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微生物L-ASPARAGINASE:现在和未来的预期

Rati Sinha1H R辛格2年代K Jha2
  1. 研究学者,贝拉理工学院,生物技术学系Mesra,兰契,恰尔肯德邦,印度
  2. 贝拉副教授,生物技术,技术研究所、Mesra,兰契,恰尔肯德邦,印度
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国际创新研究期刊》的研究在科学、工程和技术

文摘

L-Asparaginase是众所周知的化疗属性自催化水解酶的氨基酸天冬酰胺为天冬氨酸和氨对肿瘤细胞至关重要。它已经被有效地用于治疗急性淋巴细胞白血病、淋巴肉瘤的几十年。本文包括一个简短的介绍关于酶,其来源、作用机制以及对其生产和净化的详细信息。本文还提供了一些最近更新的信息结构,理化和酶的动力学性质以及关于修改如克隆酶重组形式,制定和酶的固定化。

关键字
天冬酰胺酶,化疗、生化的动能,固定

我的介绍。
重组治疗性蛋白质产生了革命性的影响在医疗领域作为这些疾病是具体的,安全的和更有效的药物。他们有一个广泛的各种各样的具体用途:溶瘤,抗凝血剂或溶栓和替代代谢缺陷。目前大约50重组thereapeutic产品已在全球范围内批准的商业使用超过四分之一已经生产到印度和获得政府批准了他们的营销在中国[1]。治疗的主要潜在应用酶在癌症的治疗。L-Asparaginase就是这样一个治疗重组酶已被证明是有希望治疗急性淋巴细胞白血病,一个儿童癌症的变换结果的克隆细胞从骨髓早期淋巴前体细胞增殖并取代骨髓的正常细胞。

二世。的作用机制
L-Asparaginase催化非必需胺基酸的水解L-Asparagine L-Aspartate和氨。Lasparagine是一个重要的胺基酸对肿瘤细胞的生长,而正常细胞的生长是独立于它的要求。大多数正常组织中合成L-asparagine量足以满足其代谢需要的酶,L-asparagine合成酶但恶性细胞需要外部源的L-asparagine增长和乘法。在L-Asparaginase面前,肿瘤细胞失去了一个重要的生长因子,无法生存。这一事实表明这种酶的发展作为一种有效的抗肿瘤药物或anti-leukemic [3]。
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三世。应用程序
作为一种抗肿瘤药物——L-asparaginase是一个治疗重要的蛋白质结合其他药物治疗各种类型的血液癌症,如急性淋巴细胞性白血病(所有,主要发生在儿童)、霍奇金病、急性粒细胞白血病、急性myelomonocytic白血病、慢性淋巴细胞白血病、淋巴肉瘤治疗,reticulosarcoma,黑肉瘤周围组织。的第一个研究表明,腹腔内注射豚鼠血清内有有高含量的天冬酰胺酶产生明显的回归加德纳淋巴肉瘤小鼠[4],[5]。后来L-Asparaginase在大肠杆菌被发现的有力来源[6]。此后L-Aparaginase来源于E。杆菌。2has been used for treating human malignant disease.
胺基酸代谢作用——L-Asparaginase也起到了非常重要的作用在生物合成天冬氨酸的氨基酸的家庭。棒状杆菌——生产氨基酸工业的利益,因为他们排泄大量的各种氨基酸[7]。赖氨酸、苏氨酸和蛋氨酸,商业上重要的氨基酸由C。glutamicum,来自天冬氨酸,在正常生理条件下,可能会限制对赖氨酸和苏氨酸的生物合成。除了克雷布斯循环(使用谷氨酸胺基酸供体)、天冬氨酸天冬酰胺的形成天冬酰胺酶的作用。L-Asparaginase产生持续及其可能的溢出作用的酶,将天过剩转化为天冬氨酸,赖氨酸和苏氨酸的直接前体。一个非常活跃的L-asparagine被发现在c . glutamicum赖氨酸生产发酵条件[8]。
使生物传感器- L -天冬酰胺酶也被用来制造一个诊断生物传感器的数量产生的氨酶的作用直接相关的病人的血液中L -天冬酰胺[9]。
在食品加工,此外还用于食品加工。最近的食品技术的进步证明了油炸和烘烤食物(特别是油炸土豆)含有大量的丙烯酰胺(一种致癌毒物)[10];[11],由天冬酰胺的反应与还原糖[12]。与天冬酰胺酶预处理的土豆片和面包面团煎或烤之前防止丙烯酰胺的形成[13][14]。在这方面,从米曲霉和天冬酰胺酶a尼日尔用于烘焙行业[15]。这些酶在40 - 6.0 - -7.0ºC和pH值优化工作。自烘烤温度经常去120ºC,是理想的酶,是稳定和活跃在广泛的温度和博士因此,L-asparaginases从各种来源(细菌、真菌、植物和动物)进行了研究。所以,L-asparaginases都治疗和工业应用。

第四,L-ASPARAGINASE来源
微生物的重要性随着L-asparaginase来源被认为是自从它首次被发现从E。杆菌和其抗肿瘤的活性在豚鼠血清。[5];[6];[16];[17]。微生物如细菌、真菌、酵母、放线菌和藻类已被证明是精通这种酶的来源。虽然也被孤立的植物和一些动物来源。但微生物更好的来源L-asparaginase因为以下的优势:
一个¯‚·批量生产能力
一个¯‚·经济
一个¯‚·微生物很容易操纵获得酶所需的特色。
一个¯‚·容易提取酶和净化。
细菌来源- - - - - -细菌来源证明是L-asparaginase的丰富来源,因为他们很容易被操纵。这些包括E。杆菌、欧文氏菌cartovora,假单胞菌flouroscens AG)[18],分枝杆菌phlei[19],葡萄球菌[20],四膜虫pyriformis[21],水生栖热菌[22],[23]海洋发光细菌和栖热菌属酸奶被报告为不水解谷酰胺[24]和一个新的欧文氏菌sp。[25]
酵母的来源,酵母来源包括红酵母sp,红色不完美的酵母,Rhodosporoidum toruloides生产为酶已经被Ramakrishnan报道[26]。
放线菌资源从诺卡氏菌属sp - L-Asparaginase生产。[27]和链霉菌属sp.已经被报道。
