酸橙内生菌Bacillus thuringiensis Bt028几丁质酶的分离纯化及其酶学性质

为揭示酸橙内生菌Bacillus thuringiensis Bt028几丁质酶的基本酶学性质,采用离心、硫酸铵盐析、葡聚糖凝胶G-100层析及SDS-聚丙烯酰胺凝胶电泳等方法对菌株Bt028发酵液中的几丁质酶进行分离纯化鉴定,并考察该几丁质酶的最适温度、最适pH、催化动力学参数等性质。结果表明,Bt028菌株发酵液经离心、硫酸铵盐析、葡聚糖凝胶G-100层析分离纯化后获得电泳纯几丁质酶比活力为681.78 U/mg,纯化倍数为3.21,回收率15.52%。SDS-聚丙烯酰胺凝胶电泳结果表明,几丁质酶的分子质量为65 kDa。酶学性质研究结果表明,该酶的最适反应温度为60 ℃,最适反应pH为6.5,在温度低于60 ℃、pH5.5~7.5时有较好的稳定性;Mg2+、Ca2+、Hg2+、Co2+对该酶活力有抑制作用,而Cu2+和Fe3+有一定的促进作用;低浓度的甲醇、乙醇、正丙醇和二甲亚砜使酶的活性增加,但当浓度继续增大,反而会抑制酶的活性;丙酮对几丁质酶有激活作用,而甲醛对几丁质酶有抑制作用。在最适催化条件下,几丁质酶催化反应的米氏常数Km、最大反应速率Vmax、酶的转换数Kcat分别为29.533 mg/mL、108.696 μmoL/(L·min)和0.527/min。研究结果可为该酶的实际应用提供必要的工艺参数。     

一株产几丁质脱乙酰酶海洋细菌的Rhodococcus hoagii筛选、鉴定及酶学性质

从连云港高公岛海域采集的海泥中使用平板变色圈法初筛和摇瓶发酵复筛后,获得一株产几丁质脱乙酰酶的菌株MCDAⅡ-2。综合形态学特征以及16SrDNA序列分析最终将菌株MCDAⅡ-2鉴定为Rhodococcus hoagii。进而研究其酶学性质,得该几丁质脱乙酰酶最适催化温度为35℃,最适pH为8.0。Na^+、Mg^2+、Li+、Sr^2+对酶促反应起到促进作用,Ca^2+、Ba^2+、Co^2+、Cd^2+、Fe^2+及EDTA对酶促反应起到了抑制作用,而K^+对酶促反应影响较小。酶在25℃~35℃时较稳定,在中性和弱碱性条件下稳定性较好。本次研究的结果将为几丁质脱乙酰酶的工业化应用奠定基础。     

一种海洋几丁质酶产生菌的筛选及酶学性质研究

海洋是自然界几丁质主要来源地，滋养出种类繁多可降解几丁质的微生物，因此从海洋中筛选产高活性几丁质酶的细菌，是获得高产几丁质酶微生物的有效途径。该文以胶体几丁质为唯一碳源，从渤海滩涂筛选到一株具有降解几丁质特性的细菌Y-8,对其进行鉴定并研究其几丁质酶酶学性质。Y-8菌株经分子鉴定为副溶血性弧菌(Vibrio parahaemolyticus),发酵上清液经SDS-PAGE及蛋白质谱分析，发现其存在2种几丁质酶，分别为Chi1几丁质外切酶，氨基酸残基数为848,理论分子质量为87.6 kDa; Chi2几丁质内切酶，氨基酸残基数为1 054,理论分子质量为112.9 kDa。Y-8所产生的几丁质酶能够高效降解胶体几丁质，获得单一产物N-乙酰氨基葡萄糖，其最适反应温度为55℃,45℃保温1 h,仍能保持70%以上活性；最适pH为6.0,在pH 4.0～9.0、37℃保温1 h后相对酶活性保留55%以上，具有较好的稳定性。10 mmol/L的Mn²⁺能使几丁质酶活性提高362%,而EDTA以及SDS、吐温-20、吐温-80对酶活力具有抑制作用。     

微生物几丁质酶的研究概况

几丁质酶（Chtinase，EC3．2．1．14）是一种能够催化水解N-乙酰-D-葡萄糖胺糖苷键的酶。本文从几丁质酶的用途、特点、理化性质等方面对几丁质酶作一简单介绍并对其进一步研究提出了展望。     

