一株产几丁质酶菌株的筛选及其产酶条件的研究

采用平板透明圈法从土壤中分离筛选到一株产几丁质酶放线菌株L12,用250mL摇瓶发酵初筛和复筛,酶活力为0.63U/mL。通过产酶条件实验,初步确定了该菌株较适产酶培养基和摇瓶发酵条件。条件优化后,30℃、250mL摇瓶发酵48h,几丁质酶活力达到1.06U/mL。     

卡拉胶固定粘质赛氏菌产碱性蛋白酶的研究

将粘质赛氏菌(Seratia marcescens)包埋于卡拉胶中,发现2．5％的卡拉胶适于固定该菌产碱性蛋白酶。固定化细胞在其较适宜产酶培养基中发酵,酶活力一般可达400u/ml,在卡拉胶中添加3％玉米粉和1%豆饼粉或2％砂子制备固定化细胞,其产酶能力分别提高了25%和23．9％;固定化细胞颗粒越小,其产酶能力越高。采用摇瓶半连续发酵。其产酶半衰期为14次(24小时为一个周期);而用环流器进行半连续发酵,其产酶半衰期为52次(12小时为一个周期),产酶效率分别比游离细胞摇瓶发酵的产酶效率高11．8％和45．07％,而环流器半连续发酵的产酶效率比摇瓶半连续发酵高29．7％。     

两株高产纤维素酶细菌的筛选、鉴定及酶学特性

从腐烂枯叶及附近土壤筛选分离得到2株产纤维素酶的菌株。经细菌形态观察、生理生化实验并结合16S rRNA序列分析,将其初步鉴定为地衣芽孢杆菌CT1(Bacillus licheniformis CT1)和枯草芽孢杆菌CM2(Bacillus subtilis CM2)。经摇瓶发酵,测定其CMCase、FPA酶活力,结果表明CT1和CM2在液体摇瓶培养4 d后的CMC酶活最大,分别可达163.3 U/mL和167.17 U/mL;CT1摇瓶培养2 d后,FPA酶活达到了211.17 U/mL,CM2摇瓶培养3 d后,FPA酶活为207.83 U/mL。进行不同碳源对菌株产酶能力影响的试验,并通过SDS-聚丙烯酰胺凝胶电泳、银染后初步分析纤维素酶谱条带,发现菌株对不同来源纤维素的降解能力及产纤维素酶的种类均有所不同。     

等离子体法诱变淡紫拟青霉产几丁质酶菌种的筛选

采用菌株诱变技术,提高生防用淡紫拟青霉菌株产几丁质酶的能力。通过常温常压等离子体诱变技术(MPMS)对淡紫拟青霉进行诱变育种处理,对处理的菌种先采用透明圈法进行初筛,然后采用发酵方法进行复筛。采用MPMS法诱变淡紫拟青霉产几丁质酶菌种时,温度25℃,处理时间30 s,样品处理量60μL,诱变菌的致死率为30.33%时,正突变率为14%。采用摇瓶分批发酵培养,诱变菌种的几丁质酶活为0.17 U/mL。结果表明,经过对淡紫拟青霉的诱变处理,获得高活性几丁质酶产生菌株,几丁质酶酶活提高180%。     

高温中性蛋白酶及其产生菌的初步研究

对高温中性蛋白酶产生菌耐热芽孢杆菌(Thermophilic Bacillus)XJT9503的产酶条件及酶的提取进行了初步研究。耐热芽孢杆菌XJT503产酶的摇瓶培养基最适碳源为葡萄糖,最适氮源为豆饼粉,100r／min,46℃培养60h酶活最高。实验结果表明高温中性蛋白酶提取可采用硫酸铵分级沉淀法。     