植物来源在植物L-Asparaginase据报道从苔藓植物门生产水藓fallax[28],卢平araboreus &卢平angustiplius[29]和Lupinus leuteus [30]。据说L-Asparaginase活动也被发现在土壤的根松果体松树和松果体辐射动物由于ectomycorhizal真菌在西澳大利亚的小麦带[31]。从拟南芥K +端依赖L-asparaginase At3g16150特征[32]。
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诉L-ASPARAGINASE从各种发酵方法生产
从各种微生物来源L-Asparaginase已报告。生产L-asparaginase二世从E。杆菌被雪松和其他报道[81]。获得更高收益率L-asparaginase二世当细菌在厌氧条件下在摇瓶培养与高浓度的各种氨基酸,但显示贫困产生葡萄糖的存在。Bilimoria[82]报道L-asparaginase2生产由大肠杆菌细菌的培养条件。28大肠杆菌群,5株E。杆菌L-asparaginase2生产。发现温柔的曝气证明适用于良好的增长以及酶含量高而生机勃勃的曝气给不错的增长,但酶含量低的细胞和固定的文化给穷人增长。L-asparaginase生产肠杆菌aerogenes透明颤菌血红蛋白高效表达描述了耗氧量Geckil和Gencer [83] L-asparaginase活动显示低于野生型。曝气和搅拌酶生产的决定因素,低曝气和搅拌显示更高的收益率,而充分的有氧和无氧条件下减少酶产量。也的影响不同浓度葡萄糖的分解代谢的镇压酶生产。 Glucose at 1% completely inhibit the enzyme activity while at 0.1 %, it showed slightly stimulatory effect on enzyme production. Kumar and others [84] reported the localization and production of novel Lasparaginase from Pectobacterium carotovorum MTCC1428. The cell fractionation technique showed the enzyme to be localized in the cytoplasm. The enzyme activity was found to be 85 and 77% grown on a medium containing Lasparagine and combination of L-asparagine and glucose respectively which were also found to be most suitable carbon sources to maximize L-asparaginase production. The maximum enzyme production was observed to be 14.56IU/ml at 4 and 2g/l of L-asparagine and glucose respectively. Enhanced production of novel glutaminase free L-asparaginase from Pectobacterium carotovorum MTCC 1428 as reported by Kumar and others [85] by the optimization of the physical process parameters (pH, temperature, rpm of the shaking incubator and inoculums size) using central composite design technique which enhanced the enzyme production and productivity by 26.39% and 10.19% respectively. Maximum production was achieved at 12h under optimum level of physical process parameters in shake flask level. Moharam and others [86] described the production of intracellular L-asparaginase from Bacillus sp. R36 and its immobilization. Optimum culture condition were found to be 9:1 air:medium ratio, 55x105 CFU/ml inoculum size, 24 hr incubation period with shaking growth conditions and an initial pH value of 5.6.Addition of 1% lactose or 1% raffinose doubled the enzyme productivity. Immobilization is done by covalent binding with the activated carbon which greatly enhance the thermal stability of the enzyme. Ebrahiminezhad and others [87] reported L-asparaginase production by moderate halophilic bacteria isolated from Maharloo Salt lake. The halophilic bacteria was screened for L-asparaginase producing ability and found to produce intra and extracellular L-asparaginase. Bacillus sp BCCS 034 was found to produce the highest L-asparaginase (1.64 IU/ml supernatant) extracellularly.