大肠杆菌表达几丁质酶对不同底物的酶学性质及其降解产物分析

采用重组大肠杆菌大量表达几丁质酶LICHI18A,经Ni-NTA柱纯化后,对不同底物的酶学性质进行了研究,以Lineweaver-Burk双倒数作图法测定米氏常数,采用薄层色谱(TLC)和高效液相色谱(HPLC)对降解产物进行分析。结果表明,该几丁质酶分子量为54 k Da,对水溶性壳聚糖的Km和Vmax分别为4.04 mg/m L和222.2μmol/(min·mg);对水溶性壳聚糖的反应最适温度和p H分别为60℃和7.0;对胶体几丁质的Km和Vmax分别为2.913 mg/m L和2.836μmol/(min·mg),反应最适温度和p H分别为40℃和5.0。Ni2+、Mg2+、Ca2+、Zn2+对胶体几丁质的降解有明显的促进作用,但对水溶性壳聚糖的降解没有促进作用,且Ni2+和Zn2+有一定的抑制作用。Tween-80对降解胶体几丁质起一定的促进作用,而不同浓度的SDS有抑制酶活作用,且浓度越高抑制作用越强。TLC和HPLC分析结果表明,该酶降解胶体几丁质的产物主要为几丁二糖和单糖,而水溶性壳聚糖的降解产物较为复杂,主要有单糖、几丁二糖和甲壳寡糖。     

重组内切几丁质酶的纯化及其催化壳聚糖降解的研究

目的:研究重组内切几丁质酶的分离纯化及其催化壳聚糖制备壳寡糖的反应条件优化。方法:重组菌发酵液经PEG20000浓缩和DEAE-Sepharose FF离子交换层析后,测定总蛋白含量和内切几丁质酶活力。用纯化的内切几丁质酶催化壳聚糖制备壳寡糖,探讨其最佳的反应条件。结果:纯化的内切几丁质酶比活力41.34U/mg,纯化倍数2.49,酶总回收率89.63%。内切几丁质酶催化壳聚糖降解制备壳寡糖的最优化反应条件是:壳聚糖质量分数4%,p H7.0,温度30℃,时间12 h。结论:本研究结果为内切几丁质酶和壳寡糖的产业化应用奠定了良好基础。     

金龟子绿僵菌(Metarhizium anisopliae Ma83)几丁质酶的纯化及性质

由金龟子绿僵茵Ma83菌株产生的几丁质酶经硫酸铵盐盐析,Sephadex G-100柱层析,DEAE-纤维素柱层析分离纯化后,得到SDS-PAGE均一样品.酶的最适反应温度为50℃,半失活温度为65℃;酶的最适反应pH值为5.0,酶在pH4.0～7.0范围内较稳定.Ag+、Co2+、K+、Mg2+对Ma83几丁质酶有激活作用,而Hg2+、Zn2+、Pb2+对几丁质酶活力有抑制作用.经计算Ma83菌株几丁质酶对胶体几丁质的Km值为1.05 mg/mL.     

单胞菌产几丁质酶的酶学性质研究

对从海水中分离得到的嗜水气单胞菌所产的几丁质酶的酶学性质进行研究.其最适pH为7.0左右,最适反应温度为45 ℃左右.在40 ℃以下有良好的热稳定性,12 h仍有50%残余酶活.Mg2 对酶有激活作用,Cu2 和Al3 对酶有明显的抑制作用.该酶对底物的亲和力很大,水解几丁质的Km值为29.15 g/L.     

绿僵菌Ma83固态发酵几丁质酶和部分酶学性质的初步研究

研究了金龟子绿僵菌MetarhiziumanisopliaeMa83菌株产几丁质酶的固态发酵条件和粗酶液的酶学性质。研究结果显示 ,当以麸皮∶蚕蛹粉为 3∶1作为培养基 ,最适氮源为1%NaNO3,起始pH为 6 5 ,发酵温度为 31℃ ,接种量为 2mL液态种子时酶活力最高。此外 ,添加稻壳对Ma83产几丁质酶有促进作用。该菌株在生长 2d时 ,酶活力达 33 9U /g干培养基。酶的最适反应温度为 5 0℃ ,最适反应 pH为 6 0 ;不同温度保温 1h后 ,酶的半失活温度为 42℃。不同 pH值的缓冲溶液中 (2 5℃ )放置 1h后 ,酶在pH 5 0～ 6 0条件下稳定性最高     

海洋产几丁质酶厦门加西利亚单胞菌Chi34的筛选、鉴定及酶学性质分析

目的：海洋是自然界中含几丁质最多的生态系统,孵育了大量可降解利用几丁质的微生物,因此利用海洋微生物几丁质酶降解几丁质,是获得高附加值几丁寡糖的重要方法。本试验以连云港海域海泥为样品,以期筛选获得稳定的产几丁质酶菌株。方法：利用平板筛选法和摇瓶发酵复筛法筛选获得产几丁质酶菌株Chi34,利用形态学分析和16S r DNA序列分析对菌株进行鉴定,同时还研究了温度、pH、金属离子、EDTA及SDS等因素对Chi34几丁质酶的影响,最后通过TLC实验分析了Chi34几丁质酶降解几丁质胶体的产物。结果：菌株Chi34鉴定为厦门加西利亚单胞菌(Gallaecimonas xiamenensis),其几丁质酶酶活力为0.631 U/m L,最适反应温度为35℃,最适反应p H为6.0,Na₊、Ca₂₊、Mn₂₊、K₊对Chi34几丁质酶具有显著的激活作用,Cu₂₊、Fe₃₊、Ba₂₊、Zn₂₊、Cd₂₊、...     

