毕赤酵母表达解淀粉芽孢杆菌壳聚糖酶及其水解制备可控结构壳寡糖

优化并全合成解淀粉芽孢杆菌（Bacillus amyloliquefaciens）壳聚糖酶编码基因并在毕赤酵母（Pichia pastoris）中实现分泌表达,表达产物的蛋白质量浓度达到0.23 mg/mL。壳聚糖水解酶的最适pH值为5.0,最适温度为45 ℃,比活力达52.2 U/mL。该酶在50 ℃以下较稳定。利用该酶水解低脱乙酰度壳聚糖并对产物进行了组成及结构分析。基质辅助激光解吸电离飞行时间质谱分析结果显示,酶解产物中包含聚合度3～15、不同脱乙酰度的壳寡糖。核磁共振鉴定结果显示,壳寡糖组分的还原末端及非还原末端均主要由氨基葡萄糖组成。综上,本研究高效表达了来源于解淀粉芽孢杆菌的壳聚糖酶,并制备了确定末端结构的壳寡糖,为壳寡糖的结构与功能关系研究提供理论支持。     

纤维素酶制备低脱乙酰度壳寡糖及其组成分析

使用具有壳聚糖水解活性商品纤维素酶对制备的低脱乙酰度壳聚糖进行水解,进而对产物的组成及结构进行分析。分子排阻高效液相色谱(Size exclusion high performance liquid chromatography,SE-HPLC)结果显示,水解产物主要为相对分子质量1000~5000的低聚合度壳聚糖及壳寡糖。超高效液相色谱-四极杆飞行时间质谱(Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry,UPLC-QTOF MS)可对水解物中小分子组分进行有效分离及鉴定,根据一级质谱信息,推测其中含有N-乙酰氨基葡萄糖及17种不同结构的壳寡糖。使用核磁共振(Nuclear magnetic resonance,NMR)对其进行分析,结果显示,产物壳寡糖的还原端主要为N-乙酰氨基葡萄糖,非还原端基本为氨基葡萄糖。综合上述测定结果,推测纤维素酶中发挥壳聚糖水解作用的主要为几丁质酶。     

微生物几丁质酶研究概况及其在食品工程中的应用

几丁质酶主要产生于微生物,其降解产物应用极其广泛,降解几丁质,产生的氨基寡糖素可调节植物细胞生命代谢活动;产生的N-乙酰几丁寡糖和壳聚几丁寡糖可用作食品添加剂和生长促进因子.另外在抗病基因工程中,对几丁质酶的研究比较多,它在害虫防治中具有增效作用.本文主要就微生物几丁质酶的特点、理化性质、结构和分类,及其在食品工程中的应用展开论述.     

1 株高产几丁质脱乙酰酶红球菌的基因组测序及其应用潜力分析

为高效利用虾蟹壳资源,推动壳聚糖的几丁质脱乙酰酶法生产,采用Illumina测序技术,对1 株高产几丁质脱乙酰酶的红球菌菌株11-3进行基因组测序,并进行系统的生物信息学分析,主要包括基因本体论（Gene Ontology,GO）、直系同源群集（Cluster of Orthologous Group,COG）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes,KEGG）功能注释,碳水化合物活性酶注释以及几丁质降解相关酶基因的生物信息学分析。研究发现,红球菌11-3的基因组为6 089 866 bp,共5 904 个编码基因,GC含量为70.514%。经碳水化合物活性酶注释共识别了165 个基因,包括59 个碳水化合物酯酶基因,42 个糖基转移酶基因,36 个糖苷水解酶基因和28 个辅助氧化还原酶基因,其中,鉴定出1 个几丁质脱乙酰酶基因（gene4907）,4 个几丁质酶（EC 3.2.1.14）基因（gene1286、gene1287、gene3810、gene4754）和2 个壳聚糖酶（EC 3.2.1.132）基因（gene4921、gene5362）。gene4907与已报道的几丁质脱乙酰酶基因的序列一致性为26.60%～32.43%,为一种新的几丁质脱乙酰酶。因此,红球菌11-3菌株在几丁质资源的开发领域具有极大潜力。     

