高产漆酶平菇的筛选及其在降解黄曲霉毒素B1中的应用

通过选择培养基平板培养法和液体发酵培养法筛选得到2株高产漆酶的平菇菌株P1和P2,并对平菇菌株产漆酶的培养基进行筛选,得到产漆酶的最适培养基为最低盐MSM培养基。菌株P1不仅产漆酶能力最高,而且降解黄曲霉毒素的能力也最好。在MSM培养基中培养10d时,产漆酶量高达769.44U/L,在800μl的反应体系中,790μl粗酶液可以将1000ng黄曲霉毒素B₁降解到222.62ng,降解率为77.74%,并且平菇粗酶液降解黄曲霉毒素B₁的能力与其中漆酶的含量呈一定的正相关性。     

曲霉Aspergillus sojae壳聚糖酶的诱导及特性

曲霉(Aspergillus sojae)在Czapek-Dox培养基中加入2％N-乙酰氨基葡糖、1％蛋白胨和0．5％酵母膏，26℃、150r／min摇瓶培养下，在60h诱导产生较强的壳聚糖酶活性(达12．34mU／mL)，同时形成了4种壳聚糖同工酶，CHA1、CHA2、CHA3和cHA4，蛋白质分子量约为38．2、30．8、27．7和25．0kD。对诱导的壳聚糖酶部分特性初步研究表明，该酶属于壳聚糖内切酶，不能分解胶体几丁质、乙二醇几丁质、干粉几丁质和羧甲基纤维素，但能随乙酰化程度的增加有效分解壳聚糖。利用薄层层析表明，该酶可将壳聚糖分解得到的产物为壳聚二糖。     

一株产高温蛋白酶耐热菌BY25的产酶条件与酶学性质研究

对菌株BY25的生长条件、产酶条件及其产生的蛋白酶的酶学性质进行了研究。结果发现,BY25的最高生长温度为55℃,最适生长温度为50℃,最佳产酶温度为30℃,最佳培养起始pH为8.0,最佳C源为葡萄糖,高通气量明显提高菌株产酶能力。在以上条件下培养52 h,上清液蛋白酶活力达4 170 U/ml。酶学性质研究表明：该蛋白酶为高温中性金属蛋白酶,最适反应pH为7.0,最适反应温度为55℃,具有良好的pH耐受性和较好的热稳定性;EDTA能强烈抑制酶活力,而Fe2+、Cu2+对酶活力也具有一定抑制作用。     

胰蛋白酶对壳聚糖的降解研究

用粘度法和吸光度法，研究胰蛋白酶非专一性降解壳聚糖过程中温度、PH值、反应时间、酶浓度、底物浓度。金属离子对胰蛋白酶降解壳聚糖反应的影响，确定了以壳聚糖为底物的胰蛋白酶的一些催化特性：最适温度为30℃，最适PH值为5.0,10-180min内酶反应速度恒定，酶浓度在0.1-0.5g/L的范围内，酶反应速度与酶浓度呈线性关系，米氏常数Km为9.54g/L。     

响应面法优化白腐菌Pleurotus eryngii-Co007产木质素降解酶条件

为提高Pleurotus eryngii-Co007产木质素降解酶能力,考察了初始pH、秸秆浓度、Cu2+浓度、吐温-80含量对其产木质素降解酶的影响.采用单因素试验和响应面分析法的Central Composite进行试验设计,得到Pleurotus eryngii-Co007产木质素降解酶的最佳条件:培养基初始pH...     

纤维素酶高产菌株的诱变选育及产酶条件研究

本研究通过γ射线照射和亚硝基胍交替处理 ,诱变出一株纤维素酶高产菌株T80 1 ,与出发菌株相比 ,其产酶能力提高 1 77倍。通过对诱变菌株产纤维素酶条件研究发现 ,以稻草粉为碳源、蛋白胨为氮源时 ,菌株产酶最高。该菌株产酶最适培养温度为 2 8℃～ 30℃ ,最适培养 pH为 4 8～ 5 0 ,在此条件下发酵五天达到产酶高峰。该诱变株产酶能力高于国内外一些已知的纤维素酶高产菌株如QM941 4等 ,具有重大的实用价值。     

