储存条件对菌株11-3几丁质脱乙酰酶稳定性的研究

实验室保藏一株产几丁质脱乙酰酶(CDA)的菌株11-3,研究发现该菌所产CDA为胞内酶,在储存过程中其酶活稳定性很差,易失活。为保证该酶的后续研究,从几个方面考察了酶稳定性的影响因素,从而得出酶的最佳储存条件:干菌体储存于-20℃,2个月后酶活损失率仅为0.98%。     

几丁质脱乙酰酶的研究进展

几丁质脱乙酰酶(chitin deacetylase,CDA)是可以催化几丁质脱乙酰基反应生成壳聚糖的酶类。壳聚糖因其具有抗菌、抗癌、抗病毒等特性,在医药、化工、食品等行业具有广阔的应用前景。对CDA的研究概况进行了综述,包括酶学性质、催化机理、分离纯化的方法等,同时对CDA产生菌的筛选方法及过程做了简要的阐述,并对CDA今后的重点研究方向进行了展望。     

红球菌利用小龙虾壳粉产几丁质脱乙酰酶的培养条件优化

为了利用小龙虾壳和降低几丁质脱乙酰酶的生产成本,以小龙虾壳粉作为培养基主要成分,接种红球菌11-3菌株发酵生产几丁质脱乙酰酶.首先对培养基进行蛋白酶水解预处理;然后,通过单因素试验、Plackett-Burman设计、最陡爬坡试验及Box-Behnken试验,对补充营养成分和环境条件进行优化.结果表明:19％虾壳粉起始...     

1株高产几丁质脱乙酰酶红球菌的基因组测序及其应用潜力分析

为高效利用虾蟹壳资源,推动壳聚糖的几丁质脱乙酰酶法生产,采用Illumina测序技术,对1株高产几丁质脱乙酰酶的红球菌菌株11-3进行基因组测序,并进行系统的生物信息学分析,主要包括基因本体论(Gene Ontology,GO)、直系同源群集(Cluster of Orthologous Group,COG)、京都基因...     

几丁质脱乙酰酶（CDA）的研究进展

综述了国内外对几丁质脱乙酰酶（CDA）的研究概况,包括酶的微生物来源、性质、酶的底物特性、酶的生物学功能和酶的基因等,并对CDA潜在的应用价值进行了展望。     

细胞破碎方法对几丁质脱乙酰酶活力的影响

实验室保藏一株产几丁质脱乙酰酶（CDA）的菌种,经过验证该酶属于胞内酶,为进一步分离纯化,需对细胞进行破碎。本文以细胞破碎后上清液酶活为指标,通过比较几种常用细胞破碎方法,最终确定破碎该细胞提取CDA的最佳方法：发酵液洗涤三次,用pH8．0 Tris—Hcl缓冲液悬浮菌体,先进行反复冻融,然后再用玻璃匀浆器处理6min,离心测上清液酶活,比破碎前上清液酶活提高了5．58倍。     

几丁质脱乙酰酶产生菌的筛选

采用显色平板法和测定脱乙酰度(Deacetylation degree,DD)相结合的方法,从浅海污泥中筛选到一株产几丁质脱乙酰酶(chitin deacetylase,简称CDA)活力较高的细菌菌株S-1,并对其产酶条件和发酵进程进行了初步研究.S-1适宜产酶条件:碳源为蔗糖,氮源为酵母膏,添加无机盐NaH2P04,培养时间54h.     

蜡状芽胞杆菌促进红球菌发酵产甲壳素脱乙酰酶

以胶体甲壳素作为产酶诱导物,利用蜡状芽胞杆菌和红球菌共同发酵提高甲壳素脱乙酰酶(chitin deacetylase, CDA)的产量。通过监测2菌株混合发酵时甲壳素酶和CDA酶活力随时间的变化,验证了双菌株协同发酵产脱乙酰酶的作用。在此基础上,采用单因素实验考察两菌株配比、发酵周期、温度、接种量、装液量和摇瓶转速等因素对脱乙酰酶活力的影响,并进一步设计Plackett-Burman(PB)实验、最陡爬坡实验、中心组合设计实验和响应面优化实验,确定适宜2菌株协同发酵的工艺条件。结果显示,2菌株接种量最佳配比为2∶8,最佳产酶培养基组分(g/L):蛋白胨0.25、K₂HPO₄ 0.6、MgSO₄ 1.5、酵母浸粉3.0、NaCl 8.5、KH₂PO₄ 0.8、胶体甲壳素8.0。优化后菌株产CDA的活力约为优化前的1.96倍,研究结果可为工业化产CDA提供参考。     

