碱性纤维素酶及其应用的研究进展

碱性纤维素酶在碱性条件下具有内切β-1,4葡萄糖苷酶活力,在加酶洗涤剂等行业具有重要的应用价值。目前已发现由芽孢杆菌和链霉菌产生的多种碱性纤维素酶,其中大部分的基因已被克隆、测序和表达,其催化反应特性、反应机理和应用也得到了较深入的研究。从碱性纤维素酶的产生菌、酶反应特性和反应机理、碱性纤维素酶的基因克隆和表达,以及碱性纤维素酶的应用等几个方面,介绍了有关碱性纤维素酶的最新研究进展。     

碱性纤维素酶及其去污机理

纤维素酶的研究,已有四五十年的历史。但是,一直是以木霉（Trichodern。a）、曲霉U印eryillus）属等真菌产生的酸性纤维素酶为研究对象,以将木质纤维素转化成葡萄糖为主要研究方向进行的。近年来,碱性纤维素酶在洗涤剂工业上的成功应用,改变了传统的去污机制,建立了一套新的去污机理,被洗涤剂工业称之为一次技术大革命,使碱性纤维素酶成为世界各国普遍重视的一种极具生命力的新型酶制剂。三产碱性纤维素酶的微生物及其酶的性质碱性微生物可以分为嗜碱菌和耐碱菌。只有在pHS以上才能生长的被称之为嗜碱菌;最适pH是中性,但在碱性…     

洗涤剂用碱性纤维素酶的研究进展

迄今报道的绝大多数纤维素酶的最适pH都在酸性和中性范围 ,当添加到洗涤剂中 ,由于处于碱性环境而无活力 ,不能发挥作用。近年来 ,国内外对由碱性芽孢杆菌 (Bacillussp )产生的碱性纤维素酶 (CMCase,endo β1 ,4 glucanase,EC 3 2 1 4)进行了广泛的研究。对该酶产生菌株的筛选和培养条件、酶学性质 ,以及该酶基因的克隆和表达等方面的研究进行综述 ;并对我国目前未能实现该酶工业化原因进行了初步分析 ,并提出解决途径。     

洗涤剂的新型生物添加剂—碱性酶

目前,碱性酶作为新型生物添加剂应用于洗涤剂行业的现象已越来越普遍,在西欧、日本和美国等一些发达国家中,加酶洗涤剂占洗涤剂市场的８０％～９５％,在国内加酶洗涤剂所占比例为２０％左右,市场上的许多高档洗衣粉产品都添加了多种酶。从发展趋势上看,国内加酶洗涤...     

碱性纤维素酶革兰氏阴性菌株筛选及酶学性质研究

用CMC平板筛选方法,从造纸厂碱性淤泥中获得透明圈直径大于30mm的产碱性纤维素酶革兰氏阴性菌H8005。液体摇瓶培养产生碱性CMC酶活力高达4．2IU／mL。酶学性质初步研究显示,H8005产生的CMC酶反应的pH值以8．0左右为适;在碱性条件下具有较高的酶活和一定的稳定性;反应温度以55℃左右为宜;且具有较好的温度稳定性。Mn^2＋与Fe^3＋对酶反应有促进作用,Cu^2＋和Pb^2＋对酶反应有抑制作用。该菌产生的纤维素酶在棉织品的水洗整理及洗涤剂工业中具有非常良好的应用前景。     

碱性淀粉酶的发酵生产及其应用研究进展

碱性淀粉酶是诸多碱性酶中的一种,指的是其最适稳定及反应pH值在碱性范围内,在碱性环境中可以保持稳定且可高效催化降解淀粉。碱性淀粉酶在纺织、洗涤剂、医药、食品等领域具有广泛应用。利用嗜碱微生物可以生产碱性淀粉酶。以下综述了碱性淀粉酶的发酵生产及其应用的研究进展。     

产碱性纤维素酶兼性厌氧菌株的筛选和酶学性质的初步研究

用CMC平板筛选方法, 从造纸厂碱性土壤中获得产碱性纤维素酶兼性厌氧菌株LZ-5, 革兰氏阴性, 经鉴定为弧菌属(Vibrio sp.)中的一个种。液体摇瓶培养产生碱性纤维素酶活力高达5.8 U/mL。酶学性质初步研究显示, LZ-5产生的纤维素酶最适反应温度为40℃, 最适反应pH值为9.0, 在碱性条件下具有较高的酶活性和较好的稳定性。经传代培养证实该菌株遗传性能稳定。     

