Evaluation of In Vitro Enzymatic Degradation of Various Thiomers and Cross-Linked Thiomers

ABSTRACT

The aim of this study was to examine the biodegradability of thiomers and cross-linked thiomers in comparison with unmodified polymers. Disulfide-cross-linked conjugates were prepared by air oxidation at room temperature. Thiomers were investigated by viscosity measurements and spectrophotometric assays. The influence of different factors on the hydrolysis rate, such as the degree of modification of thiomers, structure of the conjugates, pH value of the reaction medium, and the impact of the process of cross-linking were evaluated. Due to the modification, thiolated chitosans degraded 12.9–24.7% less than unmodified chitosan in the framework of viscosity measurements. In addition, the hydrolysis degree of thiolated alginates and modified carboxymethylcelluloses was 25.6–32.4% and 18.4–27.0% lower, respectively, in comparison to the corresponding unmodified polymers. Conjugates with higher coupling rate of thiol groups were degraded even more slowly. Moreover, the cross-linking process via disulfide bonds additionally reduced the rate of thiomer degradation. The range of degradation rates achieved in vitro could be modified by alterations of the contents of thiol and disulfide groups, as well as by suitable design of the polymer structure and ligands used.

These results represent helpful basic information for the development of mucoadhesive drug delivery systems, implantable delivery systems and tissue engineering constructs.     

Purification and characterisation of two enzymes related to endogenous formaldehyde in Lentinula edodes

In this study, γ-glutamyl transpeptidase (GGT) and l-cysteine sulphoxide lyase (C-S lyase) were purified from the fruiting body of Lentinula edodes in three steps and then characterised. We found that GGT together with C-S lyase caused the generation of endogenous formaldehyde in L. edodes. GGT was composed of a large subunit of 41 kDa and a small subunit of 25 kDa, and C-S lyase was composed of two identical subunits of 46 kDa, as determined by SDS–PAGE. GGT was stable at pH 8.0–10.0 with an optimum pH of 8.8, and was stable at 20–50 °C with an optimum activity at 37 °C. C-S lyase was stable at pH 8.0–9.0 with an optimum pH of 8.5, and was stable at 20–60 °C with an optimum activity at 40 °C. The present work supports the study of the mechanism of endogenous formaldehyde in L. edodes.     

马尾藻发酵生产生物乙醇的两步法糖化预处理

马尾藻是典型的大型褐藻,含有大量的碳水化合物,通过糖化处理后能够用于生物乙醇的发酵生产。研究了马尾藻的两步水解方法,优化了两步水解法中酸水解和酶水解的最适水解条件。试验发现在料液比为6%(w/v),H2SO4浓度1.5%(v/v),时间40 min,温度为120℃的水解条件下马尾藻的第1步酸水解效率可以达到(31.05±0.32)%(w/w)。进一步用海藻酸盐裂解酶进行第2步水解,最适水解条件:料液比5%(w/v)、酶用量0.0025%(w/v)、时间2 h,第2步酶水解后马尾藻的水解效率可以在第1步水解的基础上提高9.68%(w/w)。最终两步法的总水解效率可以达到38%～40%(w/w),达到理论产量的89%。用工业酿酒酵母R1-11对水解液进行发酵实验,在未优化发酵条件的情况下每千克马尾藻生产乙醇60.75 g,当水解液补充1%(w/v)(NH4)2SO4时乙醇的产量可以达到每千克马尾藻生产乙醇91.9 g。     

5-氨基乙酰丙酸对苹果幼果多酚含量、苯丙氨酸解氨酶活性及其基因表达的影响

探究5-氨基乙酰丙酸（aminolevulinic acid,ALA）处理对苹果幼果酚类物质、苯丙氨酸解氨酶（phenylalanin ammonia-lyase,PAL）活性及其基因表达量的影响。在苹果疏果期前,利用不同质量浓度ALA处理苹果幼果,采用紫外分光光度法测定ALA处理后12 d内苹果多酚和PAL活性,利用荧光定量法测定苹果幼果PAL基因表达量。结果表明：ALA处理质量浓度在300 mg/L以内,随着质量浓度提高,苹果多酚含量、PAL酶活性及其基因表达量均升高,到400 mg/L时各项指标均表现出下降趋势;相同质量浓度ALA处理苹果幼果后,幼果的上述3 项指标均随时间延长而明显提高,在第9天时达到最高值,12 d后开始下降。为提高苹果疏果多酚含量,实现疏果的最大利用率,生产中需要选择适宜的ALA处理浓度和时间采集幼果。     

Accumulation of lignin and involvement of enzymes in bamboo shoot during storage

Fresh bamboo (Phyllostachys praecox f. prevernalis.) shoots were harvested, and the changes of firmness, electrical conductivity (EC), respiration rate, ethylene production, total sugars (TS), reducing sugars (RS), lignin and cellulose content and the activity of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase POD were investigated during storage at 2 or 20°C for 30 or 12 days, respectively. EC increased quickly, with a more rapid rate at higher temperature, but it increased sharply after 24 days at 2°C. Respiration rate increased slowly at 20°C, while at low temperature (2°C) respiration rate and ethylene production significantly decreased. There was a quickly decrease in TS content at 20°C. Shoot firmness, lignin and cellulose increased and accelerated by higher storage temperature during storage at 2 or 20°C. Shoots firmness showed a positive correlation with accumulation of lignin and cellulose in the flesh. Among the enzymes associated with lignin synthesis, PAL and CAD activity showed a persistent rise over the whole 12 days, while POD activity increased rapidly within 6 days during storage at 20°C, followed by a slowly decrease. Accumulation of lignin in flesh tissue was also positively correlated to activity of PAL, CAD and POD. Our results suggest that the increase in firmness of bamboo shoots during storage is a consequence of tissue lignification, a process associated with increases in PAL, CAD and POD activity.     

采收成熟度与冷藏枇杷果实木质化关系研究

以七成、八成和九成熟的“解放钟”枇杷(Eriobotrya japonica Lindl. cv. Jiefangzhong)果实为试材,于4℃贮藏期间测定3 种不同成熟度果实的品质指标及木质化相关酶活性的变化,确定适宜于冷藏的枇杷果实采收成熟度,以减缓冷藏期间果实木质化败坏的发生,延长其贮藏期。结果表明：果实冷藏至35d,成熟度越高,果肉木质化越严重,但七成与八成熟果实木质素含量差异不明显(P ＞ 0.05);木质化程度相对较低;八成熟果实的总糖和可溶性固形物(TSS)含量明显高于七成熟和九成熟果实(P ＜ 0.05)。不同成熟度果实可滴定酸(TA)含量以及4- 香豆酸辅酶A 连接酶(4CL)、肉桂醇脱氢酶(CAD)和过氧化物酶(POD)活性差异不显著(P ＞ 0.05),九成熟果实苯丙氨酸解氨酶(PAL)酶活性明显高于七成和八成熟(P ＜ 0.05),PAL 活性与木质素含量之间具有较高的正相关性(r=0.89)。不同采收成熟度与冷藏枇杷果实木质化密切相关,适于冷藏的枇杷果实以八成熟采收为宜。