一个细胞内L-asparaginase枸橼酸杆菌属的菌株已报告[40]。产生的酶在发酵桶与最高产量高达2700 1 corn-steep酒中9.2% (w / v) 37°C。L-Asparaginase产生的粘质沙雷氏菌深层发酵据海等人[88]和自溶酵母提取物中产量最高,26°C的孵化温度和曝气与零的溶解氧水平有限在发酵。当酶收率最高升至8.5的pH值发酵周期。4 IU的天冬酰胺酶的产量/毫升的细胞悬液一贯获得40到42人力资源从10-liter卷(500毫升/瓶4-liter)产生往复振动器。扩大到60-liter发酵罐产生3.1 IU / ml 35小时。彼得森和其他[89]报道L-asparaginase生产欧文氏菌aroideae最高产量1250 IU / g。Liuand等[90]报道最大的酶活性欧文氏菌aroideae获得了1%的乳糖作为碳源和1.5%的酵母提取物作为氮源,而葡萄糖抑制作用。两个批处理和连续发酵酶用于大规模生产。分批发酵给更高的收益率(4国际单位/毫升)连续发酵相比除了在稀释率0.1人力资源1。 The optimum temperature was 24°C for both. L-Asparaginase production by isolated Staphylococcus sp.-6A has been reported by Prakasham and others [91]. Evaluation of various fermentation process parameters were done to optimize nutritional (carbon and nitrogen sources), physiological (incubation temperature, medium pH, aeration and agitation) and microbial (inoculum level) fermentation factors. They reported that incubation temperature, inoculun concentration, pH and nutritional sources (glucose and ammonium chloride) had major impact at individual level and contributed to more than 60% of L-asparaginase production, while aeration, agitation and incubation time showed influence at interactive level. Murthy and others [92] reported production of extracellular L-asparaginase from a soil isolate of Bacillus sp. by the method of submerged fermentation using two different carbon sources, glucose and maltose, of which glucose was found to be the better carbon source. Sarquis and others [93] reported the production of L-asparaginase by Filamentous fungi under nitrogen regulation. The two fungi Aspergillus tamarii and Aspergillus terreus were cultivated in a medium containing different nitrogen sources. Aspergillus terreus showed the highest Lasparaginase production in 2% proline medium, while lowest enzyme production levels in both fungi were found in glutamine and urea as nitrogen source. Narayana and others [94] reported L-asparaginase production by Streptomyces albidoflavus which were optimized under submerged fermentations. Enhanced level of L-asparaginase was obtained with maltose and yeast extract as carbon and nitrogen source. The optimum pH and temperature were found to be 7.5 and 35°C. Amena and others [65] reported the production of extracellular L-asparaginase by Streptomyces gulbargensis using groundnut cake extract medium with maltose (0.5%) and L-asparagine as the best carbon and nitrogen source respectively. The optimum conditions for enzyme production was found to be as follows- pH 8.5, temperature 40°C, inoculum size 1x108 spores/ml and agitation speed 200 rev/min. After purification the optimum pH and temperature for enzyme production were 9 and 40°C respectively. The enzyme was more stable at alkaline pH than acidic one and retained 55% of activity at 80°C for 60 min. L-asparaginase production by strain improvement (i.e. mutation) and whole-cell immobilization of Streptomyces gulbargensis was described by Amena and others [95]. The strain Streptomyces gulbargensis was subjected to mutagenesis in order to isolate strains capable of producing higher yields of L-asparaginase. S.gulbargensis mu24 was isolated and found to be most potent and showed the maximum Lasparaginase activity (44.7IU) at 120 h of fermentation using ground cake extract medium. Thus mutation enhanced the enzyme activity by 1.49 fold. Also the whole-cell immobilization of the mutant and the prototype strain in gelatin enhanced enzyme production by 1.10 fold with fermentation carried out at 40°C at 200 rev/min. Dharmaraj [96] studied L-asparaginase production by Streptomyces noursei MTCC 10469 isolated from marine sponge Callyspongia diffusa. This marine actinomycete was isolated using specific ISP medium. Production was carried out by submerged fermentation using Tryptone Glucose Yeast extract (TGY) broth. The enzyme was purified 98.23 fold and showed a final specific activity of 78.88 IU/mg with 2.14% yield. The optimum pH, temperature and incubation time was found to be 8, 50°C and 35 min respectively.