蠼螋肠道中碱性内切葡聚糖酶基因的克隆表达及功能分析

【目的】从蠼螋肠道细菌菌株Q5中获得一个新型耐碱纤维素酶基因,通过异源表达、酶学性质及功能分析,旨在为以后进一步研究开发高温碱性纤维素酶提供一些理论参考。【方法】采用刚果红平板初筛法,从河南南阳宝天曼国家级自然保护区落叶堆下的昆虫蠼螋肠道中,获得具有分泌较高活性碱性纤维素酶的细菌菌株。基于该菌株的形态学、生理学及16S rRNA序列特征等对高活性菌株进行分类鉴定。并通过设计简并引物,从高活性菌株中克隆出该菌株的纤维素酶基因,并进行序列分析,并导入大肠杆菌BL21中表达。【结果】获得1株具有分泌较高活性碱性内切葡聚糖酶的细菌菌株Q5,经鉴定为甲基营养型芽孢杆菌,进一步从Q5菌株中成功克隆出该菌株的一个全长1500 bp的内切葡聚糖酶基因(GenBank KR067575),在NCBI比对后发现该基因的氨基酸序列与芽孢杆菌菌株LM 4-2的耐碱性β-1,4-内切葡聚糖酶基因(AKE23721.1)有98%的同源性。重组菌经优化培养,细胞破碎后上清液中的酶活力可达3.46 U/mL,是出发菌株Q5(2.05 U/mL)的1.69倍。经正交实验优化后的酶活力为4.99 U/mL。酶学性质研究表明:该酶的最适反应温度与pH值分别为50°C与pH 8.5,在pH 8.0和9.0保温48 h,其酶活力仍然维持到最高酶活的82%和81%;该酶在50°C以下较稳定,60°C以上酶活迅速降低。10 mmol/L的Ca~(2+)和Mg~(2+)对酶的活性有明显促进作用,重组酶Ega5的K_m和V_(max)分别是2.217 mol/mL和9.606μmol/(min·L)。该重组酶对棉花黄萎病病原菌大丽轮枝菌具有显著抑制作用。【结论】本文首次从蠼螋肠道中筛选到了一株产碱性内切葡聚糖酶的细菌菌株并从中克隆出了一个碱性纤维素酶基因,为该酶在碱性条件的应用奠定了理论基础。     

地衣芽胞杆菌碱性蛋白酶基因克隆与表达特性

目的建立高产量和高活力的地衣芽胞杆菌碱性蛋白酶基因表达体系。方法采用PCR技术克隆获得目的基因,将其连入表达质粒pET-32 a构建原核表达重组质粒,经测序鉴定后,转化BL21大肠埃希菌,不同温度下IPTG诱导表达融合蛋白,测定酶活;进一步对该基因和编码蛋白进行同源性比较和酶学性质分析。结果碱性蛋白酶基因序列全长1 149 bp,编码382个氨基酸,同源性为99%,融合蛋白分子质量为62 kD,蛋白酶酶活为29 000 U/mL,并且在25℃时是以可溶蛋白形式表达,37℃时部分蛋白以包涵体形式存在。结论此种表达体系可以成功表达具有生物活性的碱性蛋白酶,诱导温度对蛋白酶存在形式具有较大影响。     

短小芽孢杆菌碱性蛋白酶基因启动子的克隆、鉴定及其应用

利用TAIL-PCR(Thermal asymmetric interlaced PCR)从短小芽孢杆菌基因组中扩增到碱性蛋白酶基因编码区上游的启动子片段。对该片段的序列测定和分析表明, 此片段长797 bp, 但与基因表达有关的序列长约390 bp。对启动子片段进行不同长度的缺失突变, 以获得最小的基因启动子片段, 结果表明, 该基因起始密码子上游约160 bp的DNA片段就可以启动基因的表达。将含有该片段的碱性蛋白酶基因WApQ3插入大肠杆菌-芽孢杆菌穿梭质粒载体pSUGV4中, 构建了碱性蛋白酶基因表达质粒pSUBpWApQ3。将该质粒分别转入枯草芽孢杆菌和短小芽孢杆菌中表达, 可在胞外检测到碱性蛋白酶活性, 最高酶活分别为466.5 U/mL和3060 U/mL。     

Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures

The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detegents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, α-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed. Received: January 22, 1998 / Accepted: February 16, 1998     

