Effects of Aliphatic Alcohols on Formation of Cyclodextrin from Soluble Starch by Bacillus macerans Cyclodextrin Glucanotransferase

Effects of aliphatic alcohols such as ethanol, n-butanol and n-decanol on the formation of cyclodextrin (CD) from soluble starch by Bacillus macerans cyclodextrin glucanotransferase (CGTase) were experimentally studied at 50°C and pH 6.0. The addition of such aliphatic alcohols caused an increase in α-CD yield, which extent was remarkable in n-butanol. From the incubation of α-CD with CGTase, such an increase was found to be due to a restriction on further transglycosylation of α-CD into β-CD by CGTase imposed by the aliphatic alcohols.     

Coupling Reaction of Bacillus macerans Cyclodextrin Glucanotransferase on Glucosyl-α-cyclodextrin and Glucose

Treatment of glucosyl-α-cyclodextrin with radioactive glucose in the presence of Bacillus macerans cyclodextrin glucanotransferase, yielded radioactive maltose, maltotriose, and four kinds of glucosyl branched oligosaccharides. The branched oligosaccharides formed had identical Rf values as those of branched oligosaccharides of 5-8 glucose unit on paper chromatography using the solvent system of 1-butanol: pyridine: water (6/4/4), (Fractions B₅-B₈). Individual oligosaccharides were isolated and their structures determined using the action of porcine pancreatic α-amylase, β-amylase, pullulanase, isopullulanase and glucoamylase. The structure of the main component of the fraction B₅ was 6⁴-O-α-glucosylmaltotetraose, B₆ was 6⁴-O-α-glucosylmatopentaose, B₇ was 6⁵-O-α-glucosylmaltohexaose, and B₈ was 6⁶-O-α-glucosylmaltoheptaose. Fraction B₈, which formed at the initial stage of the reaction, contained 6⁴, 6⁵ and 6⁶-O-α-glucosylmaltoheptaose. Reducing end ¹⁴C-labelled 6⁴-O-α-glucosylmaltotetraose was degraded to 6³-O-α-glucosylmaltotriose and radioactive glucose by the exhaustive action of Bacillus macerans cycloamylose glucanotransferase, and 6³-O-α-glucosylmaltotriose was completely non-reactive to the enzyme. From these results, we proposed a model of the active site of the enzyme.     

Bacillussp.HA-1产环糊精葡萄糖基转移酶发酵工艺研究

在10 L 发酵罐中,研究了环糊精葡萄糖基转移酶产生菌 Bacillus sp．HA-1的发酵规律,结果表明,在菌体对数增长期,发酵液溶氧处于较低状态,发酵液 pH 在对数增长前期持续下降,中期后回升,菌体产酶呈增长趋势；当菌体增长进入稳定期,发酵液溶氧迅速回升至初始状态,pH 也回升至起始值,菌体产酶接近峰值。根据发酵规律优化发酵工艺,通过有效增加菌体对数增长期发酵液溶氧,使菌株产酶到达高峰的时间缩短了 50%以上。应用该菌株在5 m~3生产罐中发酵,24 h 产酶水平达到6 800 IU/mL。     

环状糊精葡萄糖基转移酶的研究进展

环状糊精葡萄糖基转移酶是催化淀粉等合成环状糊精的酶,环状糊精由于具有特殊的理化性质,能将多种化学物质包接在其环形空隙中,因此在食品等领域有着广泛的应用前景。本文对环状糊精葡萄糖基转移酶的来源、酶学性质、作用机理、筛选及分子生物学方面进行了系统的综述。     

环麦芽糊精葡聚糖转移酶高活性的土壤微生物菌种筛选

根据环麦芽糊精葡聚糖转移酶(CGTase)催化淀粉和糊精生成环状糊精(CD),环状糊精可以吸附筛选培养基中的刚果红和甲基橙使菌落周围形成透明圈,且透明圈越大,透明度越好,CGTase的转糖基活性越高的酶催化特性,设计筛选模版,进行产酶菌株分离筛选。经初筛、复筛得到了YS-19和YS-40的2株转糖基活性较高CGTase菌种,酶活分别为2.90U/mL和3.15U/mL。     