Abdel-Fateh报道L-asparaginase生产到50071年铜绿假单胞菌在固体文化。使用Plackett-Burman阶乘设计培养条件进行了优化。pH值、水解酪蛋白和corn-steep酒被发现改善酶生产的最重要的因素及其最优值作为确定框- Behnken设计被发现是pH值7.9,酪蛋白水解物,3.11%,corn-steep酒,3.6%。优化后酶活性(142.81 iu)增强了超过五倍比基础培养基。天冬酰胺酶生产从50071年铜绿假单胞菌固态发酵也被阿什拉夫和其他人[97]。他们净化(106倍)的酶给一个特定的活动1900 iu / mg与43%的收益率和其最大活动pH值9孵化时37¢30分钟°C。最大L-asparaginase生产从芽孢杆菌circulans通过固态发酵使用红克皮(一个农业废弃物)[35]。优化各种社保基金参数使用部分!中心合成设计(FFCCD)。Venil等[98]报道L-asparaginase生产粘质沙雷氏菌SB08米糠,因为它是便宜和容易获得来源的酶生产固态培养。最高产量(79.84 IU / g)实现与初始含水率50%米糠和补充酵母提取物0.5% (w / v)和36小时孵化时间,但米糠结合其他淀粉类物质大大减少酶产量。优化L-asparaginase生产的粘质沙雷氏菌SB08 Venil报道和其他[99]是由联合法Plackett-Burman设计和响应面法在这4个因素——蔗糖,蛋白胨,KH2PO4和孵化时间筛选最佳媒介因素及其最优值由中心合成设计被发现是12.50 g / l, 4.5 g / l,分别为4.0 g / l和51 h。 Siddalingeshwara and others [100] reported the production of L-asparaginase from Aspergillus terreus KLS2 by SSF using carob pod as a substrate and showed 6.05IU enzyme activity after optimizing the fermentation parameters. Hosamani and others [101] reported L-asparaginase production by Fusarium equiseti using soya bean meal under solid state fermentation. Optimized fermentation parameters for maximum enzyme yield (8.51IU) were as follows – incubation period (48 hrs), initial moisture content (70% v/w), particle size (3mm), inoculum volume (20%), supplemented with glucose (0.5% w/v), ammonium sulphate (0.5 % w/v) and yeast extract (0.5 % w/v).
一个细胞外L-asparaginase酶从海洋Actionmycetes报道了岜沙和其他[102]。酶的生产是由固态和深层发酵。酶的生产进行了三种不同的媒体,即固态媒体、胰蛋白胨酵母提取物(TGY)肉汤和葡萄糖果糖胰蛋白胨酵母提取物(TFY)汤。隔离S3、S4和K8 L-asparaginase活动和生产潜力的酶产量从24.6到49.2国际单位/毫升。S3显示生产力的49.2国际单位/毫升65μg /毫升的蛋白质含量和最佳活动7.5 pH值和温度在50¢°C。王妃等人[103]所述隔离和筛选L-asparaginase生产真菌使用Czapek琼脂媒体从土壤样本。曲霉属真菌sp (KUFS20)是孤立的粉色区域的基础上形成。酶是由水下和固态发酵生产使用20个不同基质的橙皮显示最大酶生产。

第六,克隆L-ASPARAGINASE
L-Asparaginase是一个重要的酶用于治疗白血病的治疗。临床试验表明,这两种细菌E。杆菌和欧文氏菌sp.作为酶的潜在来源L-asparaginase,用作抗肿瘤药物。但各种副作用出现由于其内在谷氨酰胺酶活动,导致血液中lglutamine损耗以及抗肿瘤药物的成本很高。因此,新的天冬酰胺酶谷氨酰胺酶活性较低,更少的副作用和高活动向l-asparagine降低治疗成本是非常理想的癌症治疗的更好的选择。因此有需要实现重组技术来提高酶的性质和降低最终产品的成本。产生的重组酶分子克隆技术和基因工程的方法。
Khushoo等[104]报道L-Asparaginase II的细胞外表达E。杆菌重组形成的天冬酰胺酶的基因编码一种有效融合pelB领袖序列和一个氨基6 x组氨酸标签T7lac启动子克隆。纯化后的重组蛋白给整体收益率95 mg / L的纯化蛋白,恢复86%,高约八之前报道的文献中的数据。纯化蛋白具有类似的荧光光谱,分析规模和特定的活动所指的天然蛋白,蛋白质正确折叠和四聚物的形式存在于其活跃。
Kotzia等[105]报道L-asparaginase的克隆和表达E。通过执行PCR基因序列的胡萝卜,插入合适的表达载体,转化表达宿主E。大肠杆菌BL21 (DE3) pLysS。由此产生的重组酶显示的高天冬酰胺酶活动的具体活动0.72 u /毫克的蛋白质。2006年他们还报道欧文氏菌的克隆和表达L-asparaginase chrysanthemi 3937 (ErL-ASNase)在大肠杆菌BL21 (DE3) pLysS。酶纯化和表征后有效地固定在epoxy-activated琼脂糖CL-6B保留大部分的活动(60%),显示了高稳定在4度c的方法提供了可能设计一个ErL-ASNase生物反应器,可以在很长一段时间的运营效率高,可用于白血病治疗。
为了减少大肠杆菌的抗原性L-asparaginase (L-ASP)策略称为alanine-scanning诱变是应用识别氨基酸残基负责抗原性。三个连续的碱性残留物,195 rkh197,阿拉巴马州选择性突变。四个突变重组L-ASPs构建和表达E。杆菌,然后纯化。由竞争ELISA方法发现酶的抗原性大大降低[106]。