一株产几丁质脱乙酰酶丝状真菌的发酵条件优化研究

针对已筛选出的一株能生产几丁质脱乙酰酶(CDA)的海洋丝状真菌,考察其最佳发酵培养基和发酵条件。通过单因素与正交实验得出该菌的最佳产酶条件为:初始p H为6.0,发酵温度为30℃,发酵时间为108 h,葡萄糖浓度7 g/L,酵母浸膏浓度7 g/L,氯化钙浓度0.75 g/L,几丁质浓度1.25 g/L,Na Cl浓度为1.4%。通过酶活的测定,发现该丝状真菌合成的几丁质脱乙酰酶主要是胞外酶。在最佳发酵条件下该丝状真菌的最高CDA胞外酶活为21.37 U/m L。是一株具有开发潜力的几丁质脱乙酰酶生产菌株。      

桔橙小单孢菌产几丁质脱乙酰酶的酶学性质研究

从海边红树林虾场土壤中筛选的一株产几丁质脱乙酰酶（CDA）的放线菌桔橙小单孢菌（Micromonospora aurantiaca）,研究其产CDA的酶学性质。结果表明,CDA的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳结果显示为单一条带,分子质量为81.8 ku。最适pH值为7.0;最适温度为40 ℃;Ca2+对此CDA酶活有促进作用,而Cu2+、Zn2+、Mg2+表现出了抑制作用。CDA对虾壳来源的几丁质有明显的脱乙酰效果,红外光谱测得样品脱乙酰度由39.03%提高至78.40%。扫描电镜发现CDA酶解过的几丁质样品表面疏松多孔、出现凹槽、晶体消失,进一步印证了该酶的良好脱乙酰效果,也力证了该酶拥有较好的特性。     

Characterization of Nalpha-benzyloxycarbonyl-L-lysine oxidizing enzyme from Rhodococcus sp. AIU Z-35-1

An oxidase catalyzing conversion of N(alpha)-benzyloxycarbonyl-L-lysine (N(alpha)-Z-L-lysine) to N(alpha)-benzyloxycarbonyl-L-aminoadipate-delta-semialdehyde (N(alpha)-Z-L-AASA) was purified from Rhodococcus sp. AIU Z-35-1, and its properties were revealed. This enzyme catalyzed an oxidative deamination of the varepsilon-amino group of N(alpha)-acyl-L-lysine and the alpha-amino group of N(varepsilon)-acyl-L-lysine. The apparent K(m) value for N(alpha)-acetyl-L-lysine was much larger than that for N(varepsilon)-acetyl-L-lysine. The peptidyl L-lysines, L-lysine and many other L-amino acids were also oxidized, but N(alpha)-acyl-D-lysine, N(varepsilon)-acyl-D-lysine and D-amino acids were not. Thus, the conversion of N(alpha)-Z-L-lysine into N(alpha)-Z-L-AASA was catalyzed by the L-amino acid oxidase with broad substrate specificity. This enzyme, a flavoprotein with a molecular mass of 100 kDa, consisted of two identical subunits of 51 kDa.     

Novel catalytic activity of nitrile hydratase from Rhodococcus sp. N771

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme catalyzing the hydration of various nitriles to the corresponding amides. We report a novel catalytic activity of NHase. When NHase was incubated with an large excess of commercially available isovaleronitrile, the charge transfer band from the sulfur ligand to the Fe atom shifted from 710 nm to 820 nm, but recovered within 4 min. Similar UV-Vis absorption changes were observed after the addition of isobutylisonitrile (iBuNC), a major impurity in commercially available isovaleronitrile, suggesting that NHase catalyzes the conversion of iBuNC to other compounds. The reaction product was identified as isobutylamine (iBuNH(2)) by liquid chromatography tandem mass spectrometry. NHase also converts t-butylisonitrile and 1,1,3,3,-tetramethylbutylisonitrile to the corresponding amines. Kinetic analysis of the conversion of iBuNC to iBuNH(2) showed a K(m) value comparable to that for nitriles, while the V(max) value was more than 10(5) times smaller than that for methacrylonitrile. This is the first report suggesting that NHase is a bifunctional enzyme catalyzing a reaction other than the hydration of nitriles.     

全自动发酵罐小试生产胆固醇氧化酶发酵条件的研究

目的:为了将胆固醇氧化酶应用于低胆固醇系列食品开发和胆固醇测定中,研究优化了菌株Rhodococcus SP.R14-2发酵生产胆固醇氧化酶(COX)的工艺条件.方法:通过测定比较COX酶活力,利用全自动发酵罐小试生产COX,研究发酵温度、pH、乳化剂、通风量和搅拌速率等因素对COX产量的影响.结果:Rhodococcus SP.R14-2的生物生长量和产酶是部分偶联的,胞外COX酶的合成高峰比生物量高峰滞后约12h.发酵过程中分段控制搅拌转速和通气量分别为:发酵前10h,200r/min和4L/min;10～50h,300r/min和5L/min:50h后,250r/min和4.5L/min,COX达到1.58u/mL的最高量.结论:发酵温度、培养基pH、通气量及搅拌速度等因素影响其生物量和COX酶合成,采用分段式控制,有利于COX产量的提高.     