环糊精葡萄糖基转移酶分离纯化及酶学性质研究

通过筛选和诱变获得一株高产环糊精葡萄糖基转移酶芽孢杆菌菌株,研究该菌所产环糊精葡萄糖基转移酶的分离纯化及酶学性质,结果显示：发酵液经离心处理后,采用硫酸铵溶液分步盐析、DEAE-cellulose 52 离子交换层析、Sephadex G-200 凝胶过滤层析方法得到电泳级环糊精葡萄糖基转移酶,SDS-PAGE 电泳显示该酶分子量为33kD,纯化倍数为10,得率为14.4%。该酶反应的最适温度为50℃,在40～60℃基本稳定,最适pH 值为8.0,在pH6.0～10.0 范围内基本稳定。Fe2+、Cu2+、Mg2+ 对该酶活力有明显的抑制作用。     

利用来源于Paenibacillus macerans的α-CGTase突变体Y89D生产α-环糊精

对α- 环糊精葡萄糖基转移酶(α-CGTase)突变体Y89D 制备α- 环糊精的影响因素进行初步研究。其因素包括淀粉种类(马铃薯淀粉、玉米淀粉、木薯淀粉、可溶性淀粉)、加酶量、反应时间、pH 值、有机溶剂(乙醇、异丙醇、正丁醇、正癸醇)和温度。结果表明：选用5g/100mL 马铃薯淀粉、pH5.0、温度30℃、加酶量控制在 每克淀粉5U 左右,反应体系中加入体积分数5% 的正癸醇,反应6h 后,淀粉总转化率可达70%,其中α- 环糊精在产物中质量分数约为85%,转化产物中含有少量β- 环糊精(15%),而极少生成γ- 环糊精。因此,α-CGTase突变体Y89D 在制备α- 环糊精工艺中具有很好的工业化应用前景。     

Kinetic Characterization of Chimeric Cyclomaltodextrin Glucanotransferase from Genes of Two Alkalophilic Bacillus

The chimeric CGTase, which was constructed genetically with two β-CD forming type enzyme genes of different species of alkalophilic Bacillus, was characterized by comparing with the actions of parent enzymes. The final composition of CDs in the reaction digest from starch by the chimeric enzyme, especially in the yield of α-CD, was different from those by parent enzymes, and total yields of CDs by these enzymes were almost the same. The chimeric enzyme catalyzed the formation of β-CD as a major action from starch, the rapid synthesis of α-CD from other CDs and maltooligosaccharides and the slow degradation and interconversion of α-CD to others. From these results, the role of second and third segments in the enzyme molecules was discussed.     

嗜碱芽孢杆菌产环糊精葡萄糖基转移酶发酵条件的优化

对一株嗜碱芽孢杆菌产环糊精糖基转移酶的发酵条件进行了研究。利用单因素试验和正交试验获得该菌株产环糊精糖基转移酶的最佳条件为：接种量3％；培养温度30℃；pH10．5；发酵培养基的组成为玉米粉2％，酵母膏1．5％，玉米浆5％；250ml三角瓶装液量为30ml；270r/min振荡培养3d，其发酵液酶活可达5400U／ml左右。10L罐发酵时酶活可达5820U／ml。     

姜黄素超分子包合物对乙醇诱导LO2细胞损伤的保护作用

本文考察了姜黄素超分子包合物（Curcumin/Cyclodextrin polymer inclusion complex, CUR/CDP）对乙醇诱导LO2细胞损伤的保护作用。采用MTT法建立乙醇诱导LO2细胞损伤的模型,通过谷氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-PX）、丙二醛（MDA）和活性氧（ROS）等试剂盒考察了CUR/CDP对乙醇诱导LO2细胞损伤的保护效果。结果表明,经CUR/CDP（80 μg/mL）处理后,LO2细胞的存活率从54.75%±8.97%（模型对照组）提高到85.27%±2.64%,且细胞培养液中ALT、AST和LDH活力分别从（26.47±0.90）、（41.02±4.41）、（63.77±4.95）U/L（模型对照组）降低到（16.17±0.42）、（22.62±0.79）、（32.25±1.69）U/L（P<0.01）,LO2细胞内GSH-PX、SOD活力分别从（21.82±1.34）、（8.45±1.11）U/mg prot（模型对照组）升高到（36.70±0.56）、（16.47±1.27）U/mg prot,LO2细胞内MDA和ROS含量分别从（1.19±0.15）nmol/mg prot、（198.02%±11.76%）（模型对照组）降低到（0.72±0.05）nmol/mg prot、（110.87%±10.22%）（P<0.01）。综上所述,CUR/CDP通过提高LO2细胞相关抗氧化酶的活力和降低细胞内ROS含量来改善乙醇诱导LO2细胞的损伤。     