Characterization of Clostridium paraputrificum chitinase A from a recombinant Escherichia coli

Clostridium paraputrificum chitinase A (ChiA) was purified from a recombinant Escherichia coli. ChiA was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. ChiA showed maximum activity at pH 6.0 and its optimum temperature was 45 degrees C. ChiA was stable between pH 6.0 and 9.0 and at temperatures up to 40 degrees C. The K(m) and V(max) values of ChiA for 4-MU-(GlcNAc)2 were estimated to be 6.9 microM and 43 micromol/min/mg, respectively. Thin-layer chromatography indicated that ChiA hydrolyzes chitooligosaccharides to mainly chitobiose. ChiA was found to adsorb not only chitinous polymers but also cellulosic polymers.     

Conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum M-21

The chitinolytic bacterium Clostridium paraputrificum strain M-21 produced 2.2 and 1.5 mol hydrogen gas from 1 mol N-acetyl-D-glucosamine (GlcNAc) and ball-milled chitin equivalent to 1 mol of GlcNAc, respectively, at pH 6.0. In addition, strain M-21 efficiently degraded and fermented ball-milled raw shrimp and lobster shells to produce hydrogen gas: 11.4 mmol H2 from 2.6 g of the former and 7.8 mmol H2 from 1.5 g of the latter. Hydrogen evolution from these shell wastes were enhanced two fold by employing acid and alkali pretreatment. Waste from the starch industry was also converted to hydrogen. When C. paraputrificum M-21 was cultivated on ball-milled chitin and ball-milled shrimp shells for 14 and 12 h, respectively, chitinases ChiA and/or ChiB were detected as the major chitinase species in the supernatant of the cultures, suggesting that the play a critical role in the degradation of chitinous materials.     

Saccharification of chitin using solid-state culture of Aspergillus sp. S1-13 with shellfish waste as a substrate

Saccharification of chitin was performed in a suspension (mash) of a solid-state culture of chitinase-producing Aspergillus sp. Sl-13 with acid-treated shellfish waste as a substrate. The conditions for the saccharifying reaction and the solid-state cultivation were examined from the viewpoint of saccharification in the mash. Optimum cultivation conditions were defined: a solid-state medium consisting of 5 g of 10% lactic acid-treated crab shells (0.50-2.36 mm in size) and 3 ml of a basal medium (0.028% KH2PO4 0.007% CaCl2.2H2O, and 0.025% MgSO4.7H2O) supplemented with 0.3% peptone was inoculated with 4 ml of spore suspension (1 x 10(7) spores/ml), and the water content of the medium was adjusted to 75%; static cultivation at 37 degrees C for 7 d. When a culture obtained under the optimum conditions was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 4.0) and incubated at 45 degrees C for 11-13 d, 55 mM N-acetylglucosamine (GlcNAc) was formed in the solid-state culture mash, indicating that at least 33% of the initial chitin in the solid material was hydrolyzed. Through the experiments, the amounts of G1cNAc formed in the solid-state culture mash varied in a way similar to that of the water-extractable pnitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in the culture, but not to that of the colloidal chitin-hydrolyzing enzyme. G1cNAc-assimilating lactic acid bacteria, which were inoculated into the mash after or at the start of the saccharification, formed lactic acid with decreasing GlcNAc.     

Two catalytic domains of Chlorella virus CVK2 chitinase

VChti-1 chitinase encoded by the Chlorella virus CVK2 contained two catalytic domains belonging to family 18 glycosyl hydrolases. The first catalytic domain on a C-terminal-truncated derivative of vChti-1 generated exclusively chitobiose from chitotetraose, chitohexaose, and colloidal high-molecular mass chitin in the enzyme reaction, a typical characteristic of an exochitinase. In contrast, N-acetylglucosamine was produced from chitobiose as well as from chitooligosaccharides by the second catalytic domain on an N-terminal-truncated derivative of vChti-1. Therefore, the second domain possessed N-acetylglucosaminidase activity as well as endochitinase activity. The presence of two catalytic domains with different enzymatic properties in the viral enzyme seems to be necessary for hydrolyzing natural substrates in a cooperative fashion.     