表皮短杆菌ZJB-07021产R-酰胺酶培养条件的优化

采用响应面分析法(RSM)对R-酰胺酶产生菌Brevibacterium epidermidis ZJB-07021的发酵培养基进行了优化.首先运用了单因子试验筛选出了发酵培养的最佳pH与温度,在此基础上采用Plackett-Burman(PB)设计法,对 8 种影响产酶的因素进行评价,实验结果表明,葡萄糖、酵母粉与乙酰胺含量对菌株产酰胺酶的活力具有显著的影响.通过旋转中心组合实验考察了葡萄糖、酵母粉和乙酰胺这三个主要因素对菌株所产酰胺酶活力的影响.发酵培养基优化结果为葡萄糖 17.00 g/L,酵母粉 15.74 g/L,乙酰胺 7.05 g/L,采用优化后的发酵培养条件进行摇瓶发酵培养,酰胺酶的酶活达到 72.14 U/L,比优化前的初始发酵培养条件下的酶活提高了73.3%.     

产青霉素酶枯草芽孢杆菌发酵条件的优化

目的:对一株产青霉素酶的重组枯草芽孢杆菌(Bacillus subtilis DB104/pWB-pemp)的摇瓶发酵条件进行优化.方法:利用单因素实验及正交实验等方法考查重组菌摇瓶发酵条件,研究不同浓度的碳源、氮源、金属离子、磷酸盐及不同pH、接种量、装液量和温度等条件对产青霉素酶的影响.结果:重组菌株摇瓶最适发酵条件为pH 7.0、接种量3％、装液量14％、发酵温度37℃,培养基成分为2％蔗糖、3.5％酵母膏、0.1mmol/L镁离子、0.4％磷酸盐.结论:最优条件下,发酵36h,产酶活力达到2227.8U/mL,为先前研究结果(1 580 U/mL)的1.41倍,为重组菌的大规模发酵生产奠定了基础.     

亚硝基胍诱变选育低温β-半乳糖苷酶高产菌

以野生低温β-半乳糖苷酶产生菌水生拉恩菌(Rahnella aquatilis)14-1为出发菌株.通过亚硝基胍(NTG)诱变及低温驯化,采用选择性平板初筛和摇瓶复筛,筛选出一株产酶活力比原始菌株提高54%的突变株,该突变株经传5代培养,产酶特性稳定.     

盾壳霉产生几丁质酶的条件研究

本实验采用摇瓶培养研究了盾壳霉(Coniothyrium minitans)产生几丁质酶的条件。改良的天然马铃薯葡萄糖培养基(mPDB)较合成培养基(SMCS)更适宜作为盾壳霉产生几丁质酶的基础培养基。添加9种不同碳源试验表明葡萄糖较适宜于盾壳霉产几丁质酶;氮源试验表明硝酸钾是产酶的较适宜氮源。盾壳霉形成几丁质酶的培养时间以15 d为佳,培养的适宜pH值为6.5。     

低温脂肪酶产生菌的筛选、产酶发酵及粗酶性质研究

利用含有Tween 80的琼脂平板和摇瓶发酵法,从若尔盖高原土壤中筛选产脂肪酶菌株.通过菌落形态和菌体特征观察初步对菌种进行鉴定,得到一株产低温脂肪酶的适冷菌Pseudomonassp.DL-B,并设计正交试验对该菌株的产酶发酵培养条件进行了优化.摇瓶实验表明,该菌株最适产酶发酵培养基为:蔗糖10 g/L,蛋白胨20 ...     

碱性果胶裂解酶摇瓶发酵条件的研究

利用碱性果胶裂解酶生产菌株Bacillus subtilis WSH02-02进行摇瓶发酵优化,确定了最适种子斜面培养基、种子摇瓶培养基和发酵摇瓶培养基等培养条件,经14h的摇瓶发酵,酶活最高达到8．29U／mL。     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