Tryptophan Residues of Bacillus Cycloamylose Glucanotransferase: Effect of Modification with N-Bromosuccini-mide on the Enzyme-catalysed Synthesis of Cyclomaltoheptaose from Maltotriose

Cycloamylose glucanotransferase(cyclomalto-oligosaccharide glu-canotransferase, CGTase), obtained from Bacillus stearotheromophilus, was found to catalyze the synthesis of cyclomalto-oligosaccharides from maltotriose (G₃) as a substrate. The synthetic reaction is one of the CGTase-catalysed reactions, so called “cyclisation”. A main saccharide produced by the cyclisation reaction under the experimental conditions employed was confirmed to be cyclomaltoheptaose CG₇. The kinetic parameters, the Michaelis constant Km and the molar activity k₀ were evaluated for the cyclisation reaction. Modification of tryptophan residues with N-bromosuccinimide (NBS) was found to effect on Km, but not on k₀ of the cyclisation reaction, suggesting that the tryptophan residue (Trp⁹⁷) carries an essential role in the binding (disproportionation process) of maltotriose.     

Production and Characterization of Cyclodextrin Glycosyltransferase from Bacillus lentus

A new alkalophilic Bacillus. which produced exceptionally high activity of cyclodextrin glycosyltransferase, was isolated from soil. This strain of Bacillus was identified as Bacillus lentus. The enzyme was purified and characterized. It was found that optimum pH and temperature for cyclodextrin forming activity were 6.5–8.5 and 45–55°C. Thermal stability of the enzyme was remarkably increased by the addition of calcium ions. The maximum conversion of starch to cyclodextrin by the enzyme was 65%. The cyclodextrins formed after 24 h of enzymatic reaction consisted of α-. β- and -y-cyclodextrins in proportion of 1:67 : 1.6.     

短小芽孢杆菌热稳定环糊精葡萄糖基转移酶粗酶酶学特性研究

从土壤分离物中筛选到一株环糊精葡萄糖基转移酶(cyclodextrin glucanotransferase,CGTase)产生菌CGT01.16SrDNA序列分析表明该菌与短小芽孢杆菌(Bacillus pumnilus)的同源性为99%.同时根据其生理生化特征,该菌株被鉴定为短小芽孢杆菌(B.pumilus).命名为B.pumilus.CGT01菌株.产生CGTase的最适初始pH为6.5,最适培养温度为37℃.菌株所产CGlke的最适pH值和最适反应温度分别为pH 6.5和60℃,并有较高的热稳定性.60℃条件下保温,酶活基本稳定,半衰期为35 min.酶液中添加1 mmol/L Ca2+能明显提高CGTase的稳定性,60 ℃保温1 h后,剩余酶活仍达80%以上.SDS-PAGE检测表明酶的分子量约为78 ku左右.经高效液相色谱分析表明,CGTase作用于可溶性淀粉后的主要产物为葡萄糖、麦芽糖和β-环糊精,没有检出α和γ型环糊精产物,因此所产环糊精为单一类型,不同于同种属的其他菌株所产CGTase.     