一株烟草秸秆降解菌的分离、鉴定及酶学性质研究

从皖南地区烟稻轮作田土壤中经CMC-Na初筛获得5株纤维素降解菌,经DNS法测定纤维素酶活性复筛得到一株降解活性较高的降解菌YC-2。根据该菌16S r DNA序列比对结果,结合形态和生理生化特征,确定YC-2为枯草芽孢杆菌(Bacillus subtilis)。对YC-2酶学性质进行相关研究,并分析YC-2对烟碱的耐受情况及对烟草秸秆的降解情况,结果表明:在7天内,YC-2对烟草秸秆降解率为10.14%;酶学特性表现为最适反应温度为60℃且在15~60℃之间具有稳定性;最适反应pH为7.0,在pH 4.0~7.5范围内稳定性较好;YC-2在浓度为1~2 g/L烟碱中能快速生长,而在高浓度的烟碱中生长受到抑制。因此,菌株YC-2产纤维素酶活性较高、相对耐热耐碱且对烟杆有一定分解作用,通过进一步诱变选育和发酵条件优化有较好的田间应用潜力。     

聚丙烯二氧化钛负载膜固定化农药降解酶的研究

利用聚丙烯负载二氧化钛膜固定农药降解酶(EC3.1. 8. 2)，研究了酶固定化的条件，选择农药甲基对硫磷进行了降解试验。结果表明，酶固定化最佳时间为1.5 h，最适固定温度为20℃，最佳固定化酶液浓度为1.6 mg/mL。吸附农药降解酶的聚丙烯负载二氧化钛酶膜，通过与游离酶的比较，固定化酶最适反应温度有所偏移，提高了5℃，酶降解农药的最适pH值为8.5，对农药甲基对硫磷的降解率30 min内达到70%以上，并且固定后的二氧化钛酶膜具有良好的稳定性。     

产β-葡萄糖苷酶的菌种的筛选,鉴定及其酶学特性

β-葡萄糖苷酶来源广泛,几乎存在于所有的生物体中,而不同来源的β-葡萄糖苷酶其性质也各不同。本文利用七叶苷分离培养基从土样中分离筛选出产6种β-葡萄糖苷酶时间较快的菌种,其中发现菌种WGEA1酶活性较高,随后对菌种WGEA1进行初步的鉴定并且采用DNS法测该菌株所产粗酶液的酶学特性。酶学特性表明,WGEA1产的β-葡萄糖苷酶最适温度是在50～55℃之间,最适pH在6～7之间;在低于50℃条件下,pH为5～8时,酶活较稳定,同时在最适反应时间30min下,金属离子和有机溶剂都对酶活性影响很大,这些发现都为在非水相体系中酶法合成烷基糖苷奠定了一定的基础。     

秸秆纤维素降解真菌QSH3-3的筛选及其特性研究

为了获得高效降解秸秆纤维素的微生物菌株,采用滤纸降解法和刚果红染色法从含纤维素类物质的土壤中筛选到一株产纤维素酶菌株QSH3-3,通过形态观察和ITS序列分析,鉴定为草酸青霉Penicillium oxalicum QSH3-3。摇瓶产酶试验结果表明,该菌株的最佳产酶条件为:碳源为0.5%的碱处理过的玉米秸秆粉,氮源为0.2% 硫酸铵,起始pH为7,接种量为5%,产酶温度为30℃,培养时间为4 d。最佳产酶条件下,滤纸酶（FPase）、内切酶（CMCase）和木聚糖酶（Xylanase）分别为12 U、33 U、605 U（U为酶活性单位）;在15℃,其残余酶活力可达70%～80%;在pH 4～9 范围内,其残余酶活力可达70%以上。酶学稳定性研究表明,FPase、CMCase和Xylanase在pH 4～9范围残余酶活力达85%以上,具有较强的酸碱适应能力;FPase、CMCase和Xylanase在45℃以上酶活力迅速下降,耐热性较差。该菌株具有较高的木聚糖酶活力以及较强的低温、pH的耐受力,因而该菌株在田间温差大、土壤偏碱性等复杂条件下对秸秆纤维素类物质的降解具有较高的应用潜力。     

Performing a three-step process for conversion of chitosan to its oligomers using a unique bipolar membrane electrodialysis system

Chitosan, a linear polysaccharide composed of beta-1,4 linked d-glucosamine residues, can be depolymerized into oligomers by enzymatic reaction with chitosanase. Recently, bipolar membrane electrodialysis (BMED) has been used for chitosan solubilization and for terminating the enzymatic reaction by action of electrogenerated acid and base, respectively. The aim of the present study was to test a complete "3-in-1" process using a three-compartment BMED configuration to perform simultaneously the solubilization of chitosan, the inactivation of chitosanase, and the demineralization of the oligomers. In addition, the BMED process was compared to a conventional process using chemical acid and base. The BMED method was found to be as effective as the conventional method for solubilizing the chitosan and for inactivating the chitosanase. Furthermore, the use of BMED allowed a demineralization rate of 53% of the chito-oligomer solution in the diluate compartment. A global process of chitosan hydrolysis into its oligomers using a BMED system was proposed. This technology has great potential for industrial application in chitosan oligomer preparation, because it is convenient and ecological and it produces chito-oligomers with a lower mineral content compared with the conventional method.     

