印尼热泉菌Bacillus sp. BI-3产琼胶酶的发酵条件优化

采用响应面法对1株高产琼胶酶的印度尼西亚热泉菌Bacillus sp. BI-3的发酵条件进行优化。首先以温度、pH、不同碳源、不同氮源、不同金属离子及接种量作为唯一变量进行单因素实验, 筛选出对酶活有显著影响的单因素取值范围; 参考单因素实验结果, 采用Plackett-Burman实验设计筛选出影响酶活主要因素, 再利用Box-Behnken设计及响应面分析法进行回归分析以确定最佳发酵条件。结果表明, D-甘露糖、氯化锶和氯化钙与酶活存在着显著的相关性, 通过求解回归方程得到Bacillus sp. BI-3的发酵条件系统优化的结果为: 酵母粉0.3%、D-甘露糖0.66%、蛋白胨0.6%、氯化钙7.21mmol/L、氯化锶6.00mmol/L、氯化钠添加量6‰、培养温度为55℃、接种量1%、初始pH 6.0, 优化后发酵上清液的酶活达到6.0U/mL, 比优化前提高了2.4倍。     

海洋弧菌QJH-12发酵产琼胶酶条件的优化

从中国南海海域分离到了一株具有较高琼胶降解活性的弧菌Vibrio sp.QJH-12,该菌株为革兰氏阴性菌,呈弯曲杆状,极生单根鞭毛,氧化酶阳性,过氧化氢酶阳性,O/129试验阳性。在琼胶平板上生长可软化琼胶,并在菌落周围产生凹陷。通过单因子实验和正交实验确定了培养基的最佳组成:琼胶2.0‰,柠檬酸三铵1.0‰,K2HPO41.0‰,NaCl1.0%。确定该菌株的最适发酵产酶条件:装液量为250mL的三角瓶中装入125mL的液体发酵培养基,培养基起始pH6.5,摇瓶转速150r/min,30℃发酵培养26h,发酵液的酶活力达到332U/mL。结果表明,该菌株从产酶活力和发酵特点等各方面具备工业化开发的潜力。     

一株海藻多糖降解菌的分离鉴定及产酶条件优化

从大型褐藻藻体分离获得一株具有高效降解琼胶和褐藻胶能力的革兰氏阴性菌菌株ST-6。16S rDNA序列分析结果表明,该菌株与海绵假单胞菌的相似度达到99%,NJ法构建系统进化树也与海绵假单胞菌归为一类,鉴定为海绵假单胞菌Pseudomonas pachastrellae ST-6。在2216E培养基、30℃培养条件下,海绵假单胞菌ST-6的生长曲线表明,接种10~48 h为菌株的指数生长期,48~72 h为生长稳定期。产酶结果表明,菌株ST-6在指数生长期时菌液的胞外琼胶酶相对酶活力较高,在接种48 h时,菌液的琼胶酶相对酶活力最高为249.15 U/m L。此外,发现菌株ST-6的琼胶酶和褐藻胶酶的分泌类型分别为非诱导型和诱导型。采用单因素分析法对其生长和产酶条件进行分析,结果表明,海绵假单胞菌ST-6最适生长条件为:温度25~35℃,33值5~9;最适产琼胶酶条件为:温度30℃,33值为7,琼胶浓度0.3%。最适产褐藻胶酶条件为:温度35℃,33值为9。在温度30℃、339和褐藻酸钠浓度0.15%的培养条件下,海绵假单胞菌ST-6获得最高胞外褐藻胶酶相对酶活力为135.54 U/m L。     

厦门沿岸海域杂色鲍中产琼胶酶菌株的筛选及其酶学性质的研究

琼胶酶在食品工业中的多糖降解中有着重要的作用,其经济价值日益凸显,从海洋生物中筛选琼胶酶菌株是获得琼胶酶的一种重要途径.从厦门沿岸海域养殖杂色鲍鱼体内分离得到5株产高效琼胶酶的菌株,其中最高的酶活力达到133.5 U/dm3.经16S rRNA序列分析表明这5株菌株分别属于弧菌属（Vibrio）和假交替单胞菌属（Pseudoa lteromonas）.采用DNS法对这些菌株所产的琼胶酶进行酶学性质研究,结果显示这些酶的最适作用温度均为50℃,最适作用pH值为7.0;Na＋可使A017菌株所产的琼胶酶酶活力提高5倍,Fe2＋对A007、A008、A010、A021菌株所产的琼胶酶酶活力有明显的抑制作用.     

4株琼胶降解菌的分离、鉴定及产酶条件分析

从海藻及海参中分离筛选得到4株琼胶降解菌HD、JL、QJ和HS,均为革兰氏阴性菌,确定了它们发酵产酶的最佳条件均为：2216E海水培养基、初始 Ph 7.5、琼胶底物浓度0.30%、培养温度28℃、培养时间36 h。大部分酶分泌到细菌细胞外。生理生化检测、16SrDNA序列同源性及聚类分析结果表明, HD、JL、HS为白色噬琼胶菌(Agarivorans albus);QJ的16SrDNA序列与Simiduia sp.,糖噬胞菌属( Saccharophagus sp.)和船蛆杆菌(Teredinibacter sp.)的相似性为93%~94%,在进化树上单独形成一个分支,但生理生化特征与它们有所不同,分类地位需要进一步确定。从HD、JL和HS中能克隆到β-琼胶酶基因,它们与其他物种的β-琼胶酶基因序列的相似性为97%~98%。     