橡实淀粉生料发酵生产燃料酒精工艺研究

对以橡实淀粉为原料生料发酵生产燃料酒精的工艺条件进行了研究。结果表明:采用超声波助提法可有效脱除单宁,提高橡实淀粉的燃料酒精转化率。单因素试验和正交试验得出的橡实淀粉生料发酵最佳工艺参数为料水比16∶10,α-淀粉酶50 U/g,糖化酶190 U/g,发酵温度25℃,发酵时间7 d。     

利用低温蒸煮工艺进行高浓度酒精发酵

本研究利用低温蒸煮工艺,探讨了玉米淀粉的高浓度酒精发酵。在80℃下对玉米粉进行蒸煮15min,同时加α—淀粉酶进行液化。经冷却后,在55℃下加糖化酶糖化30min。再度冷却到30℃,加酵母菌悬液发酵70h。并利用这一技术研究了产高浓度酒精酵母菌株的最佳发酵条件。在加糖化酶量200～300u/g原料。酵母接种量为3％(v/v),发酵温度为30℃,pH4～4.5,发酵周期为70h,原料水比为1:2条件下,所选用的菌株之一W_4可以产生17.5％(v/v)乙醇。发现该菌株在发酵速度和耐酒精能力方面明显地优于国内酒精厂常用的酵母菌株1300。在发酵结束时,成熟发酵醪的pH值为4.2。外观糖度为0(Bx),残还原糖为0.19％,残总糖为3.6％,淀粉利用率为90％。     

茶叶乙醇提取物对蜡质玉米淀粉回生性质的影响

以蜡质玉米淀粉(waxy corn starch,WCS)为原料,研究不同添加量(1.0％、2.5％、5.0％、10.0％)茶叶乙醇提取物(ethanol extract of tea,EET)与WCS共糊化后的相互作用以及对其透光率、老化度、溶解度、膨胀度、凝胶强度、糊化及回生特性、结晶结构、微观结构及体外消化性能的...     

乙醇生产的技术进步——新型酶技术给乙醇生料发酵生产带来的突破

由于能源价格的增长和乙醇的广泛应用,发酵乙醇日益具有竞争力。文中对发酵法生产乙醇的状况和技术进行了简单回顾,着重介绍了新型生料水解酶的作用原理及其研究进展,并对该项技术的使用和对生产带来的益处进行了分析。     

Raw Starch Digestion by Bacillus polymyxa beta-Amylase

Unlike plant ß-amylases, Bacillus polymyxa ß-amylase digests raw starch at a significant rate. The action of this bacterial ß-amylase on raw starch is stimulated greatly by the presence of the debranching enzyme pullulanase.     

超声波对甘薯回生抗性淀粉生成的作用

以甘薯淀粉为原料,研究超声波作用时间、作用温度、作用顺序、盐离子以及淀粉乳浓度对回生抗性淀粉制备产率的影响。研究结果表明,超声波作用下制备回生抗性淀粉的最佳工艺条件为:淀粉乳浓度20%,NaCl的最佳加入量为每100 mL淀粉乳2.0 g,α-淀粉酶加入量200 U/100 mL,酶解时间30 min,酶解温度95℃,超声波作用在酶解和高压之间,超声波作用时间60 min,作用温度30℃,压热温度120℃,压热时间30 min,老化时间12 h,在这种工艺条件下,甘薯回生抗性淀粉产率最高为8.2%,比未经超声波作用的2.5%提高了2.28倍。     

洋葱皮乙醇提取物对玉米淀粉回生性质的影响

研究洋葱皮乙醇提取物(Ethanol Extract of Onion Skin, EEOS)对玉米淀粉热力学性质、结晶结构和微观结构的影响,综合评价EEOS对玉米淀粉回生的抑制作用以及对玉米淀粉体外消化性能的影响。结果表明,与对照组相比,添加2.5%、5%、10%的EEOS的玉米淀粉,在4℃回生5 d后,熔融焓值分别降低了29.77%、21.66%和15.26%,且随着EEOS添加量的增加,玉米淀粉的熔融焓值呈现先下降后上升的趋势;添加2.5%EEOS的淀粉,在4℃回生15 d后,其1 047/1 022的比值从下降了6.31%;与对照组相比,添加不同比例的EEOS的淀粉的相对结晶度有所下降;添加EEOS的玉米淀粉在4℃回生15 d后,表面光滑无破损现象;添加2.5%、5%、10%EEOS的玉米淀粉,在4℃回生5 d后,RDS的含量分别比对照组下降了21.36%、33.22%和29.87%,说明EEOS的添加对玉米淀粉中RDS的消耗有抑制作用。