Stabilization of alkaline proteinase and cellulases via complex formation with chitosan for use as detergent components

An effective approach to the stabilization of hydrolytic enzymes (alkaline proteinase and cellulases) via the complex formation with chitosan for their further use as detergent components has been developed. Interaction with chitosan results in a 35–50% increase in the level of catalytic activity of the enzymes after incubation for 60 min under the conditions of detergent use (alkaline pH, increased temperature, the presence of anionic surfactants) as compared to the system in the absence of chitosan both due to the enzyme stabilization and the increase of the starting level of catalytic activity. A twofold decrease of the enzyme inactivation constant is observed under the aforementioned conditions in the case of alkaline proteinase. In the case of cellulase preparation, the method for the control of the concentration of the active enzyme in the system modeling synthetic detergents has been suggested. The method is based on the enzymatic destruction of the stabilizing agent, chitosan, by enzymes of the cellulase complex. The destruction of chitosan removed the stabilizing effect, thus resulting in the inactivation of cellulases. The developed approaches allow for the widening of the field of the possible application of enzymes as detergent components.     

Desorption of Trichoderma reesei cellulase from cellulose by a range of desorbents

The desorption of Trichoderma reesei cellulase from Avicel by a wide range of desorbents was measured. Emphasis was placed on desorption at alkaline pH. A maximum desorption of 65-68% Avicelase activity was achieved by contact with NaOH, pH 10.0, at 40 degrees C for 5 min in the presence of 0.005% Triton X-100 or Tween 80. The design of a suitable desorption process using these conditions is discussed. Glycerol was also effective as a desorbent either alone or in combination with alkali and detergent. However, relatively high concentrations of glycerol were needed and the maximum desorption achieved, 68%, was not significantly greater than that with only alkali and detergent.     

Treatment of cotton with an alkaline Bacillus spp cellulase: activity towards crystalline cellulose

We analysed the influence of several enzymatic treatment processes using an alkaline cellulase enzyme from Bacillus spp. on the sorption properties of cotton fabrics. Although cellulases are commonly applied in detergent formulations due to their anti-redeposition and depilling benefits, determining the mechanism of action of alkaline cellulases on cotton fibres requires a deeper understanding of the morphology and structure of cotton fibres in terms of fibre cleaning. The accessibility of cellulose fibres was studied by evaluating the iodine sorption value and by fluorescent-labelled enzyme microscopy; the surface morphology of fabrics was analysed by scanning microscopy. The action of enzyme hydrolysis over short time periods can produce fibrillation on cotton fibre surface without any release of cellulosic material. The results indicate that several short consecutive treatments were more effective in increasing the fibre accessibility than one long treatment. In addition, no detectable hydrolytic activity, in terms of reducing sugar production, was found.     

海洋微生物产纤维素酶及其应用研究进展

纤维素是地球上最古老、最丰富的天然高分子,是天然可再生资源。纤维素酶广泛存在于自然界的生物体中,细菌、真菌和动物体内都能产生纤维素酶。微生物产纤维素酶已有较多报道,并在食品、医药、饲料、洗涤、纺织和造纸工业等领域有广阔的应用前景。海洋是一个巨大的资源库,海洋微生物产纤维素酶已经受到了广泛的关注。对产纤维素酶海洋微生物的种群、来源及基因筛选、海洋微生物产纤维素酶的酶学特性,以及纤维素酶的应用领域等方面的研究进展进行了简要综述,并对海洋微生物产纤维素酶的研究进行了展望。     

Directed evolution for engineering pH profile of endoglucanase III from Trichoderma reesei

The potential of cellulase has been revealed not only in biomass conversion but also in various industrial processes, including food, textiles, laundry, pulp, and paper. Due to the need for alkali-tolerant cellulase with high specific activity at alkaline pH, for example, for application in detergent industry an error-prone PCR approach was employed for enhancing the alkali-tolerant ability of endoglucanase III (EG III) from Trichoderma reesei by error-prone PCR. One mutant (N321T) which exhibited an optimal activity at pH 5.4, corresponded to a basic shift of 0.6 pH unit compared to the wild-type enzyme, was selected and characterized. In addition, two site-directed mutations, N321D and N321H, were designed to study the role of residue at position 321. As expected, the N321D mutation changed enzyme's optimal activity to pH 4.0, resulting in a large decrease in the specific activity. However, the N321H mutated enzyme was active over a broader pH range compared to the wild type, with no much change in the specific activity. These properties suggest that the residue at position 321 is important amino acid residue in determining the pH activity profile of the EG III from T. reesei.     