南极磷虾壳聚糖、壳寡糖的制备与品质鉴定

本研究以南极磷虾壳为原料,制备较高品质的壳聚糖与壳寡糖,并对二者的品质进行鉴定。南极磷虾壳经脱钙、脱蛋白处理,探索脱乙酰反应条件（碱溶液浓度、反应温度与反应时间）,制备具有较高脱乙酰度的南极磷虾壳聚糖,并对壳聚糖的理化指标进行鉴定;探索酶法降解条件（壳聚糖酶添加量、酶解时间）,制备较高纯度的南极磷虾壳寡糖,并对壳寡糖的结构特征进行鉴定。结果表明,使用60%的氢氧化钠于110 ℃脱乙酰处理4 h制备的南极磷虾壳聚糖脱乙酰度为85.74%,粘均分子量为 305.65 kDa,水分含量4.66%,灰分含量0.98%,酸不溶物含量0.40%,各项理化指标均符合食品级壳聚糖的要求;使用壳聚糖酶水解南极磷虾壳聚糖制备壳寡糖,在壳聚糖酶添加量为0.2% （m/V）,酶解16 h条件下,南极磷虾壳寡糖产品得率为46.0%,红外光谱与NMR谱图显示了表征壳寡糖结构的全部特征峰,质谱结果显示南极磷虾壳寡糖主要由二糖(GlcN)₂、三糖(GlcN)₂-GlcNAc与四糖(GlcN)₃-GlcNAc构成。本研究通过制备较高品质的壳聚糖与壳寡糖,为南极磷虾壳的高值综合利用与南极磷虾新产品开发提供了技术支持。     

壳聚糖酶解产物的功能研究

为了了解壳聚糖酶解产物的组分及功能,利用蜡状芽孢杆菌发酵所得到的壳聚糖酶,经盐析,透析,蛋白质纯化等步骤精制后所得酶液,对壳聚糖商品进行酶解,进而利用液质联用分析壳聚糖酶解产物的组分,并对酶解产物抗氧化的能力,以及螯合重金属的功能进行了研究。发现利用酶法降解壳聚糖所得的酶解产物中的组分分为4部分,分别为N-乙酰氨基葡萄糖、壳二糖、壳三糖、壳四糖,除N-乙酰氨基葡萄糖外,其余均为低聚合度(2～4)的壳寡糖。壳寡糖具有抗氧化的能力,且浓度越高,抗氧化的效果越显著。壳寡糖具有螯合Zn~(2+)和Cu~(2+)的能力,主要是通过分子内的羟基、氨基和乙酰氨基三个活性官能团与Zn~(2+)和Cu~(2+)发生配位反应。     

酶法降解壳聚糖及壳寡糖的初步研究

利用Mitsuaria sp.141-2发酵所得的粗酶液降解壳聚糖,研究了反应时间、底物浓度、pH、温度、加酶量、脱乙酰度对酶促反应的影响。结果表明,该酶降解壳聚糖的最适条件为:底物浓度3%,pH 5.2～5.6,温度65℃,加酶量7 U/g壳聚糖。利用薄层层析法对酶解产物进行分析,酶解产物大部分为三糖和四糖,单糖的含量随酶解时间延长而逐步增多。酶解3 h后可得到平均聚合度小于10的壳寡糖混合物。     

酶解蟹壳制备几丁寡糖的初步研究

烟曲霉YJ-407产生的外泌几丁质酶具有内切、外切、转糖苷活性及良好的热稳定性.旨在充分利用该几丁质酶独特的作用方式,诱导其大肠杆菌基因工程菌表达几丁质酶,直接将其酶解乳化蟹壳并分析其酶解产物,旨在分析以蟹壳为原料利用该几丁质酶酶解生产几丁寡糖的可行性.证明乳化蟹壳是该几丁质酶的较好底物,酶解产物经HPAEC-PAD及TLC分析得出主要是几丁二糖、N-乙酰葡萄糖胺和几丁三糖.充分利用食品工业废弃物螃蟹壳,避开酸碱化学处理,通过该几丁质酶酶解生产几丁寡糖是可行的.     

球磨、超声和盐酸处理对几丁质的微观结构和酶促脱乙酰效率的影响

几丁质是地球上第二丰富的天然多糖,可经过脱乙酰生成壳聚糖。但是,其结晶度高、溶解性差,使其酶促脱乙酰效率很低。为研究不同处理对几丁质的微观结构和酶促脱乙酰效率的影响,本实验分别对几丁质进行了球磨、超声和盐酸改性处理。首先确定球磨和超声改性的最佳条件;然后使用傅里叶变换红外光谱、元素分析、X射线衍射、热重-差示扫描量热法和扫描电子显微镜对改性几丁质的微观结构进行表征;最后测定红球菌11-3的几丁质脱乙酰酶对改性几丁质的脱乙酰效率。结果表明,球磨和超声改性的最佳条件分别为1 800 r/min、45 min和400 W、45 min。3种几丁质改性方法中,球磨几丁质改性效果最好,与原几丁质相比,黏均分子质量降低了88.14%;几丁质分子间氢键网络被破坏,部分糖苷键断裂;脱乙酰度有所增加;结晶度由96.30%降低至73.04%;热稳定性被破坏;结构呈现堆叠现象,变得疏松多孔。此外,几丁质脱乙酰酶对球磨几丁质的脱乙酰效率更高,乙酸产量较原几丁质提高了2.40倍。综上,球磨处理可以更有效地改变天然几丁质的理化性质和微观结构,从而提高几丁质的酶促脱乙酰效率。     

