The role of carboxyl groups on the chitosanase and CMCase activity of a bifunctional enzyme purified from a commercial cellulase with EDC modification

The carboxyl groups of the bifunctional cellulase–chitosanase (CCBE), purified from a commercial cellulase prepared from Trichoderma viride were modified using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC). The EDC modified CCBE lost 80–90% of its chitosnase activity and 20% of its carboxylmethyl cellulase (CMCase) activity; meanwhile, its conformation changed slightly, which altered the substrate binding affinity to chitosan, without affecting its binding to CMC. However, the modification did not alter the structure integrity. The dynamic analysis of modification indicated that the CCBE possessed two carboxylates essential for its chitosanase activity and one carboxyl group for its CMCase activity. One of the two carboxylates involved in chitosanase activity was deduced to be the proton donator, and the other may function for substrate recognition, while the only catalytic carboxyl group for CMCase activity probably also acted as a proton donator.     

Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production

Recently, considerable attention has been focused on chitosan oligosaccharides (COSs) due to their various biological activities. COSs can be prepared by enzymatic degradation of chitosan, which is the deacetylation product of chitin, one of the most abundant biopolymers in nature. In the current study, we recombinantly expressed a chitosanase and used it for COS preparation. A bacillus-derived GH8 family chitosanase with a 6×His tag fused at its N-terminal was expressed in the Escherichia coli strain BL21(DE3) as a soluble and active form. Its expression level could be as high as 500 mg/L. Enzymatic activity could reach approximately 140,000 U/L under our assay conditions. The recombinant chitosanase could be purified essentially to homogeneity by immobilized metal-ion affinity chromatography. The enzyme could efficiently convert chitosan into monomer-free COS: 1 g of enzyme could hydrolyze about 100 kg of chitosan. Our present work has provided a cheap chitosanase for large-scale COS production in industry.     

腐皮镰孢菌壳聚糖酶的酶学性质研究及其在酿酒酵母工业菌株中的表达

摘要：【目的】本研究旨在了解腐皮镰孢菌（Fusarium solani）壳聚糖酶的基本酶学性质及其在壳寡糖生产中的应用,构建能高效分泌表达壳聚糖酶的酿酒酵母工业菌株。【方法】采用RT-PCR扩增腐皮镰孢菌壳聚糖酶的cDNA序列;通过组氨酸标签,纯化得到E. coli表达的重组壳聚糖酶,并进行基本酶学性质研究;以薄层层析、高效液相色谱等技术对该酶的酶解产物进行分析;通过马克斯克鲁维酵母（Kluyveromyces marxianus）菊粉酶信号肽（INU1A）实现壳聚糖酶在酿酒酵母工业菌株N-27中的分泌表     

Mutation breeding of chitosanase-producing strain Bacillus sp. S65 by low-energy ion implantation

In order to obtain an industrial strain with higher chitosanase yield, the wild strain Bacillus sp. S65 cells were mutated by a novel mutagen, nitrogen ion beam, with energy of 15 keV and dose ranging from 2.6 × 10¹⁴ to 5.2 × 10¹⁵ ions/cm². One mutant, s65F5 with high yield of chitosanase was isolated. Results showed that the production of chitosanase of s65F5 was dramatically increased from 4.1 U/ml in s65 to 25 U/ml by ion beam implantation, while the fermentation time was shortened from 72 to 56 h, both of which greatly increased efficiency and reduced the cost of industrial production. Besides, the mutagenic effects of low-energy ion beam on survival rate showed characteristic down–up–down pattern, which was different from the traditional mutagens such as UV and γ-ray and the possible mutation mechanism was discussed.     

产壳聚糖酶菌株的初步筛选

通过大量的筛选,获得了产壳聚糖酶较好的菌株Y2、Y4、Y8。其发酵液所产壳聚糖酶的酶活力分别为2．0U／ml,2．1U／ml,2．2U／ml。     

土壤中产壳聚糖酶真菌的筛选

在以壳聚糖（Chitosan）为唯一碳源和氮源的培养基上,利用透明圈筛选法,从不同地区采集的105份土样中,共分离到26株产生透明圈较大的菌株,结合酶活力测定结果,获得产酶能力较高的菌株HF-1、HF-5,为进一步的研究提供了条件。     

壳聚糖酶

<正>壳聚糖(Chitosan)是由N-乙酰葡糖胺(GlcN Ac)和葡糖胺(GlcN)通过β-1,4糖苷键相连接的多糖,与甲壳素、壳寡糖均被称为甲壳素类物质,被誉为继糖类、蛋白质、脂肪、维生素等生命元素之外的第六大生命物质。壳寡糖由于其生物相容性好、易于被人体吸收等优势,其商业产品已遍布各个领域,主要涉及功能性食品、药品、环保、化工等。通过化学方法降解壳聚糖制备壳寡糖的方法具有污染环境、产物不均一等缺点。利用生物酶法     

海洋链霉菌Streptomyces olivaceus FXJ7.023来源多功能几丁质酶的克隆、表达及鉴定(英文)

碳水化合物水解酶家族在自然界碳素循环及农业废弃物中几丁质、纤维素等碳水化合物的生物质转化利用中发挥了重要作用。通过PCR技术从海洋链霉菌Streptomyces olivaceus strain FXJ 7.023的fosmid基因组文库中成功克隆得到1个全长885bp的编码295个氨基酸残基的包含1个19个氨基酸残基的N-末端信号肽的壳聚糖酶完全编码区。系统进化分析表明该基因编码蛋白与已报道的Streptomyces sp.SirexAA-E来源壳聚糖酶csnA同源性为71%,与Streptomyces coelicolor A3(2)来源的csn46A同源性为70%。将该编码区重组入原核表达质粒载体pET32a并转化大肠杆菌表达菌株BL21(DE3)plysS,添加IPTG在18℃条件下振荡诱导该蛋白表达,Ni2+-NTA亲和纯化获得分子量为50.3 kDa融合表达蛋白TrxA-SoCsn。该融合重组蛋白在最适反应条件下对底物胶体壳聚糖和羧甲基纤维素的最大酶活分别为3.673U/mg和1.302U/mg,最适反应温度分别为37℃和50℃,最适反应pH分别为pH5.0和pH6.0。TrxA-SoCsn相关的研究结果表明该酶在农业废弃物生物质转化等方面具有一定的应用潜力。     

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.     

Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by <Emphasis Type="Italic">Trichoderma</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>

Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40–50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds) and T. harzianum (1.06 IU/gds).