molecular mass of 28 kDa, a pHI of 4 and is thermolabile. Injection of Bclp causes the same cytoxic effects and alterations of the cuticule as those observed during mycosis, and may contribute to the virulence of strain 618. Comparatively, the most obvious characteristic of the weakly melanizing strain 101 is the lack of significant toxic activity of its P 200, which does not contain Bclp. However, this strain secretes other insecticidal molecules active on lepidopterans, presently unidentified. 相似文献
23.
A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 × His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria–Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap™ FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS–PAGE analysis. Detected by 4700 MALDI-TOF–TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito. 相似文献
24.
We have developed a culture system for efficient production of chitosanase by Bacillus sp. TKU004. TKU004 was cultivated by using squid pen powder as the sole carbon/nitrogen source. The effects of autoclave treatments of the medium on the production of chitosanase were investigated. Autoclave treatment of squid pen powder for 45 min remarkably promoted enzyme productivity. When the culture medium containing an initial squid pen powder concentration of 3% was autoclaved for 45 min, the chitosanase activity was optimal and reached 0.14-0.16 U/mL. In addition, extracellular surfactant-stable chitosanase was purified from the TKU004 culture supernatant. The antioxidant activity of TKU004 culture supernatant was determined through the scavenging ability of DPPH, with 70% per mL. With this method, we have shown that marine wastes can be utilized efficiently through prolonged autoclave treatments to generate a high value-added product, and have revealed its hidden potential in the production of functional foods. 相似文献
25.
DNA shuffling was carried out with two chitosanase genes belonging to glycoside hydrolase family eight from Bacillus cereus KNUC51 and B. cereus KNUC55. The shuffled products, YM18 and YM20, which showed higher activity than the parents at 40°C, were selected for further
studies. The 50 kDa chitosanases were purified using affinity chromatography with glutathione-Sepharose 4B. In general, the
specific activity of YM18 is enhanced 250% and that of YM20 is 350% compared to the parents. YM20 exhibits a shift of the
optimal pH level from 5.5 to 6.5. DNA sequence analysis revealed that YM18 and YM20 contained 2 amino acid substitutions (I13T
and A87V for YM18; K66R and N352S for YM20). We presumed that these amino acid substitutions increase the specific activity
and change the property of the two variants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
26.
Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation 总被引:1,自引:0,他引:1
Abstract From autolysed cultures of Mucor rouxii , two chitosanases, A and B, were purified to electrophoretic homogeneity. Apparent M r values of 76 000 and 58 000 and p I values of 4.9 and 4.7 were determined for A and B, respectively. Both chitosanases showed a high specificity for chitosan and chitosan derivatives. They had optimum activities at pH 5.0 and at temperatures of 55°C and 50°C for A and B, respectively. Enzyme A was inhibited by acetate ions and enzyme B by high substrate concentration. Both enzymes showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of dimer, trimer and higher molecular mass oligomers of glucosamine. Glucosamine oligosaccharides were poorly hydrolysed by these enzymes. Both enzymes extensively degraded the chitosan extracted from M. rouxii cell walls. 相似文献
27.
Trimukhe KD Bachate S Gokhale DV Varma AJ 《International journal of biological macromolecules》2007,41(5):491-496
This paper investigates the behavior of crosslinked chitosans and metal-complexed crosslinked chitosans under similar hydrolytic conditions. Crosslinked chitosans with trimellitic anhydride, diisocyanatohexane, and dibromodecane as crosslinking agents under heterogenous reaction conditions were used as metal complexing agents by equilibrating them with metal salts such as ZnCl2, MnSO4, CuSO4, CdSO4, Pb(NO3)2, and HgCl2. Crosslinked chitosan without metal complexation had the same hydrolytic behavior as uncrosslinked chitosan. However, when the crosslinked chitosans were complexed with metals, their rates of hydrolysis and extent of hydrolysis were significantly reduced. Thus, while for chitosan about 840 μg/ml reducing sugar was produced in 4 h time, and 780 μg/ml was produced for diisocyanatohexane crosslinked chitosan, only 400 μg/ml and 320 μg/ml reducing sugars were produced for cadmium sulfate with crosslinked chitosan and diisocyanatohexane crosslinked chitosan, respectively. Similar results are obtained for other crosslinking agents. Studies on preincubation of the metal with the enzyme show that of the metals studied, Mn has no effect on preincubatioin with the enzyme, Hg, Cd, Pb, and Cu completely deactivates the enzyme, while Zn reduces the enzyme activity by about 43.3%. Preincubation of the metal salts with the chitosan shows that Hg and Cu completely deactivate the molecule from enzyme hydrolysis, Cd and Zn inactivate it to the extent of 56.8% and 43.3%, respectively, while Mn has no effect. Availability of the amino functions seems to be a key feature for the chitosanase to hydrolyze the chitosan polymer. This was also proved by the significant increase in the extent of hydrolysis for chitosan samples with 88% (final value 1120 μg/ml reducing sugar) and 85% deacetylation (final value 840 μg/ml reducing sugar). HPIC studies of the products show that a variety of oligomers are produced in the chitosanase enzyme hydrolytic reaction. 相似文献
28.
几丁质酶和壳聚糖酶对部分乙酰化壳聚糖作用方式的比较 总被引:9,自引:0,他引:9
通过对几丁质酶和壳聚糖酶降解部分乙酰化壳聚糖的作用方式的比较,得到几丁质酶切断壳聚糖的GlcNAc- GlcNAc 和GlcNAc- GlcN 或GlcN- GlcNAc 糖苷键,而壳聚糖酶切断壳聚糖的GlcN- GlcN 和GlcN- GlcNAc 或GlcNAc- GlcN 糖苷键,为得到较高聚合度的壳寡糖提供理论基础。 相似文献
29.
Masakazu Ike Yohane Ko Kengo Yokoyama Jun-Ichi Sumitani Takashi Kawaguchi Wataru Ogasawara Hirofumi Okada Yasushi Morikawa 《Journal of Molecular Catalysis .B, Enzymatic》2007,47(3-4):159-163
Cellobiohydrolase CBH I (Cel7A) from the filamentous fungus Trichoderma reesei (TrCBHI), which is a member of glycoside hydrolase family (GHF) 7, was expressed in Aspergillus oryzae. We found that the recombinant enzyme showed significant chitosanase activity, as well as cellulase activity, and acted in an endo-type manner on soluble polymeric substrate. Furthermore, another GHF7 CBH I from Aspergillus aculeatus (AaCBHI) expressed in A. oryzae also had chitosanase activity, while endoglucanase EG I (Cel7B) from T. reesei had no activity towards chitosan. To our knowledge, this is the first report of GHF7 enzymes possessing chitosanase activity. 相似文献
30.
A chitosanase was purified from the culture supernatant of Serratia marcescens TKU011 with shrimp shell wastes as the sole carbon/nitrogen source. Zymogram analysis revealed the presence of chitosanolytic activity corresponding to one protein, which was purified by a combination of ion-exchange and gel-filtration chromatography. The molecular weight of the chitosanase was 21 kDa and 18 kDa estimated by SDS–PAGE and gel-filtration, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase were 5, 50 °C, pH 4–8, and <50 °C, respectively. The chitosanase was inhibited completely by EDTA, Mn2+, and Fe2+. The results of peptide mass mapping showed that three tryptic peptides of the chitosanase were identical to a chitin-binding protein Cbp21 from S. marcescens (GenBank accession number gi58177632) with 63% sequence coverage. 相似文献