组成型壳聚糖酶制备壳寡糖酶解条件的优化

主要对组成型壳聚糖酶降解壳聚糖制备低壳寡糖的工艺进行了研究。以酶解产物壳寡糖的产量(mg)为指标,酶解pH、酶解温度(℃)、底物浓度(%)、酶解时间(min)为自变量,在单因素试验的基础上,通过正交实验确定了最佳酶解工艺为:酶解pH 7.0、酶解温度50℃、酶解时间90 min、底物浓度1%,此时壳寡糖产含量为5.476 mg。     

Enzymatic preparation of chitooligosaccharides by commercial lipase

The effect of a commercial lipase on chitosan degradation was investigated. When four chitosans with various degrees of deacetylation were used as substrates, the lipase showed higher optimal pH toward chitosan with higher DD (degree of deacetylation). The optimal temperature of the lipase was 55 °C for all chitosans. The enzyme exhibited higher activity to chitosans which were 82.8% and 73.2% deacetylated. Kinetics experiments show that chitosans with DD of 82.8% and 73.2% which resulted in lower K_m values had stronger affinity for the lipase. The chitosan hydrolysis carried out at 37 °C produced larger quantity of COS (chitooligosaccharides) than that at 55 °C when the reaction time was longer than 6 h, and COS yield of 24 h hydrolysis at 37 °C was 93.8%. Products analysis results demonstrate that the enzyme produced glucosamine and chitooligosaccharides with DP (degree of polymerization) of 2–6 and above, and it acted on chitosan in both exo- and endo-hydrolytic manner.     

木瓜蛋白酶降解壳聚糖

通过粘度测定、还原端基测定和凝胶层析分子式量分布测定 ,研究了木瓜蛋白酶非专一性降解壳聚糖过程中温度、pH值、反应时间和酶用量等因素对木瓜蛋白酶降解能力的影响 ,探讨了壳聚糖脱乙酰化度和相对分子质量与木瓜蛋白酶降解反应的关系 .结果表明 :木瓜蛋白酶在温度4 0～ 4 5℃、pH 4 .0～ 5 .0和酶用量 0 .5 %～ 10 %范围内 ,对壳聚糖的降解将随酶用量的增加而加快 ,但随脱乙酰度的升高而减慢 ;木瓜蛋白酶适合作用于数均分子式量在 2 0～ 5万的虾壳聚糖 .     

Process Characteristics of Hydrolysis of Chitosan in a Continuous Enzymatic Membrane Reactor

ABSTRACT: Crude enzyme from Bacillus cereus NTU-FC-4 was used to hydrolyze chitosan of 66% deacetylation in a membrane reactor, operated at 45 °C and pH 5, to continuously produce chitooligosaccharides. Major oligomers in the product from the reactor were chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose. When the membrane reactor was operated at an enzyme/substrate ratio of 0.2 (unit/mg) and residence time of 100 min, it reached steady state in 2.5 h. The system could be operated for 15 h and still maintained a stable product composition. When the volume replacement exceeded 2.5, the productivity of the membrane reactor became higher than that of the batch reactor, and the difference between them became even greater when the volume replacement was further increased. The apparent Michaelis constant (K_m) for the enzyme in the membrane reactor was 18.8 mg/mL, but the apparent K_m was 5.4 mg/mL for the batch reactor, suggesting that the affinity of the enzyme for chitosan was lower in the membrane reactor compared with the enzyme in the batch reactor. The estimated values of apparent V_max were 0.18 and 0.20 mg reducing sugar/mL/min for the enzyme in the membrane reactor and in the batch reactor, respectively, indicating that the enzyme activity was not greatly altered when used in the membrane reactor.     

Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326

Aspergillus CJ22-326, a fungi strain capable of utilizing chitosan as a carbon source, was isolated from soil samples. Two types of chitosanase (ChiA and ChiB) produced from the culture supernatant of Aspergillus CJ22-326 were purified to an apparent homogeneity identified by SDS–PAGE through ammonium sulfate precipitation, CM-Sepharose FF chromatography, and Sephacryl S-200 gel filtration. Molecular weights of the enzymes were 109 kDa (ChiA) and 29 kDa (ChiB). Optimum pH values and temperature of ChiA were 4.0 and 50 °C, respectively, those of ChiB were 6.0 and 65 °C. The enzyme activities of ChiA and ChiB were increased by about 0.5-fold and 1.5-fold, respectively, by the addition of 1 mM Mn²⁺. However, 2.5 mM Ag⁺, Hg²⁺ and Fe³⁺strongly inhibited ChiA and ChiB activities. Viscosimetric assay and analysis of reaction products of these enzymes, using chitosan as a substrate, by TLC indicated endo- and exo-type cleavage of chitosan by ChiB and ChiA, respectively. ChiB catalysed the hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, and chitosan with a low degree of acetylation (0–30%), and formed chitotriose with chitohexaose as the major products. ChiA released a single glucosamine residue from chitosan and glucosamine oligomers. Both of the activities of ChiA and ChiB increased with the degree of deacetylation of chitosan. The enzyme ChiB had a useful reactivity and a high specific activity for producing functional chitooligosaccharides with high degree of polymerization.     

枯草芽孢杆菌壳聚糖酶在毕赤酵母中的高效表达及其酶解特性

研究枯草芽孢杆菌壳聚糖酶基因（BsCsn46）在巴斯德毕赤酵母（Pichia pastoris）GS115中的高效表达、重组酶性质及其酶解特性。重组菌在5 L发酵罐高密度发酵后胞外酶活力高达50 370 U/mL,蛋白质量浓度15.7 mg/mL。粗酶经强阴离子交换层析纯化,纯酶比活力为4 065.7 U/mg,最适pH 6.0,最适温度55 ℃,在45 ℃以下保持稳定。该酶水解3 g/100 mL壳聚糖得到主产物为二糖、三糖和四糖的壳寡糖,水解率为92.8%,壳寡糖得率为90.9%。本研究的重组壳聚糖酶产酶水平和水解效率高,为工业化制备壳聚糖酶及大规模制备壳寡糖的应用提供了理论支持。     

Production of high degree polymerized chitooligosaccharides in a membrane reactor using purified chitosanase from Bacillus cereus

Crude chitosanase from Bacillus cereus NTU-FC-4 was separated by a cation exchanger to three fractions named CBCI, CBCII, and CBCIII. The CBCI hydrolyzed chitosan to yield dimers. The primary hydrolytic products of CBCII were low degree polymerized (DP) chitooligosaccharides. The CBCIII had the fastest reaction rate and yielded high DP chitooligosaccharides (heptamer and higher DP oligomers). When CBCIII was used in the ultrafiltration membrane reactor with enzyme/substrate ratio 0.06 unit/mg and 100 min of residence time (RT), the concentration of high DP oligomers was 9.78 mg/mL which occupied ca. 48% of total oligomers in the final product as compared to ca. 29% resulted from the crude enzyme. Decrease of RT to 50 min and 33 min, the high DP oligomers in the products were ca. 61% and 69%, respectively. This system could be operated for at least 24 h and kept a constant permeate flux and product output rate.     

壳寡糖席夫碱纳米银溶液对烟草花叶病防治及烟叶色素的影响

为开发防治烟草花叶病的新型药剂,采用大田试验研究了壳寡糖纳米银溶液、壳寡糖席夫碱纳米银溶液及宁南霉素对烟草花叶病的田间防治效果。结果表明,壳寡糖和壳寡糖席夫碱的纳米银溶液对烟草花叶病的防治效果均优于宁南霉素,以预防和治疗效果分别达到53.32%、24.95%的壳寡糖席夫碱纳米银溶液为最好,能够减轻病株叶绿素和类胡萝卜素含量的下降幅度,增强烟株的光合作用强度,使烟株的抗病性得到提高。壳寡糖及其衍生物的纳米银溶液对烟草花叶病具有较好的防治效果,为今后开发绿色、高效的抗TMV药剂提供了新思路。     

响应面法优化微波辅助果胶酶制备壳寡糖的工艺

为优化壳寡糖制备工艺,提高壳寡糖得率,以壳聚糖为原料,采用微波法辅助果胶酶酶解壳聚糖制备壳寡糖,以还原糖含量作为壳寡糖的产率依据,通过单因素实验和响应面实验确定最佳工艺条件为:果胶酶加酶量2100 U/g,微波功率510 W,pH4.4,反应温度50℃,在此工艺条件下获得的降解产物中还原糖浓度为1.964 mg/mL,与单一果胶酶酶解法(1.747 mg/mL)相比提高了12%,与单一微波法(1.671 mg/mL)相比提高了18%。