耐热芽孢杆菌E2菌株纤维素酶基因克隆的研究

用质位ｐＢＲ３２２作载体，大肠杆菌Ｅ．ｃｏｌｉＤＨ５αＦ＇作受体，克隆到耐热芽孢杆菌Ｅ２菌株的羧甲基纤维素酶（ＣＭＣａｓｅ）基因。重组质粒ｐＢＧ３从产生ＣＭＣａｓｅ的转化子中分离得到。克隆到的ＣＭＣａｒｅ基因位于４．０ｋｂＨｉｎｄＩＩＩ片段上。功能状态ＣＭＣａｓｅ基因被亚克隆到２．４ｋｂＤＮＡ片段上。同位素杂交结果显示出克隆到的ＣＭＣａｓｅ基因与供体菌染色体ＤＮＡ的亲合性。重组质粒ｐＢＧ３所表达的ＣＭＣａｓｅ的最适作用ｐＨ为６．５，最适作用温度为５５℃，不作用于任何实验过的天然纤维素。含重组质粒ｐＢＧ３的转化子产生的纤维素酶在细胞中的分布为：胞内占８２．９％，胞外占７．９％，质壁空间（ｐｅｒｉｐｌａｓｍｉｃｓｐａｃｅ）占９．２％。

Optimization of Fermentation Conditions for Cellulase Production from Camellia Seed Shell Using Response Surface Methodology

Objective] This study was conducted to optimize cameI ia seed sheI fer-mentation conditions for ceI uIase production by Trichoderma koningi using response surface methodoIogy. [Method] Fermentation conditions for ceI uIase production from Trichoderma koningi were optimized with response surface method （RSM） by taking carboxymethyI ceI uIase （CMCase） activity as a response indicator. Three factors that affecting CMCase activity were screened out using singIe factor test among pretreatment methods of raw material, nitrogen sources, initial pH values, inocuIum voIume, fermentation time and voIume of Iiquid medium, they were fermentation time, initial pH value, and voIume of Iiquid medium. The optimum conditions of fer-mentation and interaction of the three factors were determined through Box-Behnken design and regression analysis using Design-Expert software. [Result] Pretreatment of cameI ia seed sheI with alkaline was most conducive to CMCase production. The use of 0.2% （NH4）2SO4 as nitrogen source, inocuIum size of 5%, initial pH value of 5.8 and voIume of Iiquid medium at 22 mI were the best fermentation conditions for maximizing CMCase production by T. koningi from cameI ia seed sheI . Under these conditions, 179.15 U/mI of CMCase was obtalned after 5 d of fermentation, which was improved by 24.52% compared with the maximum CMCase activity of singIe factor test. [Conclusion] The resuIts wiI provide some references for use of cameI ia seed sheI and ceI uIase production.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

In vitro Evaluation of Lysozyme-loaded Microspheres in Thermosensitive Methylcellulose-based Hydrogel

Long-terrn injectable microspheres have some inherent disadvantages such as migration of microspheres from the originalsite an.d the burst effect. In order to avoid these problems, microsphere-loaded thermosensitive, hydrogel system was designed and expected to achieve a zero-order release Of biomolecular drugs in relativehigh initial drug loadings. Lysozyme, an antibacterial protein usually used to reduce prosthetic valve endocarditis,was selected as the model drug. Poly （DL-lactide-co-glycolide） （PLGA） microspheres, prepared by solvent evaporation method, were employee to encapsulate lysozyme and dispersed into thermosensitive pre-gel solution containing methylcellulose （MC）, polyethylene glycol （PEG）, sodium citrate （SC）, and sodium alginate （SA）. The mixture could act asadrug reservoir by.performing sol-gel transition rapidly if the temperature was raised from roomtemperature to 37℃. The in vitro release results showed that the burst effect was avoided due to strengthening ofdiffusion resistance in the gel. The formulation was able.to deliver lysozy.me for over.30 daysin a nearly zero-order release profile with a rate of 32.8μg.d^-1 which exhibits its remarkable potential for effective aoolication in long-term drug delivery.

Enzymatic saccharification of cellulose using an ionic liquid [Amim] [COOH] pretreatment

In this study we used l-allyl-3-methyl imidazole formate （[Amim][COOH]） as ionic liquid to pre-treat the cellulose and determined the rate of polymerization and enzymatic hydrolysis. The results showed that pretreatment with （[Amim][COOH]） significantly decreased the cellulose polymerization. As the pretreatment temperature went up, the enzymatic hydrolysis rate was first increased and then decreased The maximal enzymatic hydrolysis rate was achieved when the pretreatment temperature was 90 ℃. Under the ultrasonic condition, the initial rate of enzmatic hydrolysis for the ionic liquid-treated cellulose was up to 11.10 gL-1h-1, which was 33% increase compared to the untreated cellulose. Scanning Electronic Microscopy （SEM） and Fourier Transform Infrared-Raman Spectroscop （FT-IR） analysis showed that ionic liquidtreated cellulose started to depolymerize. In addition, the cr3＇stallinity of the cellulose was significantly decreased after pretreatment with ionic liquid.