天冬酰胺酶的大肠杆菌重组,包括天冬酰胺酶、破伤风毒素肽(TTP)间隔片段(831 - 854),和外国胆甾醇酯转运蛋白c端片段(CETPC)的周质表达和有针对性的大肠杆菌。净化后表现出大约83%的原生酶活动,允许快速重组克隆的筛选而asparaginase-CETPC融合蛋白没有TTP间隔只有23%活动产生的原生酶。这种嵌合蛋白在老鼠细胞接种时,生成CETP-specific免疫反应。相比之下,大鼠免疫细胞表达的天冬酰胺酶只没有诱导特定anti-CETP抗体。这表明,天冬酰胺酶的大肠杆菌是一种有效的载体展示外国肽或抗原表位和TTP间隔似乎发挥重要作用在指导外国CETPC肽酶的表面。因此,嵌合酶结构融合与外国缩氨酸天冬酰胺酶通过TTP间隔可以用作快速pepscan技术抗原表位映射[107]。
L-Asparaginase从幽门螺杆菌在大肠杆菌克隆和过表达,纯化[108]。
Cappalletti等[109]克隆并表达了重组L-asparaginase从幽门螺旋杆菌的致病性毒株CCUG 17874显示谷氨酰胺酶活性低,表现出对Lglutamine s形的行为。
Krastokina等[110]克隆和表达ECAR-LANS Erwinia carotovora的L-asparaginase重组大肠杆菌被发现有一个相对较低的谷氨酰胺酶的活动。
从海床L-asparaginase furiosus在大肠杆菌克隆和表达。动力学研究表明,酶耐热性的,本地二聚的,glutaminase-free [111]。

七世。净化L-ASPARAGINASE
L-Asparaginases从不同来源已经被各种纯化方法。魏和其他[112]构造了不同突变酶的大肠杆菌以降低抗原性和有效地净化这些酶突变体,开发了一个本机制备聚丙烯酰胺凝胶电泳进一步执行免疫和生物研究400 u / mg活动。这种新方法是有效和具有成本效益的与其他方法相比,柱层析法和亲和色谱法等[112]
净化的L-Asparaginase不动杆菌calcoaceticus是由降水与链霉素、色谱deae纤维素和CM-cellulose磷酸纤维素琼脂糖凝胶过滤和色谱法。酶催化的脱氨基作用谷酰胺在相同程度上L-asparagine和显示弱肿瘤抑制权力[33]。
细菌L-asparaginases (L-ASNases)催化L-asparagine L-aspartate和氨的转换。Kotzia等[105]报道欧文氏菌的克隆和表达L-asparaginase chrysanthemi 3937 (ErLASNase)在大肠杆菌BL21 (DE3)和净化的酶是由一个单步过程涉及阳离子交换色谱在S-Sepharose FF列显示较高的活动。
测试的天冬酰胺酶大肠杆菌作为一个向量来显示每个酶亚基的线性肽表面上,构建了嵌合蛋白,并针对大肠杆菌的周质。本地野生型酶和重组突变体嵌合酶纯化从周质准备通过渗透压休克和阴离子交换色谱法。纯化嵌合酶显示出大约83%的活动的原生酶[107]。
L-Asparaginase(同工酶II)从大肠杆菌克隆和表达细胞外地。由此产生的重组蛋白纯化的单步使用Ni-NTA亲和色谱法使总收率为95 mg / L的纯化蛋白,恢复86%。这是高约8倍之前报道的数据在文献[104]。
Gladilina等[113]报道的克隆和表达重组蛋白从幽门螺杆菌J99 E。杆菌(BL21)是第一个纯化upta 1.8折,然后用细胞自由提取和酶的制备纯化细胞自由提取的可溶性部分通过色谱SP-Sepharose超过60%的收益率。
基于实验设计的统计数据来评估物理过程参数应用于最大化生产glutaminase-free L-asparaginase从Pectobacterium carotovorum MTCC 1428年净化后通过三个步骤增强L-asparaginase生产和生产力的26.39%(具体活动)和10.19%,分别为[85]。
净化L-asparaginase酶的同质性与铜绿假单胞菌50071细胞生长在固态发酵是由应用不同的纯化步骤包括硫酸铵分馏其次是g - 100交联葡聚糖凝胶过滤和分离CM-Sephadex C50crude文化滤液。酶纯化106倍,显示最后一个特定活动的1900 IU /毫克以43%的收益率[114]。
L-asparaginase重组酶的海床koshii表示在E。大肠杆菌BL21和净化通过阴离子交换色谱法和凝胶过滤其次是疏水作用色谱法和超滤[55]。L-asparaginase的链霉菌属tendae从托尔地区的红土的土壤样本中分离出粗酶纯化同质性,硫酸铵沉淀,交联葡聚糖g - 100和CM-Sephadex G-50凝胶过滤[68]。

八世。结构参数
酶结构:L-Asparaginases大多发现四聚物的形式,但当孤立从各种来源也存在二聚体,六聚物或单体形式。重组酶的大肠杆菌被发现在活跃的四聚物形成每个单元都有37 kd的分子量,揭示了sds - page凝胶过滤的纯化酶显示其分子量为150 kd [104]。张等人[115]报道大肠杆菌L-asparaginase homotetrameric形式存在与一个活跃的中心有一个分子量为138 kd。在执行L-Asparaginase来自幽门螺杆菌的sds - page,发现酶存在于homotetramer形式和37 kd每个亚基分子量凝胶过滤了分子量为140 kd [109]。的分子量L-asparaginase分离铜绿假单胞菌被发现160 kd,它存在于单体的形式由凝胶过滤sds - page [114]。分子量及其不。的亚基酶Pectobacterium carotovorum是由本地页面,凝胶过滤和MALDI-TOF质谱被发现homotetramer 144 kd分子量[85]。酶从海床furiosus被发现以二聚体形式由sds - page和质谱及其分子量被发现75.45 kd,取决于凝胶过滤和Superdex列[111]。栖热菌属thermolphilus酶存在于homohexameric形式与每个亚基分子量33 kd揭示了sds - page和分子量为200 kd由凝胶过滤[24]。时和其他[19]报道,欧文氏菌的L-asparaginase sp.有。起点周围形成六聚体结构 Liu and others [116] reported that the enzyme from Erwinia aroidea have a molecular weight of 108kD.