Characterization of a chitosanase isolated from a commercial ficin preparation

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).     

Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain

A hydrolase with chitinase and chitosanase activity was purified from commercial stem bromelain through sequential steps of SP-Sepharose ion-exchange adsorption, HiLoad Superdex 75 gel filtration, HiLoad Q Sepharose ion-exchange chromatography, and Superdex 75 HR gel filtration. The purified hydrolase was homogeneous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and 4-methylumbelliferyl beta-D-N,N',N' '-triacetylchitotrioside [4-MU-beta-(GlcNAc)(3)] and chitosanase activity for chitosan hydrolysis. For glycol chitin hydrolysis, the enzyme had an optimal pH of 4, an optimal temperature of 60 degrees C, and a K(m) of 0.2 mg/mL. For the 4-MU-beta-(GlcNAc)(3) hydrolysis, the enzyme had an optimal pH of 4 and an optimal temperature of 50 degrees C. For the chitosan hydrolysis, the enzyme had an optimal pH of 3, an optimal temperature of 50 degrees C, and a K(m) of 0.88 mg/mL. For hydrolysis of chitosans with various N-acetyl contents, the enzyme degraded 30-80% deacetylated chitosan most effectively. The enzyme split chitin or chitosan in an endo-manner. The molecular mass of the enzyme estimated by gel filtration was 31.4 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 5.9. Heavy metal ions of Hg(2+) and Ag(+), p-hydroxymercuribenzoic acid, and N-bromosuccinimide significantly inhibited the enzyme activity.     

Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain s65

For the production of oligosaccharides from chitosan, a chitosanase-producing bacterium, S65, was isolated from soil. On the basis of phylogenetic analysis of the 16S rDNA gene sequence and phenotypic analysis, S65 was identified as a Bacillus sp. strain. This bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. S65 chitosanase was homogeneously purified by DEAE Sepharose fast flow anion exchange followed by Superdex 75 size exclusion, and the molecular weight was 45 kDa according to SDS-PAGE. Enzyme analysis showed that the optimum pH and temperature of S65 were 6.0 and 65 degrees C, respectively. Catalytic activity was stable from pH 5.5-6.5 at temperatures below 40 degrees C, and the pI of chitosanase was about 6.0 as determined by a test tube method. S65 chitosanase degraded carboxymethyl cellulose (CMC) at the degree of about 5.3% relative to the value of soluble chitosan, but it cannot hydrolyze colloidal chitin and crystalline cellulose. Gene encoding was cloned and sequenced. The deduced amino acid sequence of the S65 exhibited the highest homology to those of family 8 glycanase, suggesting that the enzyme belonged to family 8.     

壳聚糖酶解产物抑制真菌活性研究

为探究并提高壳聚糖抑制真菌的活性,本研究利用蜡状芽孢杆菌ncps116发酵所制备的壳聚糖酶来酶解壳聚糖,测定不同酶解时间壳聚糖产物的抑菌活性,并监测酶解产物中还原糖的含量;之后选取抑菌活性提高最显著的壳聚糖酶解产物为研究对象,测定最低抑菌活性和最适pH,并使用电子显微镜观察酶解产物对病原真菌菌丝和孢子萌发的抑制作用。结果表明,壳聚糖经0.5~12 h酶解能够显著提高市售壳聚糖的抑菌活性,在酶解24 h内抑菌活性呈先升高后降低的趋势,而且酶解产物的抑菌活性与还原糖浓度相关,还原糖浓度过低(≤44.24 μg·mL^-1),酶解不充分,抑菌圈直径13 mm;而还原糖浓度过高(≥1 900 μg·mL^-1),酶解完全,抑菌圈直径10 mm,抑菌活性均较差。其中,酶解6 h的壳聚糖产物抑菌活性提高最显著,对3种病原真菌(棉花黄萎病菌、苹果轮纹病菌、西瓜专化型尖孢镰刀菌)的最低抑菌活性提高4~8倍,还原糖浓度为383.34 μg·mL^-1,在pH值4.0~4.7时抑菌活性最高;此外,酶解产物能导致真菌菌丝断裂、褶皱、菌丝末端囊泡等畸形生长,并长期抑制孢子生长和菌丝伸长。综上所述,酶解是提高壳聚糖抗菌活性的有效手段,这为推进亮聚糖在防腐保鲜方面的应用奠定了基础。     