海洋细菌Bacillus sp．HN07的分离鉴定及所产碱性纤维素酶性质研究

从渤海海域（121°30′00″E,40°25′00″N）海泥中筛选到一株产碱性纤维素酶的海洋细菌。通过形态观察、生理生化特性及16SrDNA序列分析,鉴定该菌株属于芽孢杆菌属（Bacillus）,命名为Bacillussp．HN07。研究表明：HN07最适生长温度30℃,适宜在pH7．0～8．0、含2．0％～3．0％NaCl的培养条件下生长和产酶,并且具有较好的遗传稳定性。酶学性质初步研究显示,HN07所产碱性纤维素酶最适反应温度为45℃,最适pH为8．0,在碱性条件下具有较好的稳定性,具有潜在的工业应用价值。     

微生物纤维素酶的应用研究

我国纤维素酶的应用研究近年来取得了很大进展。阐述了纤维素分解菌的选育 ,酶学性质以及在发酵、纺织和洗涤剂工业中的应用。     

Effects of dietary addition of cellulase and a Saccharomyces cerevisiae fermentation product on nutrient digestibility,rumen fermentation and enteric methane emissions in growing goats

This study was designed to assess the effectiveness of dietary cellulase (243 U/g, derived from Neocallimastix patriciarum) and a Saccharomyces cerevisiae fermentation product (yeast product) on ruminal fermentation characteristics, enteric methane (CH₄) emissions and methanogenic community in growing goats. The experiment was conducted in a 5 × 5 Latin square design using five Xiangdong black wether goats. The treatments included a Control and two levels of cellulase (0.8 g and 1.6 g/kg dry matter intake (DMI), i.e. 194 U/kg and 389 U/kg DMI, respectively) crossed over with two levels (6 g or 12 g/kg DMI) of the yeast product. There were no significant differences regarding feed intake, apparent digestibility of organic matter, neutral detergent fibre and acid detergent fibre among all the treatments. In comparison with the Control, the ruminal ammonia N concentration was decreased (p = 0.001) by cellulase and yeast product addition. The activities of carboxymethylcellulase and xylanase were decreased after cellulase addition. Moreover, dietary cellulase and yeast product addition led to a significant reduction (p < 0.05) of enteric CH₄ emissions although the diversity and copy numbers of methanogens among treatments were not dissimilar. The present results indicate that the combination of cellulase and yeast fermentation product can reduce the production of CH₄ energy and mitigate the enteric CH₄ emissions to a certain degree.     

重金属污染区土壤酶活性变化

从福建龙岩新罗区特钢厂污灌区农田采集土壤,测定土壤基本理化性质及脲酶、纤维素酶、碱性磷酸酶、多酚氧化酶、过氧化氢酶活性和Cu、Cd、Pb、Zn含量,探讨重金属污染和土壤性质对土壤酶活性的影响.结果表明： 4种全量或有效态重金属与土壤脲酶、纤维素酶、碱性磷酸酶和多酚氧化酶活性呈显著正相关,与过氧化氢酶活性呈显著或极显著负相关;土壤pH与碱性磷酸酶活性呈极显著正相关,粉粒含量与过氧化氢酶活性呈显著负相关.经通径分析,重金属污染刺激了脲酶、多酚氧化酶和纤维素酶活性,但对碱性磷酸酶活性的影响较小.有效态Cu、Cd、Pb、Zn对过氧化氢酶活性的直接影响并不大,但通过间接途径抑制了过氧化氢酶活性.土壤理化性质对5种土壤酶活性的影响较大,碱解氮直接抑制了脲酶活性;全磷直接刺激了碱性磷酸酶和过氧化氢酶活性,并通过有效磷刺激了纤维素酶活性;有效磷直接刺激了纤维素酶活性,直接抑制了碱性磷酸酶和过氧化氢酶活性;全钾直接抑制了碱性磷酸酶和多酚氧化酶活性;速效钾通过有效磷刺激了纤维素酶活性;土壤颗粒组成明显影响多酚氧化酶和过氧化氢酶活性.5种酶活性与土壤Cu、Cd、Pb、Zn含量之间的关系不明确,因此其活性不是指示土壤Cu、Cd、Pb、Zn污染的良好指标.