北极海洋红球菌B7740（Rhodococcus sp.）产类胡萝卜素和类异戊二烯醌的抗氧化、抗增殖活性

本实验旨在研究来自北极海洋红球菌B7740类胡萝卜素和类异戊二烯醌提取物（B7CIQE）的体外抗氧 化活性（通过β-胡萝卜素漂白测定、脂质和蛋白质氧化抑制实验、DNA氧化断裂抑制实验）、抗增殖活性（通过 抗人肝癌细胞HepG2和人口腔癌细胞KB增殖实验）和细胞内抗氧化效果。实验中使用日常饮食常见高等植物来 源类胡萝卜素（β-胡萝卜素、叶黄素、番茄红素）作为对照组,评价B7CIQE的生物活性：β-胡萝卜素氧化抑制 率为B7CIQE（70.20%）＞2,6-二叔丁基-4-甲基苯酚（66.70%）＞表没食子儿茶素没食子酸酯（17.80%）＞番茄 红素（1.90%）;油脂开始氧化的温度顺序为B7CIQE（175 ℃）＞β-胡萝卜素（165 ℃）＞叶黄素（162 ℃）＞ 番茄红素（160 ℃）;蛋白质氧化抑制率为B7CIQE（25.75%）＞β-胡萝卜素（24.97%）＞叶黄素（17.94%）＞ 番茄红素（10.40%）;HepG2细胞抗增殖实验半最大效应浓度为叶黄素（20.86 μg/mL）＜β-胡萝卜素 （124.88 μg/mL）＜B7CIQE（126.34 μg/mL）＜番茄红素（139.24 μg/mL）;KB细胞抗增殖实验半最大效应浓度为 B7CIQE（25.14 μg/mL）＜叶黄素（64.29 μg/mL）＜番茄红素（69.87 μg/mL）＜β-胡萝卜素（149.16 μg/mL）。结 果表明,与植物源类胡萝卜素相比,B7CIQE具有相对更优异的抗氧化和抗增殖活性。     

Analysis of selective production of Nalpha-benzyloxycarbonyl-L-aminoadipate-delta-semialdehyde and Nalpha-benzyloxycarbonyl-L-aminoadipic acid by Rhodococcus sp. AIU Z-35-1

The factors for selective production of N(alpha)-benzyloxycarbonyl-L-aminoadipate-delta-semialdehyde (N(alpha)-Z-L-AASA) and N(alpha)-benzyloxycarbonyl-L-aminoadipic acid (N(alpha)-Z-L-AAA) from N(alpha)-benzyloxycarbonyl-L-lysine (N(alpha)-Z-L-lysine) by Rhodococcus sp. AIU Z-35-1 were analyzed. The cultivation time was important for selective production of N(alpha)-Z-L-AASA, since N(alpha)-Z-L-lysine oxidizing enzyme reached maximum at the early stage of cell growth and then decreased. The differences of enzyme activities of N(alpha)-Z-L-lysine oxidizing enzyme and N(alpha)-Z-L-AASA dehydrogenase in pH and temperature also affected the selective production of N(alpha)-Z-L-AASA. For efficient production of N(alpha)-Z-L-AAA, it was important for cultivation time that N(alpha)-Z-L-AASA dehydrogenase activity be higher than N(alpha)-Z-L-lysine oxidizing enzyme activity, since a high concentration of N(alpha)-Z-L-AASA inhibited N(alpha)-Z-L-AASA dehydrogenase activity. The optimum pH of N(alpha)-Z-L-AAA production was affected by the instability of N(alpha)-Z-L-AASA dehydrogenase in the alkaline pH region.     