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结晶的酶也被许多科学家完成的。说明晶体结构提供洞察酶的活性部位和更好的理解L-asparaginase的结构与活性关系。Aghaiypour等[117]报道复合物的高分辨率的晶体结构与Lglutamic L-asparaginase酸、d -天门冬氨酸和琥珀酸。比较四个独立的活跃的网站在每个复杂显示独特的配体分子和特定的绑定;复合物之间的绑定方式也是类似的。缺乏alpha-NH3(+)组在往下,L-Asp相比,并不影响绑定模式。LGlu的侧链,比L-Asp,导致一些结构性扭曲时代积极的一面。氨基酸序列的比较和时代的晶体结构与其他细菌L-asparaginases表明两个活性位点残基的存在,Glu63(时代)和Ser254(时代),可能与重要的谷氨酰胺酶的活动,而他们的替代Gln和Asn,分别,可能导致最小L-glutaminase活动。Sanches等[118]E的结晶。杆菌L-asparaginase悬滴蒸气扩散法。 The X-ray structure of the enzyme, crystallized in a new form (space group C2) and refined to 1.95 A resolution is compared with that of previously determined crystal form (space group P2(1)). L-asparaginase dimer was found as he asymmetric unit of the new crystal form instead of the tetramer found in the previous crystal form. Major conformational differences are confined to the lid loop (residues 14-27). Dhavala and others [108] reported the crystal structure of Helicobacter pylori L-aparaginase in the presence of L-aspartate which was determined to 1.4 A resolution. Major differences were found in the active-site flexible loop and in the 286-297 loop from the second subunit, which is involved in active-site formation. Accordingly, Glu289, Asn255 and Gln63 appeared to play roles in modulating the accessibility of the active site.

生化的参数:
pH稳定性:L-Asparaginase从不同来源显示不同pH值稳定。一些酸性pH值稳定,在中性而有些稳定碱性博士的细胞外的天冬酰胺酶Cyberlindnera jadinii相当稳定在pH值4约10分钟50一个¢37°C和高达20 h¢°C。然而它保持稳定到一个最大的pH值10 [119]。从假单胞菌纯化酶stutzeri 405 MB的最佳pH值活动9和37°C显示一个¢最大稳定pH值7.5至9.5的范围[54]。酶来源于Helicobaterium幽门附近J99保持稳定酸碱10但迅速失去活动不可逆转地10或以上[113]。Amena等[65]报道的酶从链霉菌属gulbargensis更稳定在碱性pH值和保留其在7 - 10的pH值范围80%的活动。
温度稳定性:L-asparaginase酶从各种来源有不同的温度稳定性从37¢°C到80 A¢°C由于不同的氨基酸序列和结构。野生型和突变体酶L-asparaginase D178P从大肠杆菌保持稳定在37¢°C到4 h后,野生型酶迅速失去剩余活动相比,突变体。6小时孵化后,野生型显示35%,突变体酶显示了活动的19%的损失。孵化时间1 h,变异的97%和79%的野生型酶活性保持在45 A¢50°C,而到一个¢°C, 90%的突变体和野生型酶活性的71% [120]。张等人[115]报道,固定化酶L-asparaginase显示明显高于稳定到40-50A¢一个60°C而失去70%的活动¢°C 30分钟,成为完全灭活当孵化80¢°C 30分钟。这种酶从柱孢属obtusisporum仍然稳定在37个¢°C 30分钟而失去20%的活动40¢°C [76]。假单胞菌的酶stutzeri保持稳定在40¢1°C, h,没有重大损失的活动,虽然有一个半衰期25分钟和10分钟60 A¢65°C和A¢°C [54]。Kotzia等[50]报道,野生型酶的Tm欧文氏菌chrysanthemi及其耐热性的变异包含一个点突变(Asp133Val)是46.4一个¢55.8°C和¢°C和野生型和突变体酶的半衰期分别为2.7小时和159.7 h。酶与幽门螺杆菌保持绝对稳定的温度高达50¢°C 10分钟。但在53¢°C, 10分钟后失去50%的活动[109]。L-Asparaginase获得Azatobacter vinelandii保持稳定的温度50 a¢65°C,但得到灭活¢°C [121]。酶的链霉菌属gulbargensis保留55%的活动在80年一个¢°C 1 h在碱性pH值[65]。