Characterization of three chitosanase isozymes isolated from a commercial crude porcine pepsin preparation

Three chitosanases designated PSC-I, PSC-II, and PSC-III were purified from commercial pepsin preparation by sequentially applying pepstatin A-agarose affinity chromotography, DEAE-Sephacel ion-exchange chromatography, Mono Q column chromatography, and Mono P chromatofocusing. With respect to chitosan hydrolysis, the optimal pHs were 5.0, 5.0, and 4.0 for PSC-I, PSC-II, and PSC-III, respectively; optimal temperatures were 40, 40, and 30 degrees C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were approximately 40 kDa, as estimated by both gel filtration and SDS-PAGE, and the isoelectric points were 4.9, 4.6, and 4.4, respectively, as estimated by isoelectrofocusing electrophoresis. All three chitosanase isozymes showed activity toward chitosan polymer and N,N",N' "-triacetylchitotriose oligomer. Most effectively hydrolyzed were chitosan polymers that were 68-88% deacetylated.     

Hydrolysis of nitriles using an immobilized nitrilase: applications to the synthesis of methionine hydroxy analogue derivatives

Mild and selective hydrolysis of a large range of nitriles leading to carboxylic acids was achieved under neutral conditions by an immobilized and genetically modified enzyme preparation from Alcaligenes faecalis ATCC8750. This immobilized nitrilase has been shown to be an effective catalyst for the stereoselective hydrolysis of mandelonitrile 1a to R-(-)-mandelic acid 1c. This method is particularly useful for the production of hydroxy analogues of methionine derivatives 2c-4c that could have an interest in cattle feeding and for the transformation of compounds containing other acid- or base-sensitive groups 3a-10a. A series of aliphatic dinitriles 11a-15a was hydrolyzed to the corresponding cyano acids. The suitability of the immobilized catalyst as a robust and versatile biocatalyst is discussed, and models to account for the stereoselectivity of the enzymic hydrolysis have been proposed.     

微波辅助合成壳聚糖基高吸水性树脂

在水溶性引发剂过硫酸钾(KDS)的引发下,用微波辐照使丙烯酸在壳聚糖分子链上接枝聚合,并加入N,N’-亚甲基双丙烯酰胺进行适度交联,制备高吸水性树脂。利用FT-IR对产物结构进行定性表征,结果表明,丙烯酸在壳聚糖的分子链上发生了接枝聚合反应。研究了反应条件对产物吸液性能的影响,并通过正交试验对工艺条件进行优化。在最佳条件下合成产物的吸水倍率为815.0g/g,吸生理盐水倍率为72.2g/g,吸人工尿液倍率为67.5g/g。在微波作用下产物合成速率是传统方法的数十倍,吸液性能明显高于后者,且操作条件容易控制,后处理步骤明显简化,无污染,是一种高效的清洁生产工艺。     

Evaluation of chitosan and alginate immobilized Methylobacterium oryzae CBMB20 on tomato plant growth

Methylobacterium oryzae CBMB20, a promising plant growth promoting bacteria (PGPB) and a biocontrol agent, was immobilized in different formulations such as wet chitosan, dry chitosan, wet alginate and dry alginate and were tested for tomato plant growth promotion. Chitosan solution (1.5%) with pH 5.5–6.0 and 90 min contact time was found optimal for immobilization. The chitosan formulations showed better entrapment efficiency and good degradability resistance apart from slow release of cells under prolonged incubation. Survivability of bacteria (80%) was observed in wet chitosan formulation even after 90 days of storage at 4°C. The spermosphere survival of bacteria was high in both dry and wet chitosan formulations applied soils even after 21 days under greenhouse conditions. While the alginate formulation degraded fully, partial degradation of chitosan formulation was observed even after 30 days, indicating its ability to support the survival of M. oryzae CBMB20 in soil. Plants inoculated with wet chitosan formulation registered 1.3 fold increase in the shoot and root length and dry weight compared to other treatments. Hence, chitosan formulation supporting better plant growth compared to alginate will be a better carrier for taking bacteria to the plant rhizosphere and thereby promote plant growth.