碱性果胶酶高产菌株的构建和高密度发酵

碱性果胶酶可用于苎麻脱胶和棉织物前处理的精练工艺,与传统的高温碱煮相比,具有保护纤维、降低能耗和化学污染的优势,因此获得高表达的碱性果胶酶基因工程菌,低成本生产碱性果胶酶对于纺织工业节能减排具有重要的意义。前期研究工作已经将来源于枯草芽孢杆菌Bacillus subtilis 168的碱性果胶酶基因pel经过密码子优化后在毕赤酵母Pichia pastoris GS115中成功表达。本研究为了提高其表达量,首先利用启动子和信号肽都优化的载体pHBM905BDM进行表达,摇瓶酶活从68 U/mL增加到100 U/mL,qPCR检测转录水平提高了27%。再利用果胶底物平板筛选水解圈大的转化子进行摇瓶发酵获得菌株GS115-pHBM905BDM-pels4,摇瓶酶活为536 U/mL。随后构建重组质粒pPIC9K-pels,电转化菌株GS115-pHBM905BDM-pels4,利用抗生素G418平板进行筛选,在含4 mg/mL的G418抗性平板上得到菌株GS115-pHBM905BDM-pPIC9K-pels1,摇瓶酶活为770 U/mL,qPCR测定含7个拷贝目的基因。最后将该菌株在5 L的发酵罐中进行高密度发酵,果胶酶酶活提高至2 271 U/mL。该碱性果胶酶酶活已达到目前酵母表达的最高水平,说明其具有很好的应用于纺织工业的潜力。     

响应面法优化多杀菌素发酵培养基的研究

采用响应面分析方法,对刺糖多孢茵（Saccharopolyspora spinosa）H-2产多杀菌素的发酵培养基进行优化研究。运用单因子试验筛选出葡萄糖和棉籽粉为最适碳源和氮源,通过Plack—ett—Burman设计试验,对影响发酵培养基的8个相关因子进行评估并筛选出具有显著效应的4个因子：葡萄糖、棉籽粉、黄豆饼粉及玉米浆。通过最陡爬坡实验逼近以上4个因子的最大响应区域后,采用Box-Behnken响应面分析法,确定发酵产多杀菌素最佳培养基为葡萄糖64．5g,麦芽糖20g,玉米浆2g,大豆油40g,棉籽粉25g,黄豆饼粉2．4g,蛋白胨25g,CaCO35g,定容至1L,pH7．0。培养基优化后多杀菌素产量由278．1mg／L提高到508．7mg／L,比初始多杀茵素产量提高了1．83倍。     

Characterization of a family 3 polysaccharide lyase with broad temperature adaptability, thermo-alkali stability, and ethanol tolerance

The 774-bp pectate lyase gene plyAI4 from Bacillus sp. I4 was cloned and expressed in E. coli. The gene encodes a 257-residue polypeptide (PlyAI4, 28.3 kDa) with the highest identities of 97.3% with a putative pectate lyase from Bacillus subtilis BSn5 (ADV94306) and 60.3% with an identified pectate lyase of the polysaccharide lyase family (PL) 3 from Paenibacillus amylolyticus 27C64 (ADB78774). The purified recombinant PlyAI4 (rPlyAI4) exhibited apparently optimal activity at pH 10.5 ?? 11.0 and 50°C. Compared with the majority of reported alkaline pectate lyases, rPlyAI4 exhibited more residual enzyme activity at 20°C (??45%) or at 70°C (??50%) and better thermostability at 70°C (??60 min half-life at 70°C). In the presence of 20% (v/v) ethanol, pectate lyase activity was enhanced by 0.2 fold. After incubation in 40% (v/v) ethanol at 37°C and pH 8.5 for 1 h, the purified rPelAI4 retained more than 75% of the initial activity. Sequence analysis proposed a new signature block, A-D-G-[V/I]-H, for PL 3 pectate lyases. These properties may prove to be important with regards to PlyAI4 for basic research and industrial application.     

Cloning and expression of a pectate lyase from the oral spirochete Treponema pectinovorum ATCC 33768

The pelA gene, encoding a pectate lyase, from Treponema pectinovorum ATCC 33768 was isolated by heterologous expression of a cosmid library in Escherichia coli. In vitro transposon mutagenesis identified an open reading frame of 1293 bp capable of encoding a protein of 430 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. Analysis of the amino acid sequence suggested that it is a member of the polysaccharide lyase family 10 of which all characterized members show pectate lyase activity. An amino-terminal His-tagged recombinant form of PelA was expressed and purified from E. coli. The recombinant enzyme has characteristics common to other bacterial pectate lyases such as an alkaline pH optimum, dependence on calcium ions for activity, and inhibition by zinc ions.     