金属和离子:金属和离子酶活动的调节中发挥重要作用。L-asparaginase孤立从各种生物对不同金属离子的反应不同。L-Asparaginase从细菌芽孢杆菌获得coagulans显示快速、可逆的激活在金属离子的存在如K +、+, Na + [122]。假单胞菌的stutzeri、K +、Mg2 +和Na +原因激活L-asparaginase [54]。当1毫米,3毫米和10毫米的酶从链霉菌属sp.测试,酶活性被抑制在Cu2 +, EDTA Zn2 +激活在Mn2 + [102]。毛霉菌sp。,酶显示的最大活动存在Na + 2%氯化钠[77]。L-asparaginase获得从四膜虫thermophila显示40 - 45%的激活的Ca2 + 5毫米的浓度而Mg2 +原因5 - 10%的激活[123]。酶获得不动杆菌calcoaceticus得到刺激存在Mg2 + [124]。离子,如CN - I -和PO4 3增强了活动的L-asparaginase真菌柱孢属obtusisporum [76]。
抑制剂:有几种化合物抑制L-asparaginase活动。的抑制剂之一Lasparaginase来自大肠杆菌是5-Diazo-4-oxo-L-norvaline [125]。其他L-asparaginase抑制剂包括二甲亚砜、戊二醛固定化过程中抑制酶浓度超过0.2%,L-Asp是间接负责防止水解的酶抑制diazo-4-oxo-Lnorvaline, NH4 +导致产物抑制的pH值8.5 n和胰蛋白酶完全灭活10分钟内自由酶固定化酶保持稳定时240分钟损失30%的活动。幽门螺杆菌,5-Diazo-4-oxo-L-norvaline抑制L-asparaginase活动和影响肉汤培养滤液抑制细胞周期[126]。的假单胞菌stutzeri、5-Diazo-4-oxo-L-norvaline Ca2 +, Cd2 +, Cu2 +, Fe3 + Hg2 +碘乙酰胺,Mg2 + Mn2 + Ni2 + PCMB, SDS,尿素,Zn2 +酶的抑制剂L-asparaginase [54]。
毛霉菌sp。EDTA恰好是一个强有力的抑制剂L-asparaginase而Ca2 + 26%的损失原因的活动集中在25毫米,Cu2 +导致20毫米44%抑制浓度,Zn2 + 25毫米浓度导致25%抑制[77]。的一些抑制剂L-asparaginase获得Azatobacter vinelandii atabrin, Cu2 +,价,Hg2 +、肼、羟胺、碘乙酸,KCN, p-hydroxymercuribenzoate、铁氰化钾和Zn2 + [121]。从四膜虫L-asparaginase pyriformis, D-Asn充当竞争抑制剂逆转antiproliferating影响乳腺癌细胞的[127]。栖热菌属的酸奶,D-Asn L-Asp diethylbicarbonate作为酶的竞争性抑制剂,Mg2 + Mn2 + Ca2 +和p-hydroxymercuribenzoate L-asparaginase其他抑制剂。PMSF导致部分失活的酶和酶活性损失有40%存在Zn2 + 1.0毫米浓度[24]。L-Asp和PCMB抑制剂的酶芽孢杆菌coagulans [122]。在海床furiosus, L-asparagine 20毫米以上浓度导致酶的底物抑制[111]。
激活化合物:各种化合物负责L-asparaginase的活性增强。的假单胞菌stutzeri,化合物如2-mercaptoethanol、二硫苏糖醇、EDTA、谷胱甘肽,谷胱甘肽,LCys, L-His提高酶活性[54]虽然1-amino-6-methyl-4-phenyl-2-thioxy-1, 2, 3, 4 - tetrahydropyrimidine-5-carboxylic酸甲基酯酶的活性提高获得Pectobacterium carotovorum [128]。在四膜虫thermophila、白蛋白量仅为0.1毫克/毫升的三倍激活L-asparaginase二世在山梨糖醇浓度30% w / v导致更多than10-fold激活[123]。酶获得不动杆菌calcoaceticus刺激在柠檬酸存在[33],以防Brevibacillus短,n-dodecane和硅油L-asparaginase活动的刺激效应;IInd坚持104%和108%相比,控制在液体石蜡6% (w / v)的浓度导致增加34% L-asparaginase活动伴随着细胞群的生产增加了48%在10 l规模[2]。
储存稳定性:存储的酶L-asparaginase是一个重大关切。科学家们已经尝试了各种方法和温度存储L-asparaginase从不同的来源。从不同的微生物源酶显示变量存储稳定性。乔恩和其他[124]报道L-asparaginase B Acenetobacter calcoaceticus可以保持稳定在-20ºC小损失的活动,即使经过长时间的存储。法律和其他[122]报道,芽孢杆菌coagulans L-asparaginase可以保持稳定在冻结6个月或更长时间。罗伯特和其他[129]报道,E。杆菌L-asparaginase lyphilyzed形式可以存储5ºC和保持稳定至少几周而作为无菌酶高的解决方案特定的活动也可以存储在5ºC没有损失的活动。E。杆菌L-asparaginase酶也可以存储1000 u /毫升在等离子体解决方案在-40ºC下降1年没有活动[130]。L-asparginase从普罗透斯寻常的斗犬和其他报道[131]可以为1个月保持稳定时储存在-20ºC存储5ºC时悬挂在3.5硫酸铵可以为1年保持稳定。 Ferrara and others [132] reported that the crude asparaginase preparation from Saccharomyces cerevisiae is stable upon storage at -18ºC for several months.