Identification of a marine agarolytic bacterium Agarivorans albus QM38 and cloning and sequencing its beta-agarase genes

A total of 117 agar-decomposing cultures were isolated from coastal seawater around Qingdao,China.The phenotypic and agarolytic features of an agarolytic isolate,QM38,were investigated.The strain was gram negative,strictly aerobic,curved rod and polar flagellum.On the basis of several phenotypic characters,biochemical and morphological characters and phylogenetic analysis of the gene coding for the 16S rRNA,the strain was identified as Agarivorans albus strain QM38.This strain can liquefy the agar on the solid agar plate.An excellular agarase activity was determined in liquid culture.The enzyme exhibited maximal activity at 40℃,pH 7.6.Its activity was greatly affected by different concentrations of agarose.The highest activity 32 U/ml was achieved in the culture supernatant.The hydrolytic product was analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE).After complete hydrolysis of agarose,a series of agaro-oligosaccharides were produced.The main products of the enzymes were oligosaccharides in the degree of polymerization (DP) of 2,4,6 and 8.Three genes agaD01,agaD02 and agaD03,encoding β-agarases,had been cloned from genomic DNA of Agarivorans albus strain QM38.The open reading frame of agaD01,consisted of 2 988 bp,and shared 95.5%-98.9% identity to the β-agarase genes of some strains of Vibrio and Agarivorans.Gene agaD02 comprised 2 868 bp and encoded a 955-amino-acid protein.It showed 97.4% and 98.7% identity to the β-agarase genes of strain Vibrio sp.PO-303 and strain Vibrio sp.JT0107,respectively.Only partial sequence of agaD03 gene has been cloned.It showed 96.5% identity to β-agarase gene (agaB) of Pseudoalteromonas sp.CY24,and shared 96.8% identity to β-agarase-c gene of Vibrio sp.PO-303.     

A polysaccharide-degrading marine bacterium Flammeovirga sp. MY04 and its extracellular agarase system

Bacteria of the genus Flammeovirga can digest complex polysaccharides (CPs), but no details have been reported regarding the CP depolymerases of these bacteria. MY04, an agarolytic marine bacterium isolated from coastal sediments, has been identified as a new member of the genus Flammeovirga. The MY04 strain is able to utilize multiple CPs as a sole carbon source and grows well on agarose, mannan, or xylan. This strain produces high concentrations of extracellular proteins (490 mg L-1 ± 18.2 mg L-1 liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose. These proteins have an activity of 310 U mg-1 ± 9.6 U mg-1 proteins. The extracellular agarase system (EAS) in the crude extracellular enzymes contains at least four agarose depolymerases, which are with molecular masses of approximately 30-70 kDa. The EAS is stable at a wide range of pH values (6.0-11.0), temperatures (0-50℃), and sodium chloride (NaCl) concentrations (0- 0.9 mol L-1). Two major degradation products generated from agarose by the EAS are identified to be neoagarotetraose and neoagarohexaose, suggesting that β-agarases are the major constituents of the MY04 EAS. These results suggest that the Flammeovirga strain MY04 and its polysac-charide-degradation system hold great promise in industrial applications.     

Flammeovirga sp. SJP92琼脂酶的分离纯化及其酶学性质

琼胶酶是一类能够降解琼脂糖生成琼胶寡糖的酶类,具有广泛的应用范围.从海洋性高产琼胶酶菌株Flammeovirga sp. SJP92的发酵液经硫酸铵沉淀,DEAE 琼脂糖离子层析,分离纯化获得了一个分子量约为70KDa的琼胶酶AgaB.经过酶学性质分析,它的最适pH值为70,最适温度为40℃,在最适温度下能够保持较好的稳定性.进一步的分析表明, MgSO4和β Me对该酶有明显的促进作用,而CaCl2、MnCl2、ZnCl2、CoCl2、FeCl3、CuSO4等则会强烈抑制它的活性.此外,通过产物分析表明,该酶能够通过内切作用将琼脂糖最终降解成6糖和4糖.AgaB的分离纯化及其酶学性质分析为其工业应用奠定了基础.     

响应面法优化产琼胶酶南极菌Pseudoalteromonassp. NJ21的发酵条件

采用响应面法对1株高产琼胶酶的南极海冰菌Pseudoalteromonassp. NJ21的发酵条件进行优化。首先分别以温度、pH、不同碳源、不同氮源、不同金属离子作为唯一变量进行单因素实验, 筛选出对酶活有显著影响的单因素取值范围; 参考单因素实验结果, 采用Plackett-Burman实验设计筛选出影响酶活主要因素, 再利用Box-Behnken设计及响应面分析法进行回归分析以确定最佳发酵条件。结果表明, D-甘露糖、蛋白胨、CaCl2和培养温度与酶活存在着显著的相关性, 通过求解回归方程得到Pseudoalteromonassp. NJ21的最佳培养基组成为D-甘露糖5.52g/L; 蛋白胨7.4g/L; CaCl25.84mmol/L; 最佳发酵温度为15℃; 优化后发酵液中琼胶酶酶活由3.5U/mL提高到25.76U/mL, 比优化前提高了6.36倍。