Cloning and characterization of a 4-nitrophenol hydroxylase gene cluster from Rhodococcus sp. PN1

A 4-nitrophenol (4-NP)-degrading bacterium was isolated from activated sludge and identified as a Rhodococcus sp. This bacterium, designated as strain PN1, could utilize 4-NP as a sole carbon, nitrogen and energy source. Degradation tests of 4-NP using cell suspensions of strain PN1 revealed that the degradation was induced by 4-NP and that 4-nitrocatechol (4-NC) was one of the metabolites. A gene library was constructed from the total DNA of strain PN1 and introduced into Rhodococcus rhodochrous ATCC 12674. Two recombinant strains showed 4-NP hydroxylase activity, and a 9.1-kb DNA fragment encoding the activity was isolated from one of the strains. In addition, a 2.4-kb smaller fragment expressing the activity was subcloned from the 9.1-kb fragment and sequenced. The sequence analysis showed that the fragment encodes a two-component 4-NP hydroxylase, the predicted amino acid sequence of which exhibits significant similarity to those of phenol hydroxylases and 4-hydroxyphenylacetate 3-hydroxylases belonging to the two-component flavin diffusible monooxygenase (TC-FDM) family proposed by Galán et al. (J. Bacteriol., 182, 627-636, 2000).     

北极海洋红球菌（Rhodococcus sp.）B7740产类胡萝卜素的提取条件优化及甲基萘醌类类胡萝卜素鉴定

利用酶的高效性和专一性破裂细菌细胞壁,实现对类胡萝卜素的温和提取并鉴定。以加酶量、酶解时间和料液比作为单因素进行正交试验,得出最优提取条件为：菌粉与溶菌酶配比1∶10（mg/mL）、酶解时间80 min、料液比1∶80（mg/mL）,此条件下类胡萝卜素提取量为675 μg/g。采用液相色谱-质谱串联进行类胡萝卜素的结构鉴定,液相色谱-质谱条件为：色谱柱YMCC30（4.6 mm×150 mm,5 μm）、检测波长450 nm、大气压力化学电离源、正离子模式、离子源温度230 ℃、充电电压2 000 V、质荷比扫描范围200～1 200,鉴定出分子式分别为C51H76O2和C51H74O2的2 种甲基萘醌类胡萝卜素。     

降胆固醇红球菌F21-1的分离筛选与鉴定

利用以胆固醇为唯一碳源的选择性培养基从豚鹿粪便样品中分离出1株降解胆固醇良好的菌株.经生理生化鉴定和16SrDNA基因序列分析,该菌株为马红球菌(Rhodococcus equi.).     

Rhodococcus sp.SHZ-1腈水合酶的高酶活发酵工艺

通过研究发酵过程中溶氧、生物量、腈水合酶活力以及残糖的变化规律和搅拌速度、通气量、接种量及诱导剂对产腈水合酶的影响,确立了5L发酵罐中Rhodococcus sp.SHZ-1腈水合酶的高酶活发酵工艺参数。结果表明,发酵过程中溶氧控制在30%以上有利于菌体快速生长和腈水合酶的合成。在pH7.2,温度30℃的发酵条件下,适宜的腈水合酶合成工艺条件为:接种量10%,通气量1.0vvm,搅拌速度采用由初始的200r/min调至500r/min的变速调控方式,同时于48h补加产酶诱导剂,发酵液腈水合酶的最高酶活力达到了9500U/mL,是摇瓶培养时最高酶活力8208U/mL的1.2倍,且比未进行工艺优化时最大产酶期缩短了20～24h,其最佳产酶期为52～60h。     

来源于Anoxybacillus sp. SK3-4普鲁兰酶PulASK的固定化及酶学性质研究

普鲁兰酶在淀粉加工、多糖制备以及啤酒酿造等领域有着广泛应用。然而,该酶游离下催化产物需分离纯化和本身不可再生限制了其工业应用。磁性纳米材料具有可重复使用、磁回收性质和比表面积大等特点,有助于解决游离酶的工业化应用难题。作者将源于LAnoxybacillus sp. SK3-4的普鲁兰酶PulASK固定在纳米磁性材料Fe₃O₄@Histidine上以制备固定化酶复合体：Fe₃O₄@Histidine/PulASK,测定固定化酶的酶学性质和动力学参数。结果显示,与游离酶PulASK相比,固定化酶Fe₃O₄@Histidine/PulASK的最适反应温度由60 ℃提高到65 ℃,最适pH与游离PulASK相同;在60 ℃,pH 6.0下孵育7 h,固定化酶残余酶活为62%,而游离酶的仅为30%。分析酶动力学数据可知,在60 ℃,pH 6.0的情况下,游离酶PulASK的K_m为4.7 mmol/L,为Fe₃O₄@Histidine/PulASK的1.47倍;Fe₃O₄@Histidine/PulASK的k_cat值是350 s^-1,与PulASK的k_cat值相当,Fe₃O₄@Histidine/PulASK的k_cat/K_m是PulASK的1.57倍;并且Fe₃O₄@Histidine/PulASK在重复使用9次后,活力仍保持50%以上。与PulASK相比,Fe₃O₄@Histidine/PulASK具有良好的热稳定性且可重复使用,具有潜在应用于食品工业的价值。