Production of lyases byPhoma exigua associated with seed-rot ofVigna radiata

Phoma exigua associated with seed-rot ofVigna radiata produced lyases which varied with the media tested. The production of lyases was higher in pectin-supplemented media.Vigna seed meal medium was not suitable for induction of lyase production. The pectin lyase and pectate lyase was maximum after 11 d of incubation by which time the pH was shifted to alkaline side. Temperature of 25 °C and pH 9 was found to be optimum for the activity of pectin lyase and pectate lyase. Fungicides (antracol and panoctine), phenols (pyrocatechol and gallic acid) and growth substances (gibberellic acid and yeast extract) adversely affected the enzyme secretion.     

Erwinia chrysanthemi EC16 Produces a Second Set of Plant-Inducible Pectate Lyase Isozymes

The enterobacterium Erwinia chrysanthemi causes soft-rot diseases involving extensive tissue maceration in a wide variety of plants and secretes multiple pectic enzymes that degrade plant cell walls and middle lamellae. An E. chrysanthemi mutant with directed deletions or insertions in genes pehX, pelX, pelA, pelB, pelC, and pelE, which encode exo-poly-alpha-d-galacturonosidase, exopolygalacturonate lyase, and four isozymes of pectate lyase, respectively, was constructed by the marker exchange of a cloned pehX::TnphoA fragment into E. chrysanthemi CUCPB5010, a Delta(pelA pelE) Delta(pelB pelC)::28bp Delta(pelX)Delta4bp derivative of strain EC16. This mutant, E. chrysanthemi CUCPB5012, no longer caused pitting in a standard pectate semisolid agar medium used to detect pectolytic activity in bacteria. Nevertheless, the mutant still macerated leaves of chrysanthemum (Chrysanthemum morifolium), although with reduced virulence. The mutant was found to produce significant pectate lyase activity in rotting chrysanthemum tissue and in minimal media containing chrysanthemum extracts or cell walls as the sole carbon source. Activity-stained, ultra-thin-layer isoelectric focusing gels revealed the presence in these preparations of several pectate lyase isozymes with pIs ranging from highly acidic to highly alkaline. Sterile culture fluids containing these isozymes were able to macerate chrysanthemum leaf tissue. Unlike the products of the pelA, pelB, pelC, and pelE genes in E. chrysanthemi EC16, these plant-inducible pectate lyase isozymes were not produced in minimal medium containing pectate. The results suggest that E. chrysanthemi produces two sets of independently regulated pectate lyase isozymes that are capable of macerating plant tissues.     

响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

麻类脱胶高效菌株果胶裂解酶基因克隆与表达

【目的】克隆麻类脱胶高效菌株Dickeya sp.DCE-01的果胶裂解酶基因并进行原核表达,对表达产物进行纯化和酶学性质研究。【方法】根据该菌株全基因组序列预测的果胶裂解酶基因Q59419设计引物,PCR扩增后将该基因连接到pEASY-E1和pACYCDuet-1载体上,导入E.coli BL21(DE3)进行表达。选择酶活力高的阳性克隆子进行大量诱导表达后,采用超滤和Sephadex G-100凝胶层析两步法纯化出果胶裂解酶,研究其酶学性质。【结果】克隆到果胶裂解酶基因pel(GenBank登录号:JX964997),其序列全长1 128 bp,编码375个氨基酸。pACYCDuet-1-pel-BL表达胞外果胶裂解酶活力最高,发酵液粗酶活达298.8 IU/mL。其最适反应温度为50°C,最适pH为9.0;保温1 h,酶活稳定温度≤45°C,稳定pH为9.0?10.0。酶催化作用依赖于Ca2+,其最适作用浓度为2 mmol/L;Zn2+、Ca2+和NH4+促进酶活力,Fe3+和Pb2+严重抑制酶活力;聚半乳糖醛酸钠为该酶的最适底物。【结论】从麻类脱胶高效菌株中发掘到碱性果胶裂解酶基因,其表达产物在生物质加工过程中具有重要工业化应用前景。