动力学参数:酶的稳态动力学测量L-asparaginase perfermed了不同底物的浓度。稳态的非线性回归分析数据通常被用来计算各种动力学常数如公里,Vmax, Kcat等不翻。计算的基础上每单元一个活性部位。一个没有。可供分析的计算机程序是动态数据(如GraFit Erithacus软件有限公司,Leatherbarrow, 1998;OriginLab 8.5版本,OriginLab公司北安普敦,妈,美国)。LAsparaginase从不同来源显示动力学性能的差异,因此在公里,的值将不。等等的一些动力学参数L-asparaginase从不同来源如下:
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八世。制定和固定
一些主要的限制酶的使用是其严重的免疫反应和血清半衰期很短。修改配方和固定化酶的到一个合适的矩阵可以高雅减少酶的免疫原性;增加它的半衰期和治疗的潜力。
廉价香烟和其他[138]报道制定保利(lactide-co-glycolide)包含酶L-asparaginase(身为)纳米颗粒。他们在身为封装酶纳米粒子使用water-in-oil-in-water溶剂蒸发技术导致的交付L-asparaginase在较长一段时间。
固定的E。杆菌L-asparaginase成水凝胶基质制成的聚(乙二醇)挂钩和BSA的Km值会增加200倍和更广泛的pH值范围为酶的最佳活动有90%的活动生理pH值7.3作为本土43%的活动形式。固定化酶的半衰期也提高到50天90%活动37ºC与2天的半衰期为原生酶[139]。在另一个实验中,他们(1997)耦合的E。杆菌在生物相容的水凝胶由天冬酰胺酶在大鼠血清白蛋白和挂钩和评估其有效性耗尽体内血清L-asparagine。发现85 - 90%的血清天冬耗尽了2天5单位/鼠和酶的80%活动仍保留甚至10天后[140]。
维纳和其他[141]报道的固定L-asparaginase从Erwinia carotovora生物活性果糖不同分子质量聚合物果聚糖(75 & 2000 kDa)获得发酵单胞菌属mobilis。他们采用的方法高碘酸盐氧化还原烷基化多糖后的保留大于55%的活动,提高公里价值和酶的最佳pH值范围宽。酶的固定化也导致减少的电泳淌度,较高的热稳定性和长期存储(1月)水溶液比原生酶。
固定的另一个方法是尝试由张等人[115]。他们共价结合酶天然蚕丝丝胶蛋白质的微粒子随后与戊二醛交联保持62.5%的原始酶的活动。
一个新的策略基于multisubunit共价固定化的L-asparaginase E。杆菌在激活支持(agarose-glutaraldehyde)一直受雇于Balacao等[142]其次是固相intersubunit aldehyde-dextran交联。这导致更长的半衰期和零子单元的风险释放进入血液循环
芽孢杆菌sp。L-asparaginase被共价结合活性炭高效固定化Moharam报道和其他[86]。这提高了酶产量(73.6%)和显示33 u / g的活动载体。固定化过程大大提高酶的热稳定性显示100%活动高达80ºC相比50ºC的原生酶[86]。

第九。结论
从过去的30年里,大量的研究已经完成L-asparaginase作为一种强有力的抗肿瘤药物的作用。首次发现豚鼠血清展览一个抗癌特性。后来这种药物是隔绝E。杆菌和欧文氏菌sp.已被用于治疗急性淋巴细胞白血病。但使用这种药物的限制因素是一些副作用,由于其抗原性和很短的半衰期血液中的这种酶。其重复剂量可导致过敏反应的患者的发展。此外酶的低收益率也的一个主要问题。不仅各种模式的产品和优化条件采用隔离世界各地的不同来源的酶来提高它的生产也是各种修改这种酶被科学家试图降低其免疫原性和增加它的半衰期。利用重组技术及其配方和固定仍在进行中。仍然有很长的路要走。 Moreover application of L-asparaginase in the food industry for the elimination of cancer-causing acrylamide from baked food has been one of the eminent discoveries of mordern time. Thus a lot more is needed to explore about this amazing enzyme.

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