龙虾头壳制备低聚壳聚糖的研究

以虾仁加工的副产品龙虾头壳为原料,采用酸浸碱煮工艺制备甲壳素,得率为(16.11±0.73)%;浓碱处理脱乙酰基制得壳聚糖,得率为(68.07±1.60)%(相对于甲壳素);选用纤维素酶对壳聚糖进行降解制备低聚壳聚糖。分别研究了加酶量、pH、温度、时间、底物物浓度对壳聚糖降解为低聚壳聚糖的影响。选择0.5%的壳聚糖浓度,通过优化设计试验,确定纤维素酶降解壳聚糖最佳条件为:加酶量8IU/(g底物),pH5.0,温度60℃,时间4h。取最佳工艺条件下降解的低聚壳聚糖,采用乙醇分步沉淀,EP75为(2.36±0.15)%,平均聚合度为9;EP87为(0.85±0.15)%,平均聚合度为7;ES87为(79.84±0.10)%,平均聚合度为5。龙虾头壳制备壳聚糖,纤维素酶降解制成聚合度10以下的低聚壳聚糖,平均得率为8.8%。     

发光杆菌产几丁质酶的工艺优化

为提高海洋发光杆菌Photobacterium sp.LG-1产低温几丁质酶的酶活性,利用响应面法对其发酵产酶条件进行了优化.Plackett-Burman实验结果表明,影响菌株产酶的三个主要因素分别为发酵温度、发酵时间和酵母膏含量.菌株产酶的最适条件为:胶体几丁质浓度12.0 g/L、酵母膏浓度4.5 g/L、转速2...     

Purification of Aspergillus sp. S1-13 chitinases and their role in saccharification of chitin in mash of solid-state culture with shellfish waste

In a suspension of solid-state culture of Aspergillus sp. S1-13 containing a lactic acid-treated crab shell as the substrate, the saccharification of chitin in the shell proceeded to form N-acetylglucosamine (GlcNAc): the culture was the source of chitin and chitinases. The analysis of chitinases in the water-extract of the solid-state culture indicated occurrence of an exochitinase (Exo, MW 73 kDa) and two endochitinases. The amounts of the endochitinases suggested that one of them (Endo-1, MW 45 kDa) might be the main species in the chitin-saccharification. The amount of GlcNAc released from the LA-treated crab shell by the combined action of isolated Exo and Endo-1 was very small, predicting participation in the saccharification of other enzyme species, which might be hardly extracted with water from the solid-state culture. The re-extraction of the solid-state culture using 2 M KCl, which was extracted with water beforehand, demonstrated another endochitinase (Endo-2, MW 51 kDa). Endo-2 isolated from the salt-extract can adsorb to chitin, and can hydrolyze the chitin in the adsorbed state. The roles of these chitinases in the chitin-saccharification based on their properties and combined action were discussed.     

高产几丁质酶菌株的诱变选育及发酵条件的优化

以粘质沙雷氏菌G3-1为出发菌株,通过紫外-LiCl和微波-LiCl两轮复合诱变,得到一株产酶能力高且遗传稳定的突变株GF-21,通过单因素实验和正交实验对突变株GF-21的发酵培养基和发酵条件进行优化。结果表明,最优发酵培养基成分为:乳糖6 g/L,尿素10 g/L,KCl 1.0 mmol/L,NaCl 5 g/L,胶体几丁质10 g/L,此时,几丁质酶活力为4.73 U/mL,比优化前提高了109.3%,较出发菌株提高了470%;最优发酵条件为:培养基初始pH9.0,温度30 ℃,摇床转速220 r/min,接种龄5 h,接种量10%。本文为几丁质酶的生产应用奠定了良好的基础。     

产低温几丁质酶菌株的筛选、鉴定与产酶条件优化

目的:筛选产低温几丁质酶菌株,并对其进行鉴定及发酵条件优化。方法:本实验以渤海海域海泥为样品,以胶体几丁质为唯一碳源,通过平板筛选法筛选产低温几丁质酶细菌,对其进行形态学鉴定和分子生物学鉴定,并对其发酵产酶条件进行了单因素优化。结果:菌株鉴定结果为发光杆菌(Photobacterium sp.),单因素优化结果为:胶体几丁质12.0 g/L、酵母膏6.0 g/L、发酵温度15 ℃、初始pH7.0、转速为220 r/min、装液量75 mL/250 mL、接种量1%、发酵时间96 h,酶活力达4.566 U/mL,比优化前提高389.91%。结论:为下一步酶学性质研究及低温几丁质酶在工业中的应用提供参考。